Chemical Constituents of Anneslea fragrans and Their Antiausterity Activity against the PANC-1 Human Pancreatic Cancer Cell Line

Article abstract of DOI:10.1021/acs.jnatprod.9b00735

An ethanolic extract of Anneslea fragrans leaves showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under a nutrient-deprived condition, with a PC₅₀ value of 9.6 μg/mL. Phytochemical investigation of this active extract led to the isolation of two new secondary metabolites, fragranones A (1) and B (2), along with 15 previously reported compounds. The structure elucidation of the new compounds was achieved by HRFABMS, acid hydrolysis, NMR, and ECD spectroscopic analysis. Fragranone A (1) is the first example of a rare natural product bearing an acetonide glucose moiety. Fragranone B (2) is representative of a rare class of natural products with a threonolactone unit linked to a chalcone through an ether linkage. The isolated compounds exhibited antiausterity activity against PANC-1 cells under nutrient-deprived conditions, and betulin (14) was found to be the most potent compound tested, with a PC₅₀ value of 8.4 μM. In addition, fragranone A (1) was found to suppress PANC-1 cancer cell migration in real time.

Full text of DOI:10.1021/acs.jnatprod.9b00735

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Chemical Constituents of Anneslea fragrans and Their Antiausterity
Activity against the PANC‑1 Human Pancreatic Cancer Cell Line
†
†
†
†
‡
Ashraf M. Omar, Dya Fita Dibwe, Ahmed M. Tawila, Sijia Sun, Ampai Phrutivorapongkul,
,
†
and Suresh Awale*
†
Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand
‡
*
S Supporting Information
ABSTRACT: An ethanolic extract of Anneslea fragrans leaves
showed potent preferential cytotoxicity against PANC-1
human pancreatic cancer cells under a nutrient-deprived
condition, with a PC50 value of 9.6 μg/mL. Phytochemical
investigation of this active extract led to the isolation of two
new secondary metabolites, fragranones A (1) and B (2),
along with 15 previously reported compounds. The structure
elucidation of the new compounds was achieved by
HRFABMS, acid hydrolysis, NMR, and ECD spectroscopic
analysis. Fragranone A (1) is the first example of a rare natural
product bearing an acetonide glucose moiety. Fragranone B
(
2) is representative of a rare class of natural products with a
threonolactone unit linked to a chalcone through an ether
linkage. The isolated compounds exhibited antiausterity activity against PANC-1 cells under nutrient-deprived conditions, and
betulin (14) was found to be the most potent compound tested, with a PC value of 8.4 μM. In addition, fragranone A (1) was
5
0
found to suppress PANC-1 cancer cell migration in real time.
ancer is continuing to be a challenging health problem in
cancer cells without toxicity in nutrient-rich conditions are
classified as “antiausterity agents”. Previous studies using this
4
,5
C
the 21st century. It was responsible for over 9.6 million
1
deaths worldwide in the year 2018. Among all types of cancer,
pancreatic cancer is known to be the deadliest in having the
lowest chance of survival after initial diagnosis. It has the
strategy resulted in the discovery of several potent antiausterity
4
6
7
agents such as arctigenin, angelmarin, isopanduratin A1,
7
8
9
nicolaioidesin C, bergamotin, and ancistrolikokine E .
3
2
lowest five-year relative survival rate of less than 5%. In Japan,
In a continuing investigation, a 95% ethanolic extract of
Anneslea fragrans Wall. (Theaceae) leaves, collected from the
Chiang Mai Province of Thailand, showed discernible
preferential cytotoxicity against PANC-1 cancer cells under a
nutrient-deprived condition with a PC50 value of 9.6 μg/mL.
Therefore, a phytochemical investigation of this active extract
was carried out and resulted in the isolation of the new
dihydrochalcone glucoside fragranone A (1) and the new
chalcone fragranone B (2), together with 15 previously
reported compounds. The isolated compounds can be
classified as five dihydrochalcones, two chalcones, five flavonol
an estimated 40 000 patients were diagnosed with pancreatic
cancer in 2018 and 34 900 deaths occurred from this
2
malignancy. The incidence rate is nearly equivalent to the
mortality rate. The aggressiveness of pancreatic cancer and its
inadequate response to either chemotherapy or radiotherapy
have been complicated considerably due to both the absence of
appropriate screening tests and diagnostic markers for its early
3
detection. Therefore, there is an urgent need to find effective
new agents for the treatment of pancreatic cancer.
Pancreatic tumors are hypovascular in nature, yet they thrive
under a nutrient-deficient “austere” tumor microenvironment.
In particular, PANC-1 human pancreatic cancer cells have
shown extreme tolerance to nutrition starvation and survive for
a prolonged time even in the complete absence of glucose,
3
-O-glycosides, three triterpenoids, and two miscellaneous
compounds. The previously reported compounds were
1
0
identified as 3,4,2′,4′-tetrahydroxydihydrochalcone (3),
11
12
13
confusoside (4), vacciniifolin (5), davidioside (6), butein
14
15
16
17
19
4
(
7), afzelin (8), quercitrin (9), isoquercitrin (10),
18
amino acids, and serum. Such a phenomenon is referred to as
kaempferol 3-neohesperidoside (11), nictoflorin (12),
“
austerity”. An antiausterity strategy is an approach for
20
20
21
lupeol (13), betulin (14), spinasterol (15), (epi)catechin
discovering unique anticancer agents that can target preferen-
tially the ability of cancer cells to tolerate nutritional starvation.
Test compounds and extracts are evaluated under both
nutrient-rich and nutrient-deprived conditions, and the agents
showing preferential cytotoxicity against nutrient-deprived
22
23
(
16), and isoscopoletin (17) (Chart 1).
Received: August 2, 2019
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
RESULTS AND DISCUSSION
and H-1″/H-5″ suggested their α-axial orientation, while
correlations between H-4″/H-6″b indicated a β-axial orienta-
■
Fragranone A (1) was isolated as a white, amorphous solid. Its
tion. Furthermore, NOESY correlations between H -1‴/H-4″,
3
molecular formula was deduced by HRFABMS as C H O
2
4
28
9
+
H -1‴/H-6″b and H-4″/H-6″b suggested the trans-fusion of
3
[
m/z 461.18126 (M + H) ]. The IR spectrum of 1 showed
−
1
the β-glucose moiety with a ring-bearing acetonide unit, with
both rings adopting a chair conformation. Finally, acid
hydrolysis of 1 was carried out to confirm the absolute
configuration of the glucose moiety, which yielded a sugar with
absorption bands for hydroxy (3420 cm ) and α,β-
−
1
unsaturated carbonyl (1634 cm ) groups. The UV spectrum
of 1 showed absorption maxima at 208, 272, and 320 nm. The
H NMR spectrum of 1 (Table 1) displayed the presence of a
1
2
5
[
α]
+67.7 (c 0.06, H O) (Figure S11, Supporting
D 2
1
,4-disubstituted phenyl ring [δ 6.70 (d, J = 8.5 Hz, H-3,5)
H
Information), similar to the value reported for D-glucose of
and 7.06 (d, J = 8.5 Hz, H-2,6)] along with signals for a 1,2,4-
2
5
25
[
α]
+52 (c 0.02, H O). Thus, the structure of 1 was
D 2
trisubstituted phenyl ring [δ 6.56 (d, J = 2.4 Hz, H-3′), 6.59
H
assigned as davidigenin-4′-O-(4″,6″-O-isopropylidene-β-D-glu-
copyranoside).
(
dd, J = 2.4, 9.2 Hz, H-5′), and 7.81 (d, J = 9.2 Hz, H-6′)] and
two mutually coupled methylenes [δ 2.92 (t, J = 7.6 Hz, H -
H
2
The occurrence of an acetonide moiety among natural
β) and 3.23 (t, J = 7.6 Hz, H -α)]. These signals closely
2
26−28
2
4
1
products is rare but does exist in Nature.
A phenyl-
resembled those of davidigenin. In addition, the H NMR
spectrum showed the presence of an anomeric proton [δ 5.09
propanoid with an acetonide unit has been reported recently
H
29
from the rhizomes of Acorus gramineus. A resocyclic acid
(
(
d, J = 7.3 Hz, H-1″)] and two diastereotopic protons [δ 3.78
H
lactone with a natural acetonide group was isolated from
dd, J = 10.4, 10.5 Hz, H-6″b) and 3.91 (dd, J = 5.5, 10.5 Hz,
30
Colchiobolus lunatus. However, an acetonide incorporated in
a sugar unit has never been found from Nature before.
Therefore, in an attempt to confirm 1 as being a natural
compound, 20 mg of confusoside (4), which lacks the
acetonide unit in 1, was dissolved in acetone (5 mL) and
allowed to stir at room temperature for 1 week. Afterward, the
H-6″a)] corresponding to a β-glucose unit, in addition to two
1
3
methyl singlets [δ 1.41 (H -1‴) and 1.52 (H -3‴)]. The
C
H
3
3
and DEPT NMR spectra of 1 (Table 1) also exhibited signals
corresponding to davidigenin and a β-glucose moiety, together
with those of two methyls [δ 19.3 (C-3‴) and 29.4 (C-1‴)]
C
and an acetal carbon [δC 100.9 (C-2‴)]. In the HMBC
spectrum of Figure 1, correlations were observed from the two
methyls to the acetal carbon, which suggested the presence of
an acetonide unit. The HMBC correlation from H-6″a to the
quaternary acetal carbon was consistent with the placement of
the acetonide moiety as being attached to OH-4″ and -6″ of
the β-glucose unit. The HMBC correlation from H-1″ to C-4′
1
solvent was evaporated, and the product was subjected to H
NMR spectroscopic analysis. There was no change in the
structure of confusoside (4). Moreover, acetone was not used
throughout the extraction and isolation processes in the
present phytochemical investigation. Therefore, it is proposed
that fragranone A (1) represents the first example of a rare
natural product bearing an acetonide glucose moiety.
(
δ 164.8 Hz) confirmed the location of the sugar moiety at C-
C
4
′ of the davidigenin unit. The relative configuration of 1 was
Fragranone B (2) was isolated as a yellow oil. It showed a
1
1
+
assigned by analyzing the coupling constants and H− H
molecular ion peak at m/z 389.08816 [M + H] ,
NOESY data (Figure 2). The large coupling constant (J
=
corresponding to the molecular formula, C H O . The IR
1
″,2″
19 16 9
7
.3 Hz) suggested the trans-disposition of H-1″ (α) and H-2″
spectrum of 2 showed absorption bands due to hydroxy (3407
cm ) and α,β-unsaturated carbonyl (1602 cm ) groups. The
−
1
−1
(β). The presence of NOESY correlations between H-1″/H-3″
B
DOI: 10.1021/acs.jnatprod.9b00735
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Journal of Natural Products
Article
Table 1. H NMR (400 MHz) and 13C NMR (100 MHz)
1
Spectroscopic Data of 1 and 2
a
b
1
2
position
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
1
2
3
4
5
6
133.1, C
130.4, CH 7.45, d (1.8)
116.3, CH
156.7, C
116.3, CH 6.84, d (8.0)
113.0, C
7.06, d (8.5)
6.70, d (8.5)
117.8, CH
d
Figure 2. Most stable conformer of 1 calculated at the DFT/B3LYP/
145.4,
C
6
-31G* level of theory showing hydrogen bonds (dashed blue lines)
147.9, C
115.9, CH
124.4, CH
and key NOESY correlations (blue arrows) observed for 1.
6.70, d (8.5)
7.06, d (8.5)
130.4, CH 7.25, dd (1.8,
8
.0)
oxymethines (δ 72.1, 72.9), seven olefinic methines (δ 98.2,
C
C
d
α
3.23, t (7.6)
2.92, t (7.6)
41.3, CH2
30.8, CH2 6.64, s
206.2, C
116.1, C
165.8, C
145.5,
C
111.8, 112.8, 115.9, 117.8, 124.4, 125.6), two olefinic
quaternary carbons (δC 113.0, 123.3), five oxygenated
β
111.8, CH
181.0, C
123.3, C
166.1, C
98.2, CH
167.3, C
112.8, CH
CO
quaternary carbons (δC 145.4, 145.5, 147.9, 166.1, 167.3),
1
1′
2′
3′
4′
5′
and two carbonyl carbons (δ 175.5, 181.0). Part of the H
C
1
3
and C NMR data set resembled those of 3,4,2′,4′,α-
6.56, d (2.4)
105.0, CH 6.75, d (1.8)
164.8, C
pentahydroxychalcone, as confirmed from the COSY,
31
HMQC, and HMBC spectra of 2 (Figure 1).
6.59, dd (2.4, 9.2) 109.3, CH 6.71, dd (1.8,
.5)
The remaining carbon signals of 2 were identified as an
oxymethylene (δ 69.7, C-5″), two oxymethines (δ 72.9, C-3″
8
C
C
6
1
2
3
4
′
7.81, d (9.2)
5.09, d (7.3)
133.5, CH 7.60, d (8.5)
101.6, CH
125.6, CH
and δ 72.1, C-4″), and an ester carbonyl (δ 175.5, C-2″). In
C
C
″
″
″
″
the COSY spectrum, correlations were observed between H-
e
c
3.50−3.53
75.6 , CH
175.5, C
72.9, CH
72.1, CH
3
3
″, H-4″, and H -5″, suggesting the connectivity between C-
e
c
2
3.56−3.58
74.9 , CH 4.08, d (6.7)
″−C-4″−C-5″. In the HMBC spectrum, correlations were
e
c
3.58−3.6
74.5 , CH 4.15, ddd (6.7,
observed from H-3″, H-5″a, and H-5″b to an ester carbonyl
carbon (C-2″), suggesting the presence of an α-hydroxy-γ-
butyrolactone unit. Moreover, an HMBC correlation from H-
6
.7, 6.7)
68.7, CH 4.33, dd (6.7,
.8) 3.85, dd
6.7, 8.8)
e
5
″
″
3.45−3.50
69.7, CH2
8
(
4
″ to C-4′ (δ 167.3) suggested an ether linkage between the
C
6
3.91, dd (5.5, 10.5) 63.1, CH
lactone and the chalcone unit through C-4″−C-4′, thus
establishing the planar structure of fragranone B (2). The
relative configuration of 2 was assigned by a difference NOE
experiment (Figure 3) and coupling constant data (J3″,4″ = 6.7
3
1
.78, dd (10.4,
0.5)
1‴
2‴
3‴
1.41, s
29.4, CH3
100.9, C
19.3, CH3
Hz). Irradiation of the H-4″ (δ 4.15) signal caused the NOE
1.52, s
H
a
b
c
enhancement of H-5″a (δ 4.33, 3.6%) without any effect on
H
Measured in CD OD. Measured in DMSO-d . Interchangeable.
Interchangeable. Overlapped.
3
6
d
e
H-5″b. On the other hand, irradiation of the H-3″ (δ 4.08)
H
signal caused a NOE enhancement of H-5″b (δ 3.85, 2.6%)
H
without any effect on H-5″a, which suggested the relative
orientation of H-3″ and H-5″b on the same side of the
molecule. Therefore, the α-hydroxy-γ-butyrolactone unit in 2
was concluded to be threono-1,4-lactone. The absolute
configuration of the α-hydroxy lactones can be deduced by
employing the axial halo-ketone rule, with the orientation of
the α-hydroxy group determined from the sign of the
electronic circular dichroism (ECD) spectrum Cotton
UV spectrum of 2 exhibited absorption maxima at 208, 344,
and 376 nm. In turn, the H NMR spectrum of 2 (Table 1)
displayed signals due to the presence of two ABX-type phenyl
1
rings [δ 6.71 (dd, J = 1.8, 8.5 Hz, H-5′), 6.75 (d, J = 1.8 Hz,
H
H-3′), 7.60 (d, J = 8.5 Hz, H-6′), 6.84 (d, J = 8.0 Hz, H-5),
7
.25 (dd, J = 1.8, 8.0 Hz, H-6), and 7.45 (d, J = 1.8 Hz, H-2)]
and the signals characteristic for one AMXY system [δ 3.85
H
32,33
(
(
dd, J = 6.7, 8.8 Hz, H-5″b), 4.08 (d, J = 6.7 Hz, H-3″), 4.15
effect.
According to this rule, the S (or R) configuration
ddd, J = 6.7, 6.7, 6.7 Hz, H-4″), and 4.33 (dd, J = 6.7, 8.8 Hz,
at the α-carbon gives a positive (or a negative) Cotton effect at
220−230 nm. In the ECD spectrum of 2, a negative Cotton
effect was observed at 223 nm (Figure 4), suggesting the R-
configuration at C-3″. Therefore, the lactone was identified as
1
3
H-5″a)]. The C NMR spectrum of 2 (Table 1), in
combination with DEPT analysis, showed the presence of 19
carbons corresponding to an oxymethylene (δC 69.7), two
Figure 1. Connectivity (bold blue lines) deduced by COSY and HMQC and significant HMBC correlations (red arrows) in 1 and 2.
C
DOI: 10.1021/acs.jnatprod.9b00735
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Journal of Natural Products
Article
Figure 3. Most stable conformers of 2 calculated at the DFT/B3LYP/6-31G* level of theory. The relative populations are in parentheses. NOESY
correlations (blue arrows) and hydrogen bonds (dashed blue lines) in 2.
threonolactone with an ether linkage. The biogenetic
formation of 2 could involve the coupling of the L-threonic
acid with a 3,4,2′,4′,α-pentahydroxychalcone unit through an
ether linkage followed by the lactonization of the L-threonic
acid moiety to give fragranone B (2) (Figure 5).
All isolated compounds were tested for their preferential
cytotoxic activity against the PANC-1 cell line in both nutrient-
deprived medium (NDM) and standard nutrient-rich medium
(
Dulbecco’s modified Eagle medium, DMEM), using an
4
antiausterity strategy. The data are presented as preferential
cytotoxicity (PC ) values, which represent the concentration
5
0
of 50% cancer cell death in NDM without any toxicity in
DMEM (Table 2). Among the compounds tested, betulin (14)
Table 2. Preferential Cytotoxicity (PC ) of Compounds 1−
50
1
7 against the PANC-1 Human Pancreatic Cancer Cell Line
a
Figure 4. Experimental and calculated ECD spectra of 2.
in Nutrient-Deprived Medium (NDM)
a
a
compound
PC , μM
compound
PC , μM
50
5
0
L-threono-1,4-lactone. The absolute configuration of 2 was
further confirmed by computational ECD calculation using
density functional theory (DFT). The (3″R,4″S) configuration
for 2 was analyzed for the lowest energy conformation and
their Boltzmann distribution, followed by optimization using
DFT theory at the B3LYP/6-31+G* level. Two stable
conformers with predominating relative populations, 94%
and 5.4%, were obtained (Figure 3). DFT-optimized results
suggested the presence of four-intramolecular hydrogen
bonding in the predominating conformer “a”, resulting in the
most stable lowest energy state. The second stable conformer
1
2
3
4
5
6
7
8
9
60.9
>100
10.5
82.6
10.4
>100
67.0
8.9
10
11
12
13
14
15
16
17
>100
17.4
67.1
38.6
8.4
58.1
14.7
26.9
0.7
b
21.8
arctigenin
a
Concentration at which 50% of cells were killed preferentially in
b
NDM. Positive control.
“b”, however, had only three-intramolecular hydrogen bonding
(
Figure 3). The theoretical ECD calculations on these two
optimized geometries were carried out at the same level of
theory. The experimental ECD spectrum (Figure 4) matched
closely with the theoretical ECD spectrum for the (3″R, 4″S)
configuration, confirming the absolute configuration of 2 as
displayed the most potent preferential cytotoxicity, with a PC50
value of 8.4 μM. Lupeol (13), which contains a hydroxy-
methylene group at C-28, showed a weaker activity (PC 38.6
μM) than betulin (14). In case of the flavones, the activity
found was in the order 8 > 11 > 9 > 12 > 10. Among the
chalcones, the dihydrochalcone-4′-O-glucoside 5 and its parent
5
0
3
″R,4″S. There are only a few reports for the isolation of C-
34−36
methylated or glycosylated threonolactones from Nature.
However, this is the first report of a naturally occurring
Figure 5. Plausible biogenetic pathway for 2.
D
DOI: 10.1021/acs.jnatprod.9b00735
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Journal of Natural Products
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aglycone 3 showed PC50 values of 10.4 and 10.5 μM,
respectively.
Information). The velocity of migration in the control PANC-1
cells was found to be quite fast compared to the treated group,
and the wound area was closed to 38% within 24 h when
compared to the initial wound at 0 h (100%). PANC-1 cells
treated with fragranone A (1), on the other hand, led to
significant inhibition of migration, so that the wound area
remained open to 93% after 24 h. This difference was reduced
at 48 h, and the gap of the wound area was found to be 12%
and 81% for the control and the treated group, respectively.
After 72 h, less than 5% of the wound area remained in the
control, while the treated cells still had a 72% open wound area
During the development of pancreatic cancer, the primary
tumor mass engineers migratory cells that can invade adjacent
tissues and circulate to distant organs where cancer cells have
sufficient nutrients to grow and form new colonies. These
processes are responsible for ∼90% of pancreatic cancer deaths
3
7
in patients. Thus, finding inhibitors of pancreatic cancer cell
migration is crucial for suppressing the process of pancreatic
tumor metastasis. The uniqueness of the structure of 1 in
addition to its noncytotoxicity under nutrient-rich conditions
encouraged an evaluation of the ability of 1 to inhibit PANC-1
cell migration using a quantitative real-time cell migration
assay. Fragranone A (1) was exposed to PANC-1 cells in a
standard nutrient-rich medium (DMEM) against the mono-
layer of PANC-1 cell scratch. Parallel experiments were run
under the same culture conditions for the control and the
treated cells, and the real-time images were captured every 15
min for 72 h automatically through a digital microscope
(
Figure 6B). These results suggested that fragranone A (1) has
the ability to suppress the migration of PANC-1 cells. This
real-time cancer cell migration inhibition study has a significant
benefit over conventional end-point measurement in a
migration assay in several aspects. Conventional methods are
often subjective, and the length of the measured wound may
vary due to differences in the exact photographic location.
Since cancer cell migration is heterogeneous, conventional end
point measurement of a wound length often leads to
inaccuracy, as the length of the wound at different locations
might be different. Real-time wound areas address the issue of
heterogeneous cancer cell migration and provide reliable
quantitative information in real time without any bias, because
the wound study area remains fixed throughout the study
period within the CO2 incubator without any external
disturbances.
installed within a CO incubator (Figure 6A).
2
Altogether, 319 images were captured for each group
(
Figure S23, Supporting Information). The wound area at
38
each interval of time was quantified using a Fiji platform.
Compound 1 was found to inhibit PANC-1 cell migration in
real time compared to the control (Movie 1, Supporting
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations and ECD
spectra were measured on a JASCO P-2100 digital polarimeter and a
JASCO J-805 spectropolarimeter, respectively. UV spectra were
recorded on a U-5100 UV−visible ratio-beam spectrophotometer
(
Hitachi). IR spectra were measured with a JASCO FT/IR-460 Plus
spectrophotometer. NMR spectra were recorded using a JEOL
ECX400 Delta spectrometer with tetramethylsilane as internal
standard, and chemical shifts are expressed in δ values. HRFABMS
measurements were carried out on a JEOL JMS-AX505HAD mass
spectrometer. Medium-pressure liquid chromatography (MPLC) was
performed with a Buchi MPLC C-605 dual-gradient pump system
̈
using normal-phase silica gel (silica gel 60N, spherical, neutral, 40−50
μm) purchased from Kanto Chemical, Japan. Analytical and
preparative thin-layer chromatography (TLC) were carried out on
precoated silica gel 60F254 and RP-18F254s plates (0.25 or 0.50 mm,
Merck KGaA, Darmstadt, Germany) and precoated silica gel 70
PF254 plates (0.75 mm thickness) from Wako (Wako Pure Chemical
Industries, Ltd., Japan).
Plant Material. Leaves of A. fragrans were collected at Omkoi
district, Chiang Mai Province, Thailand, on November 2017. The
plant material was identified by Mr. Wittaya Pongamornkul, and a
voucher specimen (NDD2017-AFL) has been deposited in the
Natural Drug Discovery Laboratory, University of Toyama, Japan.
Extraction and Isolation. Air-dried leaves (1 kg) of A. fragrans
were ground to a fine powder, which was then extracted with 95%
EtOH in three cycles (3 days/cycle) at room temperature. The
extracts were combined and concentrated under reduced pressure to
yield an ethanolic extract (105 g). The extract (100 g) was suspended
in H O (200 mL) and partitioned successively with CH Cl (4 × 200
2
2
2
mL) and EtOAc (4 × 200 mL) to give CH Cl (5.4 g), EtOAc (17.3
2
2
g), and H O (74 g) soluble fractions, respectively. The EtOAc-soluble
2
Figure 6. Fragranone A (1) suppresses the migration of PANC-1 cells
in a wound-healing assay in real time. (A) The white lines indicate the
wound length at the start of the experiment. Treated and control cells
were subjected to time-lapse imaging at 15 min intervals for 72 h, and
the representative images are shown. (B) Quantification of wound
healing by measuring the open scratch area for each time point.
fraction was subjected to silica gel column chromatography using an
n-hexane−EtOAc gradient mixture (0−100%) followed by an
EtOAc−MeOH gradient system (0−100%) to give 12 fractions (Fr.
1, 3.6 g; Fr. 2, 847 mg; Fr. 3, 322 mg; Fr. 4, 110 mg; Fr. 5, 900 mg; Fr.
6, 1.2 g; Fr. 7, 215 mg; Fr. 8, 120 mg; Fr. 9, 490 mg; Fr. 10, 310 mg;
Fr. 11, 8.9 g; Fr. 12, 460 mg). Fraction 2 (847 mg) was washed with
E
DOI: 10.1021/acs.jnatprod.9b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
n-hexane to obtain lupeol (13, 396 mg). Fraction 4 (110 mg) was
rechromatographed on silica gel by MPLC using an n-hexane−EtOAc
gradient system (0−100%) to give spinasterol (15, 1 mg). Fraction 5
these optimized geometries were carried out at the same level of
41
theory in a PCM solvation model for EtOH in Gaussian 09. The
output files were summed to obtain Boltzmann-weighted spectra and
compared with the experimental spectra using SpecDis v. 1.71. A UV
correction (−18 nm) was carried out with respect to the experimental
(
900 mg) was recrystallized from an n-hexane−EtOAc solvent
mixture to give betulin needles (14, 42 mg). Fraction 7 (215 mg)
was purified by normal-phase MPLC using a CH Cl −MeOH
42
value.
2
2
gradient system (0−100%) to yield 3,4,2′,4′-tetrahydroxydihydro-
chalcone (3, 154 mg). Fraction 8 (120 mg) was rechromatographed
on silica gel using MPLC with a CH Cl −MeOH gradient mixture
Preferential Cytotoxicity Assay against PANC-1 Cells.
Preferential cytotoxicity of the 95% EtOH extract of A. fragrans and
the isolated compounds was determined by a procedure described
2
2
4
(
0−100%) followed by preparative TLC with a CH Cl −MeOH
previously.
2
2
solvent mixture (97:3) to give butein (7, 0.9 mg). Fraction 9 (490
mg) was also rechromatographed on silica gel by MPLC using a
CH Cl −MeOH gradient system (0−100%) to give five subfractions
Quantitative Real-Time Cell Migration Assay. PANC-1 cells
6
(9 × 10 cells/well) were seeded in a 35 mm Petri dish and allowed to
attach for 24 h. The monolayers were then incised with a micropipet
tip in the central area of the culture to create a wound.
2
2
(
Fr. 9-1, 11 mg; Fr. 9-2, 12 mg; Fr. 9-3, 116 mg; Fr. 9-4, 14 mg; Fr. 9-
5
, 189 mg). Subfraction 9-4 was obtained as pure fragranone B (2, 14
Cells were then treated with either DMEM alone (the control) or
DMEM containing fragranone A (1, 100 μM) and placed on the
mg). Subfraction 9-2 (12 mg) was purified by preparative TLC with a
CH Cl −MeOH solvent mixture (98:2) to yield isoscopoletin (17,
CytoSMART systems installed within a CO incubator. The images
2
2
2
0
.6 mg). Subfraction 9-5 (189 mg) was further purified by preparative
were captured every 15 min automatically. The open wound area in
each image was calculated using Fiji software, and the data were
processed using GraphPad Prism 7.
TLC with CH Cl −MeOH solvent mixture (85:15) to give a mixture
2
2
of (+)-catechin and (−)-epicatechin (16, 36 mg). Fraction 11 (8.9 g)
was rechromatographed on RP-silica gel with MPLC using an eluent
system consisting of solvent A [H O] and solvent B [CH CN−
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00735.
2
3
■
MeOH (1:1)], with a gradient (0−100% B) to give seven subfractions
*
S
(
1
(
Fr. 11-1, 205 mg; Fr. 11-2, 411 mg; Fr. 11-3, 3.2 g; Fr. 11-4, 4.5 g; Fr.
1-5, 199 mg; Fr. 11-6, 30 mg; Fr. 11-7, 335 mg). Subfraction 11-2
411 mg) was rechromatographed on silica gel by MPLC using an
EtOAc−MeOH gradient system (0−100%) followed by preparative
TLC with an EtOAc−MeOH solvent mixture (9:1) to give
davidioside (6, 60 mg), isoquercetrin (10, 4.7 mg), and kaempferol
Copies of spectroscopic data for 1 and 2 (PDF)
Captures of live imaging of real-time cell migration assay
3
-neohesperidoside (11, 16 mg). Subfraction 11-3 (3.2 g) was
(PDF)
rechromatographed on silica gel with MPLC using an EtOAc−MeOH
gradient system (0−100%) to give four subfractions (Fr. 11-3-1, 5
mg; Fr. 11-3-2, 347 mg; Fr.11-3-3, 1.2 g; Fr.11-3-4, 123 mg).
Subfractions 11-3-1 and 11-3-3 were identified as quercetrin (9, 5 mg)
and vacciniifolin (5, 1.2 g), respectively. Subfraction 11-3-4 (123 mg)
was further chromatographed on silica gel with MPLC using a
CH Cl −MeOH gradient system (0−100%) to afford nictoflorin (12,
Real-time movie showing the inhibitory effect of
fragranone A (1, 100 μM) on the migration of PANC-
1
cells (AVI)
AUTHOR INFORMATION
Corresponding Author
Tel: +81-76-434-7640. Fax: +81-76-434-7640. E-mail (S.
■
*
2
2
2
5 mg). Subfraction 11-4 (3.9 g) was rechromatographed on silica gel
by MPLC using an EtOAc−MeOH gradient system (0−100%)
Awale): suresh@inm.u-toyama.ac.jp.
followed by preparative TLC with an EtOAc−MeOH solvent mixture
(
9:1) to produce confusoside (4, 3.3 g) and afzelin (8, 27 mg).
ORCID
Subfraction 11-6 (30 mg) was further purified by preparative TLC
with an EtOAc−MeOH solvent mixture (98:2), which afforded
fragranone A (1, 5.5 mg).
Suresh Awale: 0000-0002-5299-193X
Notes
The authors declare no competing financial interest.
2
5
Fragranone A (1): white, amorphous powder; [α] −15 (c 0.03,
D
MeOH); UV (MeOH) λmax (log ε) 208 (4.24), 272 (3.84), 320
−
1
1
ACKNOWLEDGMENTS
(
3.51) nm; IR (KBr) ν 3420, 1634 cm ; H NMR (400 MHz,
3
■
max
13
CD OD) and C NMR (100 MHz, CD OD) data, see Table 1;
This work was supported by the Japanese Society for the
Promotion of Science (JSPS), Japan, Kakenhi (16K08319),
and Kobayashi International Scholarship to S.A. The authors
thank the Egyptian-Japanese Education partnership (EJEP)
fellowship for supporting A.M.O. and A.M.T.
3
+
HRFABMS m/z 461.18126 [M + H] (calcd for C H O ,
2
4
28
9
4
61.18113).
Fragranone B (2): yellow oil; [α]25D +136.8 (c 0.1, MeOH); UV
(
2
MeOH) λ (log ε) 208 (5.07), 344 (4.48), 376 (4.55) nm; ECD (c
max
.6 × 10−4 M, EtOH) λ (Δε) 223 (−2.5), 242 (+1.8); IR (KBr)
max
−
1
1
13
νmax3407, 1602 cm ; H NMR (400 MHz, DMSO-d ) and C NMR
6
REFERENCES
(
[
100 MHz, DMSO-d ) data, see Table 1; HRFABMS m/z 389.08816
■
6
+
M + H] (calcd for C H O , 389.08723).
(1) Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.;
Jemal, A. Ca-Cancer J. Clin. 2018, 68, 394−424.
1
9
16
9
Acid Hydrolysis of 1. Fragranone A (1, 1.9 mg) was heated with
6
% HCl (1 mL) for 5 h at 80 °C. The reaction mixture was extracted
(2) The Editorial Board of Cancer Statistics in Japan. Cancer
Statistics in Japan 2018; Foundation for Promotion of Cancer
Research (FPCR), 2018. http://ganjoho.jp/en/professional/
statistics/brochure/index.html.
(3) Poruk, K. E.; Firpo, M. A.; Adler, D. G.; Mulvihill, S. J. Ann. Surg.
2013, 257, 17−26.
(4) Awale, S.; Lu, J.; Kalauni, S. K.; Kurashima, Y.; Tezuka, Y.;
Kadota, S.; Esumi, H. Cancer Res. 2006, 66, 1751−1757.
(5) (a) Izuishi, K.; Kato, K.; Ogura, T.; Kinoshita, T.; Esumi, H.
Cancer Res. 2000, 60, 6201−6207. (b) Lu, J.; Kunimoto, S.; Yamazaki,
Y.; Kaminishi, M.; Esumi, H. Cancer Sci. 2004, 95, 547−552.
(c) Esumi, H.; Lu, J.; Kurashima, Y.; Hanaoka, T. Cancer Sci. 2004,
95, 685−690.
with CH Cl (4 × 3 mL), and the aqueous phase was dried by using a
Genevac EZ-2 vacuum centrifuge. The resulting residue (4.2 mg) was
2
2
purified by preparative TLC with a CH Cl −MeOH−H O solvent
2
2
2
2
5
mixture (8:5:1) to yield glucose (0.7 mg), [α] +67.7 (c 0.06, H O).
Identification of glucose was done through H NMR spectroscopic
analysis (Figure S11, Supporting Information).
D
2
1
39
ECD Calculations for 2. The conformational search was
performed on Spartan’14 by using the MMFF molecular force
4
0
field. Eight conformers with a Boltzmann distribution were
reoptimized using DFT at the B3LYP/6-31+G* level in the gas
phase, which suggested two predominant conformers having a relative
population of 94% and 5.4%. The theoretical ECD calculations on
F
DOI: 10.1021/acs.jnatprod.9b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
6) Awale, S.; Nakashima, E.M. N.; Kalauni, S. K.; Tezuka, Y.;
Kurashima, Y.; Lu, J.; Esumi, H.; Kadota, S. Bioorg. Med. Chem. Lett.
006, 16, 581−583.
7) Nguyen, N. T.; Nguyen, M. T. T.; Nguyen, H. X.; Dang, P. H.;
Dibwe, D. F.; Esumi, H.; Awale, S. J. Nat. Prod. 2017, 80, 141−148.
8) Sun, S.; Phrutivorapongkul, A.; Dibwe, D. F.; Balachandran, C.;
Awale, S. J. Nat. Prod. 2018, 81, 1877−1883.
9) Awale, S.; Dibwe, D. F.; Balachandran, C.; Fayez, S.; Feineis, D.;
Lombe, B. K.; Bringmann, G. J. Nat. Prod. 2018, 81, 2282−2291.
10) Siddaiah, V.; Rao, C. V.; Venkateswarlu, S.; Subbaraju, G. V.
Tetrahedron 2006, 62, 841−846.
11) Tanaka, T.; Kawamura, K.; Kohda, H.; Yamasaki, K.; Tanaka,
O. Chem. Pharm. Bull. 1982, 30, 2421−2423.
12) Ling, T. J.; Lin, L. D.; Wu, P.; Zhou, W. H.; Ye, H. G.; Liu, M.
F.; Wei, X. Y. Chin. Chem. Lett. 2004, 15, 1182−1184.
13) Gutmann, A.; Bungaruang, L.; Weber, H.; Leypold, M.;
Breinbauer, R.; Nidetzky, B. Green Chem. 2014, 16, 4417−4425.
14) Junior, G. M. V.; de M. Sousa, C. M.; Cavalheiro, A. J.; Lago, J.
H. G.; Chaves, M. H. Helv. Chim. Acta 2008, 91, 2159−2167.
15) Chen, C.-Y.; Hsieh, S.-L.; Hsieh, M.-M.; Hsieh, S.-F.; Hsieh, T.-
J. Chin. Pharm. J. 2004, 56, 141−146.
16) Aderogba, M. A.; Kgatle, D. T.; McGaw, L. J.; Eloff, J. N. S. Afr.
J. Bot. 2012, 79, 125−131.
17) Han, J.-T.; Bang, M.-H.; Chun, O.-K.; Kim, D.-O.; Lee, C.-Y.;
Baek, N.-I. Arch. Pharmacal Res. 2004, 27, 390−395.
18) Kazuma, K.; Noda, N.; Suzuki, M. Phytochemistry 2003, 62,
29−237.
19) Park, S. Y.; Kim, J. S.; Lee, S. Y.; Bae, K.; Kang, S. S. Nat. Prod.
Sci. 2008, 14, 281−288.
20) Mahato, S. B.; Kundu, A. P. Phytochemistry 1994, 37, 1517−
575.
21) Iida, T.; Ishikawa, T.; Tamura, T.; Matsumoto, T. J. Japan Oil
Chem. Soc. 1980, 29, 345−346.
22) Galotta, A. L. Q. A.; Boaventura, M. A. D.; Lima, L. A. R. S.
Quim. Nova 2008, 31, 1427−1430.
23) Zhao, D.-D.; Zhao, Q.-S.; Liu, L.; Chen, Z.-Q.; Zeng, W.-M.;
Lei, H.; Zhang, Y.-L. Molecules 2014, 19, 3345−3355.
24) Desire, O.; Riviere, C.; Razafindrazaka, R.; Goossens, L.;
(38) Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.;
Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.;
Schmid, B.; Tinevez, J.-Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.;
Tomancak, P.; Cardona, A. Nat. Methods 2012, 9, 676−682.
2
(
(39) Gurst, J. E. K. J. Chem. Educ. 1991, 68, 1003−1004.
(
(40) SPARTAN’14; Wavefunction: Irvine, CA, 2014.
(41) Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E.,
Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci,
B., Petersson, G. A., Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.;
Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi,
R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar,
S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox,
J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A.
D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.
Gaussian 09w (version 7.0); Gaussian, Inc: Wallingford, CT, 2009.
(
(
(
(
(
(
(
(
(
42) Bruhn, T.; Schaumlo
̈
ffel, A.; Hemberger, Y.; Pescitelli, G.
SpecDis version 1.71; Berlin, Germany, 2017.
(
(
2
(
(
1
(
(
(
(
̀
Moreau, S.; Guillon, J.; Uverg-Ratsimamanga, S.; Andriamadio, P.;
Moore, N.; Randriantsoa, A.; Raharisololalao, A. J. Ethnopharmacol.
2
(
010, 130, 320−328.
25) Kim, K. H.; Park, Y. J.; Chung, K. H.; Yip, M. R.; Clardy, J.;
Senger, D.; Cao, S. J. Nat. Prod. 2015, 78, 320−324.
26) Arriaga, F. J.; Rumbero, A.; Wollenweber, E. Z. Naturforsch., C:
J. Biosci. 1996, 51, 423−425.
27) Mamadalieva, N. Z.; Janibekov, A. A.; Girault, J.-P.; Lafont, R.
Nat. Prod. Commun. 2010, 5, 1579−1582.
28) Yamaguchi, I.; Yokota, T.; Murofushi, N.; Takahashi, N.;
Ogawa, Y. Agric. Biol. Chem. 1975, 39, 2399−2403.
29) Yu, J. S.; Moon, E.; Choi, S. U.; Kim, K. H. Tetrahedron Lett.
016, 57, 1699−1701.
30) Shao, C. L.; Wu, H. X.; Wang, C. Y.; Liu, Q. A.; Xu, Y.; Wei, M.
(
(
(
(
2
(
Y.; Qian, P. Y.; Gu, Y. C.; Zheng, C. J.; She, Z. G.; Lin, Y. C. J. Nat.
Prod. 2011, 74, 629−633.
(
31) Metuno, R.; Ngandeu, F.; Tchinda, A. T.; Ngameni, B.;
Kapche, G. D. W.F.; Djemgou, P. C.; Ngadjui, B. T.; Bezabih, M.;
Abegaz, B. M. Biochem. Syst. Ecol. 2008, 36, 148−152.
(
32) Meguro, H.; Konno, T.; Tuzimura, K. Agric. Biol. Chem. 1973,
3
(
1
(
7, 945−947.
33) Nakaminami, G.; Edo, H.; Nakagawa, M. Bull. Chem. Soc. Jpn.
973, 46, 266−269.
34) Syed, A. S.; Akram, M.; Bae, O.-N.; Kim, C. Y. Chem. Nat.
Compd. 2019, 55, 8−10.
35) Uriburu, M. L.; Gil, R. R.; Sosa, V. E.; de la Fuente, J. R. J. Arg.
Chem. Soc. 2008, 96, 55−61.
36) Lee, C.-L.; Huang, P.-C.; Hsieh, P.-W.; Hwang, T.-L.; Hou, Y.-
Y.; Chang, F.-R.; Wu, Y.-C. Planta Med. 2008, 74, 1276−1279.
37) Keleg, S.; Buchler, P.; Ludwig, R.; Buchler, M. W.; Friess, H.
Mol. Cancer 2003, 2, 1−7.
(
(
(
̈
̈
G
DOI: 10.1021/acs.jnatprod.9b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX