In this study, the methanolic extract of the leaves of Artocarpus against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions at a concen-tration of 50 µg/mL.. The is
Trang 1! Pancreatic cancer is one of the most deadly forms
of cancer associated with the lowest 5-year sur-vival rates known for cancers [1] It shows resis-tance to conventional chemotherapeutic agents such as 5-fluorouracil, paclitaxel, doxorubicin, and cisplatin [2] Pancreatic cancers are hypovas-cular [3] in nature resulting in an inadequate sup-ply of nutrition and oxygen to aggressively prolif-erating cells However, pancreatic cancer cells show an extraordinary tolerance [4] to starvation enabling them to survive in hypovascular (auster-ity) conditions Thus, development of drugs aimed at countering this tolerance to nutrient starvation is a novel antiausterity approach [5] in anticancer drug discovery Working under this hypothesis, we screened medicinal plants from widely different origins for the discovery of anti-austerity agents, using the PANC-1 human
Artocarpus altilis (Parkinson ex F A Zorn) Fors-berg (Moraceae) is a medicinal plant locally
its leaves has been used traditionally for the treat-ment of gout, hepatitis, hypertension, and diabe-tes [16] Previous work on this plant species
re-ported a number of geranyl dihydrochalcones [17] having cytotoxic activities and aurones
activ-ity [18] In our screening program, recently we found that the methanolic extract of the leaves displayed 100 % preferential cytotoxicity against PANC-1 cells in nutrient-deprived condition at
50 µg/ml A further phytochemical study on this extract furnished the isolation of eight new
these compounds and their antiausterity activity
Results and Discussion
! Sakenin A (1) was isolated as a yellow amorphous solid Its molecular formula was determined by
ex-hibited signals due to a pair of ortho-coupled
6.48 (1H, d, J = 8.1 Hz), an aromatic ABX spin
Abstract
! Human pancreatic cancer cell lines have remark-able tolerance to nutrition starvation, which en-ables them to survive under a tumor microenvir-onment The search for agents that preferentially inhibit the survival of cancer cells under low nu-trient conditions is a novel antiausterity strategy
in anticancer drug discovery In this study, the methanolic extract of the leaves of Artocarpus
against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions at a
concen-tration of 50 µg/mL Further investigation of this extract led to the isolation of eight new gerany-lated dihydrochalcones named sakenins A–H (1–
Among them, sakenins F (6) and H (8) were iden-tified as potent preferentially cytotoxic
re-spectively
Supporting information available online at http://www.thieme-connect.de/ejournals/toc/
plantamedica
Geranyl Dihydrochalcones from Artocarpus altilis and
Their Antiausteric Activity
Authors Mai Thanh Thi Nguyen 1 , Nhan Trung Nguyen 1 , Khang Duy Huu Nguyen 1 , Hien Thu Thi Dau 1 , Hai Xuan Nguyen 1 ,
Phu Hoang Dang 1 , Tam Minh Le 1 , Trong Huu Nguyen Phan 1 , Anh Hai Tran 2 , Bac Duy Nguyen 2 , Jun-ya Ueda 3 , Suresh Awale 3
Affiliations 1 Faculty of Chemistry, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam
2 Vietnam Military Medical University, Ha Noi, Vietnam
3 Frontier Research Core for Life Sciences, University of Toyama, Toyama, Japan
Key words
received July 23, 2013
revised Nov 13, 2013
accepted Nov 22, 2013
Bibliography
DOI http://dx.doi.org/
10.1055/s-0033-1360181
Published online January 15,
2014
Planta Med 2014; 80: 193–200
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Dr Suresh Awale
Frontier Research Core for Life
Sciences
University of Toyama
2630 Sugitani
Toyama 930 –0194
Japan
Phone: + 81 76 434 7640
Fax: + 81 76 434 7640
suresh@inm.u-toyama.ac.jp
Trang 2Table 1 1 H and 13 C NMR data (500 and 125 MHz) for compounds 1 –4.
Position 1 (in CD3OD) 2 (in CD3OD) 3 (in DMSO-d 6 ) 4 (in CD3OD)
3 ′ 6.15, d (2.4) 102.3 6.26, d (2.4) 103.8 6.25, d (2.3) 102.4 6.24, d (2.4) 103.8
5 ′ 6.22, dd (8.9, 2.4) 107.7 6.34, dd (8.8, 2.4) 109.3 6.35, dd (8.8, 2.3) 108.2 6.30, dd (8.9, 2.4) 109.2
6 ′ 7.53, d (8.9) 132.3 7.64, d (8.8) 133.7 7.78, d (8.8) 132.9 7.63, d (8.9) 133.9
β 2.99, t (7.4) 39.3 3.10, m 40.7 3.18, m 38.0 3.12, t (7.4) 39.9
α 2.78, t (7.4) 27.6 2.88, m 29.0 2.75, m 26.9 2.90, m 29.0
5 6.48, d (8.1) 112.3 6.59, d (8.1) 113.8 6.54, d (8.1) 115.5 6.59, d (8.1) 113.7
6 6.42,d (8.1) 121.0 6.53, d (8.1) 121.1 6.52, d (8.1) 120.3 6.57, d (8.1) 121.0
1 ′′ 3.30, d (6.5) 24.6 3.40, d (6.6) 26.1 2.95, m 33.8 2.69, dd (16.5, 5.0) 22.0
3.35, m 2.48, dd (16.5, 13.0) 2′′ 5.04, td (6.5, 1.2) 123.5 5.18, td (6.6, 1.1) 125.2 5.19, t (9.0) 84.5 1.60, dd (13.0, 5.0) 48.5
4 ′′ 1.84, t (7.4) 39.7 1.99, m 37.7 2.06, m 30.6 2.00, m 38.8
5 ′′ 1.33, m 22.2 1.61, m 30.5 2.16, m 25.8 1.77, m 28.5
6 ′′ 1.27, m 42.9 3.35, m 76.6 5.12, td (7.0, 1.4) 124.7 3.32, dd (11.2, 4.0) 78.8
8′′ 1.01, s 27.7 1.08, s 21.2 1.65, s 25.4 0.85, s 14.7
9 ′′ 1.01, s 27.7 1.03, s 20.7 1.57, s 17.5 1.12, s 20.0
10 ′′ 1.62, d (1.2) 14.9 1.74, s 16.5 4.90, s
5.14, s
109.9 1.07, s 27.8
Fig 1 Chemical structure of new compounds (1 –8) isolated from Vietnamese Artocarpus altilis.
Trang 3δH3.30, d, J = 6.5 Hz;δH1.84, t, J = 7.4 Hz;δH1.33, mδH1.27, m),
the other hand, displayed signals of 25 carbons, which could be
oxygenated carbon, six methylene carbons, and three methyl
′,4′,3,4-tetrahydroxydehydro-chalcone) previously isolated from A altilis from Micronesia [19]
′,4′,3,4-tetrahydroxydihydro-chalcone unit However, 1 showed signals due to a quaternary
group instead of signals due to an olefin moiety as in the geranyl
unit in compound AC-5-1 Therefore, the presence of a
hydroxy-geranyl unit instead of a hydroxy-geranyl unit was assumed In the HMBC
7-hydroxy-3,7-dimethyl-2-octenyl group Furthermore, the HMBC correlations from
H3-10′′/H2-1′′ (l"Fig 3) Therefore, the structure of 1 was
′,4′,3,4-tet-rahydroxydihydrochalcone
Sakenin B (2) was isolated as a yellow amorphous solid The
except for the appearance of a signal due to an oxymethine group
Therefore, the structure of 2 was concluded as
′,4′,3,4-tetrahydroxydihydrochal-cone
Sakenin C (3) was isolated as a yellow amorphous solid having
ger-anyl side chain The DEPT and HSQC spectra of 3 showed the
deduced In the HMBC spectrum, the two tertiary methyl groups
C4′′ (l"Fig 2) These data suggested that 3 contains a geranyl side chain in the form of 7-methyl-3-methyleneoct-6-enyl-2-ol Fi-nally, the location of this side chain was determined to be at C-2
Fig 2 Connectivities (bold lines) deduced by the COSY and HSQC spectra and significant HMBC correlations (solid arrows) observed for new compounds 1 –8.
Trang 4Therefore, the structure of sakenin C was concluded as
2-[2-hy-
droxy-7-methyl-3-methyleneoct-6-enyl]-2′,4′,3,4-tetrahydroxy-dihydrochalcone
Sakenin D (4) was isolated as a yellow amorphous solid Its
indicative of the presence of a
the remaining signals in 4 as three tertiary methyl groups, an
oxymethine, a quaternary oxygenated carbon, an aliphatic
meth-ine, an aliphatic quaternary carbon, and three aliphatic
methy-lene groups This data together with COSY analysis showed two
cy-clized geranyl group in 4 The relative stereochemistry of 4 was
established by coupling constant data and the NOESY spectral
indi-cated that it should be axially oriented Furthermore, NOESY
indi-cated that these groups are oriented in the same direction
(l"Fig 3) and the ring bearing hydroxyl group adopts a chair
con-formation Therefore, the structure of sakenin E was concluded as
shown
ascrib-able for a cyclized geranyl group that could be classified into
three tertiary methyl groups, two methylene groups, three
The COSY and HSQC analysis showed two partial connectivities
correlations observed in HMBC so as to get a planar structure
having a geranyl group in the form of a bicyclic ring as shown in
l"Fig 2 Its relative stereochemistry was determined by NOESY
spectral analysis NOESY correlations were observed between
Therefore, the structure of 5 was concluded as shown
Sakenin F (6) was isolated as a yellow amorphous solid Its
However, it showed a difference in signals due to the geranyl group Comparison of molecular formulae indicated that sakenin
F (6) contains two hydrogen atoms less than 1, which suggests the presence of a cyclic form of the geranyl group in 6 The partial
presence of a cyclized geranyl group Therefore, the structure of sakenin F (6) was concluded as shown
Sakenin G (7) was isolated as a yellow amorphous solid having a
ob-served was the occurrence of signals due to the presence of a benzofuran unit instead of a geranyl unit, which was confirmed
was concluded as 1-(2,4-dihydroxyphenyl)-3-(7-hydroxybenzo-furan-4-yl)propan-1-one
for the presence of a methoxyl group with the disappearance of
As a result, the structure of sakenin H (8) was concluded as 1- (2,4-dihydroxyphenyl)-3-(7-hydroxy-2-methoxy-2,3-dihydro-benzofuran-4-yl)propan-1-one
The known compounds were identified by analysis of their spectroscopic data and comparison with literature data to be 1- (2,4-dihydroxyphenyl)-3-[3,4-dihydro-3,8-dihydroxy-2-methyl-2-(4-methylpent-3-enyl)-2H-1-benzopyran-5-yl]propan-1-one (9) [17],
and cycloaltilisin (12) [22]
The isolated compounds were tested for their cytotoxic activity against a PANC-1 human pancreatic cancer cell line in normal
Fig 3 Key NOESY correlations (dashed arrow) ob-served for new compounds 1, 4, and 5.
Trang 5DMEM and nutrient-deprived medium (NDM), utilizing an
anti-austerity strategy All the tested geranyl dihydrochalcones
showed preferential cytotoxicity in a nutrient-deprived
condi-tion without apparent toxicity in a nutrient-rich condicondi-tion
preferential cell death in nutrition-deprived medium (NDM)
without cytotoxicity in normal nutrient-rich medium (DMEM),
strategy-based anticancer agent which was used as a positive control in
well-known anticancer agent, was virtually inactive Among them, 6
and 8 displayed the most potent preferential cytotoxic activity
Sakenin F (6) was further evaluated for its effect on the cell
mor-phology of PANC-1 cells in NDM The microscopic images were
analyzed under phase-contrast and fluorescence mode using
ethidium bromide/acridine orange (EB/AO) reagent AO is a cell
permeable dye and gives a green or orange fluorescence in live
cells EB is permeable to dead cells only and gives a red
stained with AO giving a green fluorescence However, when
treated with 6, the morphology of PANC-1 cells was distinctly
(red) The phase contrast image of PANC-1 cells treated with 6
showed rounding of the cell membrane, rupture, and
indicated that A altilis and its constituents could have a potential
utility for the development of drugs against pancreatic cancer
Materials and Methods
! General experimental procedures Optical rotations were recorded on a PerkinElmer 241 digital po-larimeter UV spectra were obtained on a Shimadzu UV-160A spectrophotometer IR spectra were measured with a Shimadzu
taken on a Bruker Avance III 500 spectrometer (Bruker Biospin) with tetramethylsilane (TMS) as an internal standard, and
a Micro O-QIITOF mass spectrometer (Bruker Daltonics) Analyti-cal and preparative TLC were carried out on precoated Merck
Plant material The leaves of cultivated Artocarpus altilis were collected in Binh Duong Province, Vietnam, in October 2009 and identified by Ms
Hoang Viet, Faculty of Biology, University of Science, National University Ho Chi Minh City, Vietnam A voucher specimen (AN-2964) has been deposited at the Department of Analytical Chem-istry of the University of Science, National University Ho Chi Minh City, Vietnam
Extraction and isolation Air-dried leaves of A altilis (3.5 kg) were extracted with MeOH (12 L, reflux, 3 h × 3) to yield a MeOH extract (350 g) The MeOH
Table 2 1 H and 13 C NMR data (500 and 125 MHz) for compounds 5 –8.
Position 5 (in CDCl3) 6 (in CD3COCD3) 7 (in DMSO-d 6 ) 8 (in CD3COCD3)
3 ′ 6.38, d (2.5) 103.6 6.32, d (2.4) 103.7 6.25, d (2.4) 108.3 6.32, d (2.4) 103.5
5′ 6.36, dd
(8.3, 2.5)
108.3 6.41, dd (8.8, 2.4) 109.0 6.35, dd (8.8, 2.4) 102.3 6.43, dd (8.8, 2.4) 108.7
6 ′ 7.64, d (8.3) 132.5 7.82, d (8.8) 134.0 7.77, d (8.8) 132.8 7.81, d (8.8) 133.6
β 3.17, m 39.3 3.23, m 39.2 3.25, m 38.6 3.21, m 39.0
α 2.95, m 25.5 2.87, m 28.3 3.05, m 26.7 2.86, m 27.9
1 ′′ 2.20, m 46.3 3.28, m 30.1 7.00, d (2.1) 105.3 3.38, dd (16.5, 6.5) 36.6
2 ′′ 3.93, d (6.0) 76.5 4.67, dd (9.5, 8.5) 90.0 7.90, d (2.1) 146.1 5.67, dd (6.5, 2.0) 108.3
4 ′′ 1.95, m 32.8 1.55, m 39.2
1.64, m
5′′ 1.57, m 20.9 2.17, m 22.8
1.25, m
6 ′′ 3.83, m 38.7 5.13, tt (7.2, 1.4) 126.1
8 ′′ 1.39, s 23.8 1.65, s 26.0
9 ′′ 1.42, s 30.3 1.23, s 22.4
10 ′′ 1.39, s 26.0 1.61, s 17.9
2 ′-OH 12.65, s 12.78, s 12.61, s 12.77, s
Trang 6The CHCl3 fraction (90 g) was subjected to silica gel column
ether (0 : 100, 5 : 95, 10 : 90, 20 : 80, 40 : 60, 60 : 40, 80 : 20, and
100 : 0; each 3 L) to give 6 fractions: fr 1 (5.7 g), fr 2 (18.5 g), fr 3
(11.2 g), fr 4 (15.7 g), fr 5 (12.5 g), and fr 6 (25.2 g) Fraction 2 was
rechromatographed on silica gel column (6.5 × 80 cm) with 15 %
2 was rechromatographed on silica gel column (3.5 × 60 cm) with
was further separated by silica gel column chromatography
5 (1.7 mg) Subfraction 5–3 was rechromatographed on silica gel
(1.3 mg) Fraction 6 was further separated by silica gel column
(4.3 mg) and 12 (1.3 mg)
l"Table 1; HR‑ESI‑MS m/z 451.2091 [calcd for C25H32O6Na (M +
Table 3 Preferential cytotoxicity
of compounds isolated from Arto-carpus altilis against the PANC-1 human pancreatic cancer cell line
in nutrient-deprived medium (NDM).
Compounds PC50, µM a Compounds PC50, µM
6 8.0 ± 0.9 Arctigenin b 0.8 ± 0.4
Fig 4 Morphology of PANC-1 cells in NDM:
a control, b treated with 1 µM of 6, c treated with
10 µM of 6, d treated with 10 µM of 6, phase con-trast (Live cells were stained with AO giving green fluorescence; dead cells were stained with EB giving red fluorescence).
(Color figure available online only.)
Trang 7Sakenin E (5): Yellow amorphous solid; [α]D25− 11.3 (c 1.0, CHCl3);
353.1001]
Biological material
Dulbeccoʼs phosphate-buffered saline (D‑PBS) was purchased
me-dium (DMEM) was purchased from Wako Pure Chemical Some-dium
bicarbonate, potassium chloride, magnesium sulfate, sodium
di-hydrogen phosphate, potassium didi-hydrogen phosphate, sodium
chloride, and phenol red were purchased from Wako Pure
Chem-ical HEPES was purchased from Dojindo Fetal bovine serum
(FBS) was from Nichirei Biosciences Antibiotic antimycotic
solu-tion was from Sigma-Aldrich, Inc WST-8 cell counting kit was
from Dojindo Cell culture flasks and 96-well plates were
ob-tained from Falcon Becton Dickinson Labware (BD Biosciences)
Arctigenin was isolated from the seed of Arctium lappa in
crystal-line form [5], and its purity was determined to be > 96 % from
NMR and HPLC Nutrient-deprived medium (NDM) was prepared
according to a previously described protocol [5]
Cancer cell line and cell culture
The PANC-1 (RBRC-RCB2095) human pancreatic cancer cell line
was purchased from Riken BRC cell bank and maintained in
stan-dard DMEM with 10 % FBS supplement, 100 U/mL penicillin G,
0.1 mg/mL streptomycin sulfate, and 0.25 µg/mL amphotericin B
Preferential cytotoxic activity against PANC-1 cells in
nutrient-deprived medium (NDM)
The in vitro preferential cytotoxicity of the crude extract and the
isolated compounds was determined by a previously described
procedure [6] Briefly, human pancreatic cancer cells were seeded
test samples in either DMEM or NDM with the control and blank
in each well After 24 h incubation, cells were washed twice with
kit solution was added to each well After 3 h incubation, the
ab-sorbance at 450 nm was measured (PerkinElmer EnSpire
Multila-bel Reader) Cell viability was calculated from the mean values of
data from three wells by using the following equation:
Morphological assessment of cancer cells
cells were then washed twice with D‑PSB and treated with 1 µM and 10 µM of sakenin F (6) in NDM After 24 h incubation, cells were treated with EB/AO, and morphology was observed using
an inverted Nikon Eclipse TS 100 microscope (40 × objective) with phase-contrast and fluorescent mode Microscopic images were taken with a Nikon DS‑L‑2 camera directly attached to the microscope
Supporting information
HSQC, HMBC, NOESY NMR, and MS spectra of new compounds (1–8) are available as Supporting Information
Acknowledgements
! This work was supported by a grant from the University of Sci-ence, Vietnam National University to MTTN, a Grant from Toyama Support Center for Young Principal Investigators in Advanced Life Sciences, and a grant in aid for Scientific Research (No 24510314) from the Japan Society for the Promotion of Science (JSPS) to SA
Conflict of Interest
! The authors declare no conflict of interest
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