DSpace at VNU: Constituents of the Rhizomes of Boesenbergia pandurata and Their Antiausterity Activities against the PANC-1 Human Pancreatic Cancer Line

In this investigation, a methanol extract of the rhizomes of Boesenbergia pandurata showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-de

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Constituents of the Rhizomes of Boesenbergia pandurata and Their

Line

Nhan Trung Nguyen, *, † Mai Thanh Thi Nguyen,† Hai Xuan Nguyen,† Phu Hoang Dang,†

Dya Fita Dibwe,§ Hiroyasu Esumi,‡ and Suresh Awale *, §

†Faculty of Chemistry, University of Science, Vietnam National University, Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5,

Ho Chi Minh City, Vietnam

‡Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba 278-8510, Japan

§Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

*S Supporting Information

ABSTRACT: Human pancreatic cancer cell lines have a

remarkable tolerance to nutrition starvation, which enables them

to survive under a tumor microenvironment The search for

agents that preferentially inhibit the survival of cancer cells under

low nutrient conditions represents a novel antiausterity strategy in

anticancer drug discovery In this investigation, a methanol extract

of the rhizomes of Boesenbergia pandurata showed potent

preferential cytotoxicity against PANC-1 human pancreatic cancer

cells under nutrient-deprived conditions, with a PC50value of 6.6

μg/mL Phytochemical investigation of this extract led to the

isolation of 15 compounds, including eight new cyclohexene

chalcones (1−8) The structures of the new compounds were

elucidated by NMR spectroscopic data analysis Among the isolated compounds obtained, isopanduratin A1 (14) and nicolaioidesin C (15) exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions, with PC50 values of 1.0 and 0.84μM, respectively

Pancreatic cancer is one of the deadliest forms of malignancy

and is associated with the lowest ﬁve-year survival rates

known for cancer.1It shows resistance to conventional anticancer

agents in clinical use.2 Pancreatic cancers are hypovascular in

nature, resulting in an inadequate supply of nutrition and oxygen

to aggressively proliferating cells However, pancreatic cancer

cells show an extraordinary tolerance to starvation, enabling

them to survive in hypovascular (austerity) conditions.3Thus,

the development of test compounds aimed at countering this

tolerance to nutrient deprivation is a novel antiausterity strategy

in anticancer drug discovery Working under this hypothesis,

medicinal plants of diﬀerent origin have been screened for the

discovery of antiausterity agents, using the PANC-1 human

pancreatic cancer cell line.4−12

Boesenbergia pandurata (Roxb.) Schltr is a perennial medicinal

herb belonging to the Zingiberaceae family It is cultivated in

some tropical countries in Southeast Asia including Vietnam,

Thailand, Myanmar, Indonesia, and Malaysia In Vietnam, it is

known as“Ngai bun”, and the fresh rhizomes are mainly used as a

spice.13The rhizomes are also used as traditional medicine to

cureﬂatulence, fatigue, and dysmenorrhea and to promote the

discharge of bile in Vietnam, Cambodia, Laos, and the People’s

Republic of China.14This plant contains prenylated chalcones

and otherﬂavonoids as the major bioactive constituents, with

reported preferential cytotoxicity against PANC-1 cells in nutrient-deprived medium (NDM).4,7

In the present investigation, it was found that a methanol extract of the rhizomes of B pandurata displayed potent preferential cytotoxicity against PANC-1 cells under nutrient-deprived conditions, with a PC50value of 6.6μg/mL Puriﬁcation

of this extract led to the isolation of eight new secondary metabolites (1−8), together with seven known compounds (9− 15) Reported herein are the isolation, stucture determination, and antiausterity activities of these compounds

■ RESULTS AND DISCUSSION

A methanol-soluble extract of the rhizomes of B pandurata was partitioned between CHCl3and water to give a CHCl3-soluble fraction The CHCl3fraction was subjected to a series of column chromatographic separation steps and preparative TLC to aﬀord eight new secondary metabolites (1−8), together with seven known compounds The known compounds nicolaioidesin A (9),15 panduratin A (10),16 isopanduratin A (11),17 4-hydroxypanduratin A (12),18nicolaioidesin B (13),15 isopandur-Received: August 26, 2016

pubs.acs.org/jnp

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atin A1 (14),17and nicolaioidesin C (15)15were identiﬁed by

comparing their spectroscopic data with literature values

Compound 1 was isolated as a yellowish, amorphous solid, and

its molecular formula was found to be C25H28O4by HRESIMS

The IR spectrum of 1 showed absorptions due to hydroxy (3500

cm−1), carbonyl (1640 cm−1), and phenyl (1450 cm−1) groups

The 1H NMR spectrum displayed signals corresponding to a phenyl group (δH7.25, 7.07, 6.97), two magnetically equivalent aromatic protons (δH5.74), two oleﬁnic methine protons (δH

5.58, 5.13), three aliphatic methines (δH4.85, 3.06, 2.84), two allylic methylenes (δH 2.31, 2.23, 2.15, 2.14), and three vinyl methyls (δH1.71, 1.51, 1.41) Its13C NMR spectrum revealed 25

Chart 1

Figure 1 Connectivities (bold lines) deduced by the COSY and HSQC spectra and signiﬁcant HMBC correlations (solid arrows) of compounds 1−8.

DOI: 10.1021/acs.jnatprod.6b00784

B

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carbon signals including those for a ketone carbonyl carbon (δC

210.4), 12 aromatic carbons, four oleﬁnic carbons (δC 137.2,

132.7, 123.5, 123.1), three methine carbons (δC54.8, 47.3, 46.0),

two methylenes (δC35.5, 29.8), and three vinyl methyls (δC26.0,

21.7, 18.0) These data were similar to those of nicolaioidesin A

(9),15an isolate obtained from the same extract, except for the

disappearance of signals due to a methoxy group at C-4 in 9 (δH

3.70;δC55.7) Thus, compound 1 was assigned tentatively as

4-hydroxynicolaioidesin A, which was conﬁrmed by the HMBC

spectrum (Figure 1) The relative conﬁguration of 1 was

determined from the coupling constant data and NOESY

analysis The large coupling constant between H-1′ and H-6′ (J =

11.4 Hz) and between H-1′ and H-2′ (J = 10.2 Hz) indicated that

they are in a trans-diaxial orientation This was supported by the

NOESY correlations between H-1′ and H-1″, H-1′ and

H-2‴/H-6‴, H-1′ and H-5′α, H-2′ and H-6′, and H-6′ and H-5′β (Figure

2) Therefore, the structure of compound 1 was assigned as

4-hydroxynicolaioidesin A

Compound 2 was obtained as a yellowish, amorphous solid,

and its molecular formula was determined as C26H30O5 by

HRESIMS The IR spectrum of 2 exhibited absorption bands for

hydroxy (3600 cm−1), carbonyl (1640 cm−1), and phenyl (1460

cm−1) groups The1H NMR spectrum showed signals due to a

phenyl ring (δH 7.22, 7.18, 7.05), two magnetically equivalent

aromatic protons (δH5.95), two oleﬁnic methine protons (δH

5.43, 5.19), an oxymethylene (δH3.74), three aliphatic methines

(δH4.84, 3.45, 2.34), two allylic methylenes (δH2.38, 2.34, 2.13,

2.00), two vinyl methyls (δH1.78, 1.56), and a methoxy group

(δH3.77) The13C NMR spectrum displayed 26 carbon signals

including a ketone carbonyl carbon (δC 207.5), 12 aromatic

carbons, four oleﬁnic carbons (δC137.8, 136.4, 125.3, 121.9),

one oxymethylene (δC68.7), three methine carbons (δC54.7,

43.3, 37.8), two methylenes (δC36.8, 29.2), two vinyl methyls

(δC23.1, 14.0), and one methoxy (δC55.8) These data closely

resembled those of panduratin A (10),16a major compound of

B pandurata, except for the appearance of signals for a

hydroxymethyl group in 2 instead of one of the vinyl methyls

in 10 The hydroxymethyl group was determined to be at C-4″

based on the HMBC correlations of H-2″ and H-5″ with C-4″

(Figure 1) and the downﬁeld shift of C-4″ (δH 3.74;δC68.7)

The double-bond geometry at C-2″ was assigned in the

E-conﬁguration based on the upﬁeld-shifted 13C NMR chemical

shift for a vinyl methyl carbon C-5″ (δC14.0) along with NOESY correlations of H-2″ with H-4″ and of H-1″ with H-5″ (Figure 2) Moreover, the relative conﬁguration of the cyclohexenyl unit of 2 was established by coupling constant data and NOESY spectroscopic analysis The large coupling constant between

H-1′ and H-6′ (J = 11.8 Hz) indicated that they are in a trans-diaxial orientation, and the small coupling constant between H-1′ and H-2′ (J = 4.6 Hz) showed their cis relationship This was conﬁrmed by the NOESY correlations between H-1′ and H-2′, H-1′ and H-2‴/H-6‴, H-1′ and H-5′α, H-6′ and H-1″, and H-6′ and H-5′β (Figure 2) Therefore, the structure of compound 2 was concluded to be 3″-hydroxymethylpanduratin A

Panduratin J (3) was obtained as a yellowish, amorphous solid having the molecular formula C26H30O5, as determined by HRESIMS The IR spectrum of 3 showed absorptions due to hydroxy, carbonyl, and phenyl groups The1H and13C NMR data resembled those of panduratin A (10),16isolated from the same plant extract, and indicated the presence of a substituted cyclohexene ring, a phenyl ring, two magnetically equivalent aromatic protons, and a methoxy group However, 3 showed signals corresponding to exomethylene (δH4.81, 4.61;δC150.1, 109.6) and oxymethine (δH 3.50; δC 75.4) groups instead of signals corresponding to oleﬁnic methine and vinyl methyl groups as in the prenyl unit in compound 10 Thus, the presence

of a 3-methyl-2-hydroxybut-3-enyl moiety rather than a prenyl moiety was proposed The HMBC correlations of the H-4″ exomethylene protons (δH4.81, 4.61) with the C-2″ oxymethine carbon (δC75.4) and the C-5″ methyl carbon (δC18.2) indicated that the exomethylene group occurs at C-4″ Similarly, the location of the hydroxy group was determined to be C-2″ based

on HMBC correlations from the H-2″ oxymethine proton (δH

3.50) to the C-1″ methylene carbon (δC 37.4) and the C-4″ exomethylene carbon (δC 109.6) and from the H-4″ exo-methylene protons (δH4.81, 4.61) and the H-5″ methyl proton (δH1.62) to the C-2″ oxymethine carbon (δC75.4) (Figure 1) Moreover, the partial structure C-1″−C-2″ was deduced from the COSY and HSQC spectra and the downﬁeld shift of C-2″ (δH

3.50; δC 75.4) Finally, the coupling constants and NOESY correlations suggested 3 as having the same relative conﬁguration

as 2 in the cyclohexenyl chalcone unit Therefore, the structure of panduratin J (3) was elucidated as shown

Figure 2 Key NOESY correlations observed for compounds 1−8.

C

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Panduratin K (4) was isolated as a yellowish, amorphous solid,

and its molecular formula was found to be C26H30O5 by

HRESIMS The IR spectrum of 4 displayed absorbances for

hydroxy, carbonyl, and phenyl groups The1H and13C NMR

data of 4 also resembled analogous data for panduratin A (10).16

However, they diﬀered in the signals due to the prenyl side chain

The1H NMR and HSQC spectra showed the signals of a pair of

trans-coupled double bond [δH5.54 dd (J = 15.4 and 9.4 Hz);δC

126.1 (C-1″) and δH5.37 d (J = 15.4 Hz);δC142.0 (C-2″)], a

quaternary oxygenated carbon [δC70.2 (C-3″)], and two tertiary

methyl groups [δH1.18;δC30.6 (C-4″) and δH1.17;δC30.6

(C-5″)] In the HMBC spectrum, the two H3-4″ and H3-5″ tertiary

methyl groups showed correlations with the C-3″ quaternary

oxygenated carbon and the C-2″ oleﬁnic methine carbon,

suggesting the linkage of C-4″ and C-5″ with C-2″ of the double

bond via the C-3″ quaternary oxygenated carbon (Figure 1) The

relative conﬁguration of the cyclohexenyl unit of 4 was found to

be the same as those of 2 and 3 based on the coupling constant

data and the NOESY spectroscopic analysis Therefore, the

structure of panduratin K (4) was determined as shown

Panduratin L (5) was obtained as a yellowish, amorphous

solid It showed a sodiated molecular ion at m/z 459.2163 [M +

Na]+, corresponding to the molecular formula, C27H32O5Na, in

the HRESIMS The1H and13C NMR data of 5 were similar to

those of compound 4, except for the appearance of one more

methoxy group (δH 3.00; δC 50.4), and two meta-coupled

aromatic proton signals atδH6.05 and 5.85 (J = 1.9 Hz) instead

of the singlet signal of two magnetically equivalent aromatic

protons The aromatic methoxy group was located at C-6 based

on the HMBC correlation between the methoxy proton atδH

3.99 and the C-6 oxygenated quaternary aromatic carbon atδC

164.0 The location of the aliphatic methoxy group was

determined to be at C-3″ based on the HMBC correlations

observed between the methoxy proton (δH3.00) and the C-3″

quaternary oxygenated carbon (δC75.1) (Figure 1) Analysis of

the NOESY correlations together with the coupling constants

indicated the relative conﬁguration of the cyclohexenyl moiety to

be the same as in 2−4 Therefore, the structure of panduratin L

(5) was established as shown

Panduratin M (6) was isolated as a yellowish, amorphous solid

Its molecular formula was assigned as C26H30O5by HRESIMS

nicolaioidesin B (13),15a compound isolated from the same

plant extract This compound showed the presence of a

trans-3-methyl-3-hydroxybutenyl group at C-2′ of the cyclohexenyl ring

instead of the prenyl side chain (Figure 1) On the basis of the

HMBC spectrum, the phenyl and

2,6-dihydroxy-4-methoxyben-zoyl units were assigned at C-1′ and C-6′ of the cyclohexenyl

ring, respectively, and were very diﬀerent from those of 1−5 The relative conﬁguration of 6 was established from the coupling constant data and the NOESY spectrum The large coupling constant between H-1′ and H-6′ (J = 11.4 Hz) indicated that they have a trans-diaxial orientation, while the small coupling constant between H-1′ and H-2′ (J = 5.2 Hz) is cis-oriented Furthermore, in the NOESY spectrum, correlations between

H-1′ and 2′, 1′ and 5′α, 6′ and 2‴/6‴, 6′ and H-1″, and H-6′ and H-5′β (Figure 2) were observed, suggesting their proximity, as in nicolaioidesin B (13) Therefore, the structure of panduratin M (6) was concluded as shown Panduratins N (7) and O (8) were both obtained as yellowish, amorphous solids, and they were found to possess the same molecular formula, C26H30O4, as determined by HRESIMS and HRFABMS, respectively The1H and13C NMR data of 7 and 8

isopanduratin A1 (14),17respectively Also apparent were the methoxy groups at C-4 in 7 and at C-6 in 8, as confirmed by the HSQC and HMBC spectra (Figure 1) However, 7 and 8 were observed to differ from 13 and 14 from a variation in the stereoconfiguration at C-2′ of the cyclohexenyl moiety Both the large coupling constants between H-1′ and H-6′ (J = 11.0−11.2 Hz) and between H-1′ and H-2′ (J = 10.9 Hz) indicated that they are oriented in a trans-diaxial manner This was also supported by the NOESY correlations between H-1′ and 1″, 1′ and

H-5′α, H-2′ and H-2‴/H-6‴, H-2′ and H-6′, H-6′ and H-2‴/H-6‴, andH-6′ and H-5′β (Figure 2) Therefore, the structures of panduratins N (7) and O (8) were assigned as shown

All isolated compounds were tested for their preferential cytotoxic activity against the PANC-1 human pancreatic cancer cell line, according to an antiausterity strategy.2Their PC50values (the 50% preferential cell death in NDM without cytotoxicity in DMEM) are listed in Table 4 Among the compounds tested, isopanduratin A1 (14) and nicolaioidesin C (15) exhibited the most potent preferential cytotoxicity, with PC50values of 1.0 and 0.84μM, respectively, which is comparable to that of arctigenin, a positive control (PC50value, 0.8μM)

The activity of the isolates was found to greatly depend on the nature of the substituents in the cyclohexene chalcone unit In general, compounds having a phenyl group at C-1′ and a benzoyl substituent at C-6′ of the cyclohexenyl moiety were found to have potent activity (6 > 4, 13 > 10 and 9, 14 > 11) Interestingly,

at C-1′ and C-2′ of the cyclohexenyl substituent, prenyl and benzoyl groups or prenyl and phenyl groups on the same side of the ring are more favorable than when on diﬀerent sides (12 > 1,

13> 7, 14 > 8) Moreover, at C-2′ and C-3′ of the cyclohexenyl unit, it was observed that the position of the prenyl moiety or its modiﬁed form leads to a change of activity (2 > 4 > 10 > 3, 15 >

Figure 3 Morphology of PANC-1 cells under the control and following treatment with nicolaioidesin C (15, 1.5 μM) in NDM at 24 h and stained by ethidium bromide (EB)/acridine orange (AO) Live cells were stained with AO and emitted a bright green ﬂuorescence, while dead cells were stained with EB and emitted a red ﬂuorescence Treatment with nicolaioidesin C (15) at 1.5 μM led to dramatic alteration of PANC-1 cell morphology and total death of PANC-1 cells within 24 h.

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13> 6) Furthermore, the presence of a methoxy group at C-6 of

the benzoyl moiety was found to result in more potent activity

than when a methoxy group at C-4 or a hydroxy group at C-6 is

present (11 > 10, 14 > 13, 8 > 7, 11 > 12) At C-4, a hydroxy

group was favored over a methoxy group (1 > 9, 12 > 10)

Nicolaioidesin C (15) was studied further for its eﬀects on the

morphological changes of PANC-1 using an ethidium bromide

and acridine orange (EB/AO) staining assay.12Cells treated with

nicolaioidesin C (15, 1.5 μM) showed round morphology of

PANC-1 cells and emitted a redﬂuorescence of EB, indicative of

dead cells In contrast, the control cells showed intact

suggestive of live cells (Figure 3)

■ EXPERIMENTAL SECTION

General Experimental Procedures Optical rotations were

recorded on a JASCO DIP-140 digital polarimeter IR spectra were

measured with a Shimadzu IR-408 spectrophotometer in CHCl3

solution NMR spectra were taken on a Bruker Advance III 500

spectrometer (Bruker Biospin) with tetramethylsilane as an internal

standard, and chemical shifts are expressed in δ values HRESIMS and

HRFABMS measurements were carried out on a Bruker

micrOTOF-QII mass spectrometer and JEOLJMS-AX505HAD mass spectrometer,

respectively Silica gel 60, 40−63 μm (230−400 mesh ASTM), for

column chromatography was purchased from Scharlau Analytical and

preparative TLC was carried out on precoated Merck Kieselgel 60F254or

RP-18F254plates (0.25 or 0.5 mm thickness).

Plant Material The rhizomes of Boesenbergia pandurata were

collected in Tinh Bien District of An Giang Province, Vietnam, in April

2013, and this species was identiﬁed by Ms Hoang Viet, Faculty of

Biology, University of Science, Vietnam National University, Ho Chi

Minh City (VNU-HCM) A voucher specimen (MCE0043) has been

deposited at the Division of Medicinal Chemistry, Faculty of Chemistry,

University of Science, VNU-HCM.

Extraction and Isolation Dried powdered rhizomes of B

pandur-ata (5.5 kg) were extracted with MeOH (15 L, reﬂux, 3 h × 3) to yield

680 g of a dry extract The MeOH extract was suspended in H2O (1.5 L) and then partitioned successively with CHCl3(3 × 1.5 L) and EtOAc (3

× 1.5 L) to give CHCl 3 (470 g), EtOAc (10 g), and H2O (150 g) extracts, respectively A part of the CHCl3-soluble extract (450 g) was subjected to silica gel column chromatography (9 × 120 cm), eluted with EtOAc−n-hexane gradient mixtures (0−50%), to yield 15 fractions (fr-1, 22.0 g; fr-2, 197.5 g; fr-3, 22.0 g; fr-4, 26.0 g; fr-5, 6.0 g; fr-6, 24.0 g; fr-7, 15.0 g; fr-8, 20.0 g; fr-9, 22.0 g; fr-10, 21.0 g; fr-11, 11.0 g; fr-12, 18.0 g; fr-13, 14.0 g; fr-14, 10.0 g; fr-15, 22.0 g) Fraction 3 (22.0 g) was subjected to further silica gel column chromatography (7.5 × 120 cm), eluted with EtOAc −n-hexane gradient mixtures (0−80%), to give seven subfractions (1, 160 mg; 2, 2.2 g; 3, 8.1 g; 4, 6.3 g;

fr-3-5, 3.3 g; fr-3-6, 1.5 g; fr-3-7, 1.3 g) Subfraction 3-2 was rechromato-graphed on silica gel with a CHCl 3 −n-hexane gradient system to yield four subfractions, fr-3-2-1−4 Subfraction 3-2-1 (379 mg) was chromatographed on ODS silica gel with MeOH−H 2 O gradient mixtures (0−50%) to give 10 (300 mg), followed by normal-phase preparative TLC with EtOAc−n-hexane (20:80) to aﬀord 9 (6.1 mg) Subfraction 3-2-3 (377 mg) was chromatographed on ODS silica gel with MeOH−H 2 O gradient mixtures (0−50%) and then puriﬁed by normal-phase preparative TLC with EtOAc−CHCl 3 −n-hexane (5:25:70) to give 7 (5.0 mg), 13 (5.0 mg), and 14 (6.6 mg) Subfraction 3-3 was dissolved in CHCl3−n-hexane and left overnight to give crystals

of 11 (6.0 g) Subfraction 3-6 was subjected to silica gel column chromatography with an acetone−n-hexane gradient system, to yield ﬁve subfractions, fr-3-6-1−5 Subfraction 3-6-4 (190 mg) was again separated by silica gel column chromatography with a further acetone − n-hexane gradient system, followed by reversed-phase preparative TLC with MeOH −CH 3 CN −H 2 O (10:70:20), to a ﬀord 14 (10.0 mg) Fraction 4 (26.0 g) was subjected to silica gel column (7.5 × 120 cm) chromatography, eluted with an acetone−n-hexane gradient system, to yield 14 subfractions (1, 18 mg; 2, 113 mg; 3, 127 mg;

fr-4-4, 199 mg; fr-4-5, 65 mg; fr-4-6, 34 mg; fr-4-7, 616 mg; fr-4-8, 20−23 g; fr-4-9, 269 mg; fr-4-10, 32 mg; fr-4-11, 31 mg; fr-4-12, 850 mg; fr-4-13,

303 mg; fr-4-14, 2−8 g) Subfraction 4-13 was chromatographed by silica gel column chromatography, with CHCl 3 −n-hexane gradient mixtures (0−100%), to obtain 8 (6.5 mg) Fraction 6 (24.0 g) was further separated by silica gel column (7.5 × 120 cm) chromatography,

Table 1.1H NMR Spectroscopic Data (500 MHz) of Compounds 1−5 in Acetone-d6(δ in ppm, Multiplicities, J in Hz)

1 ′ 4.85 dd (11.4, 10.2) 4.84 dd (11.8, 4.6) 4.89 dd (11.7, 4.5) 4.93 dd (11.6, 5.0) 4.71 dd (11.8, 4.9)

2 ′ 2.84 brd (10.2) 2.73 ddd (10.3, 5.0, 4.6) 2.98 ddd (10.5, 4.5, 4.3) 3.16 dd (9.4, 5.0) 3.13 dd (9.0, 4.9)

5 ′α 2.31 dd (18.2, 11.3) 2.13 dd (18.2, 11.2) 2.07 dd (18.0, 11.3) 2.07 dd (17.8, 11.5) 2.08 dd (18.0, 11.6)

5 ′β 2.14 ddd (18.2, 4.6, 4.4) 2.38 ddd (18.2, 6.4, 4.2) 2.32 ddd (18.0, 6.0, 4.6) 2.37 ddd (17.8, 4.6, 4.4) 2.37 ddd (18.0, 5.1, 5.0)

6 ′ 3.06 ddd (11.4, 11.3, 4.6) 3.45 ddd (11.8, 11.2, 6.4) 3.30 ddd (11.7, 11.3, 6.0) 3.40 ddd (11.6, 11.5, 4.6) 3.40 ddd (11.8, 11.6, 5.1)

1″ 2.23 brd (16.7) 2.00 ddd (15.5, 6.9, 5.0) 1.34 ddd (17.6, 10.6, 4.3) 5.54 dd (15.4, 9.4) 5.47 dd (15.7, 9.0)

2.15 brd (16.7) 2.34 ddd (15.5, 10.3, 6.9) 2.00 ddd (17.6, 10.5, 4.0)

4.81 s

3 ‴, 5‴ 7.07 dd (7.8, 7.4) 7.18 dd (7.4, 7.2) 7.17 dd (7.8, 7.5) 7.18 dd (7.4, 7.2) 7.18 dd (7.4, 7.2)

E

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with a MeOH −CHCl 3 gradient system, to yield 13 subfractions (fr-6-1,

464 mg; fr-6-2, 388 mg; fr-6-3, 1.6 g; fr-6-4, 3.8 g; fr-6-5, 5.0 g; fr-6-6, 576

mg; fr-6-7, 1.7 g; fr-6-8, 3.3 g; fr-6-9, 1.1 g; fr-6-10, 1.7 g; fr-6-11, 794 mg;

fr-6-12, 815 mg; fr-6-13, 442 mg) Subfraction 6-6 was also

chromatographed on silica gel with an acetone−n-hexane gradient

system, followed by normal-phase preparative TLC with

acetone−n-hexane (20:80), to give 3 (0.8 mg) Subfraction 6-7 was subjected to

silica gel chromatography, with an EtOAc−n-hexane gradient system, to

give four subfractions, fr-6-7-1−4 Of these, fr-6-7-2 (115 mg) was

chromatographed on ODS silica gel, with acetone−H 2 O gradient

mixtures (0−80%), and followed by normal-phase preparative TLC with

EtOAc −n-hexane (20:80), to aﬀord 1 (18.3 mg) Subfraction 6-9 was also chromatographed on silica gel with an EtOAc −n-hexane gradient system to give three subfractions, fr-6-9-1 −3, and then fr-6-9-2 was dissolved in EtOAc −n-hexane and left overnight to give 12 (300.0 mg) Fraction 8 (20.0 g) was chromatographed on silica gel (7.5 × 120 cm) with MeOH−CHCl 3 gradient mixtures (0−50%) to give 20 subfractions (fr-8-1, 12 mg; fr-8-2, 30 mg; fr-8-3, 40 mg; fr-8-4, 22 mg; fr-8-5, 6.9 mg; fr-8-6, 140 mg; fr-8-7, 4.2 g; fr-8-8, 4.2 g; fr-8-9, 402 mg; fr-8-10, 752 mg; fr-8-11, 216 mg; fr-8-12, 2.43 g; fr-8-13, 4.43 g; fr-8-14, 538 mg; fr-8-15,

89 mg; fr-8-16, 119 mg; fr-8-17, 414 mg; fr-8-18, 639 mg; fr-8-19, 116 mg; fr-8-20, 158 mg) Subfraction 8-9 was subjected to silica gel column

Table 2.1H NMR Spectroscopic Data (500 MHz) of Compounds 6−8 in Acetone-d6(δ in ppm, multiplicities, J in Hz)

2.15 ddd (16.2, 6.4, 5.0) 2.14 ddd (16.1, 6.4, 4.9)

Table 3.13C NMR Spectroscopic Data (125 MHz) of Compounds 1−8 in Acetone-d6

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chromatography, eluted with EtOAc−n-hexane gradient mixtures (0−

30%) and then acetone−n-hexane gradient mixtures (0−50%), to aﬀord

2 (5.0 mg) Subfraction 8-10 was subjected to silica gel column

chromatography with acetone−n-hexane gradient mixtures (0−50%) to

yield three subfractions, 10-1−3 Both 10-1 (143 mg) and

fr-8-10-3 (315 mg) were subjected to silica gel column chromatography,

eluted with acetone −n-hexane gradient mixtures (0−50%), to aﬀord six

subfractions, fr-8-10-1-1 −3 and fr-8-10-3-1−3, respectively Subfraction

8-10-1-1 (36.3 mg) was chromatographed over ODS silica gel with

MeOH−H 2 O gradient mixtures (0−80%), followed by reversed-phase

preparative TLC with MeOH−H 2 O (20:80), to give 5 (6.7 mg).

Subfraction 8-10-3-2 (45.1 mg) was subjected to normal-phase

preparative TLC with EtOAc−n-hexane (4:96) to give two subfractions.

Of these, fr-8-10-3-2-1 (25.6 mg) was recrystallized with MeOH−

CHCl 3 to aﬀord 4 (13.1 mg), while fr-8-10-3-2-2 (9.3 mg) was puriﬁed

by reversed-phase preparative TLC with acetone−H 2 O (30:70) to

aﬀord 6 (6.0 mg).

Compound 1: yellow, amorphous solid; [α] D25 +35.6 (c 1,

CH3COCH3); IR ν max (CHCl3) 3500, 1640, 1450, 1100 cm−1; 1 H

and 13 C NMR (acetone-d6, 500 MHz, see Tables 1 and 3 ); HRESIMS

m/z 415.1885 [M + Na] + (calcd for C25H28O4Na, 415.1885).

m/z 445.1991 [M + Na] + (calcd for C 26 H 30 O 5 Na, 445.1976).

CH 3 COCH 3 ); IR ν max (CHCl 3 ) 3600, 1640, 1445, 1090 cm−1; 1H

and13C NMR (acetone-d 6 , 500 MHz, see Tables 1 and 3 ); HRESIMS

m/z 445.1997 [M + Na] + (calcd for C 26 H 30 O 5 Na, 445.1991).

Compound 6: yellow, amorphous solid; [α]D25 +27.5 (c 1,

CH 3 COCH 3 ); IR ν max (CHCl 3 ) 3600, 1650, 1450, 1100 cm−1; 1H

and13C NMR (acetone-d 6 , 500 MHz, see Tables 2 and 3 ); HRESIMS

m/z 405.2075 [M − H] − [calcd for C26H29O4, 405.2066].

and 13 C NMR (acetone-d6, 500 MHz, see Tables 2 and 3 ); HRFABMS

m/z 407.22253 [M + H] + (calcd for C26H31O4, 401.22224).

Preferential Cytotoxicity Assay against PANC-1 Cells The

PANC-1 (RBRC-RCB2095) human pancreatic cancer cell line was

purchased from the Riken BRC cell bank and maintained in standard Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum supplemented and stored at 37 °C under a humidified atmosphere of 5% CO2and 95% air Brie fly, human pancreatic cancer cells were seeded in 96-well plates (1.5 × 10 4 /well) and incubated in fresh DMEM at 37 °C under 5% CO 2 and 95% air for 24 h After the cells were washed twice with phosphate-buffered saline (PBS), the medium was changed to serially diluted test samples in both nutrient-rich medium (DMEM) and nutrient-deprived medium (NDM)2 with a control and blank in each test plate The composition of the NDM was

as follows: 265 mg/L CaCl2(2 H2O), 0.1 mg/L Fe(NO3)3(9 H2O), 400 mg/L KCl, 200 mg/L MgSO4(7 H2O), 6400 mg/L NaCl, 700 mg/L NaHCO3, 125 mg/L NaH2PO4, 15 mg/L phenol red, 25 mM/L HEPES buﬀer (pH 7.4), and MEM vitamin solution (Life Technologies, Inc., Rockville, MD, USA); the ﬁnal pH was adjusted to 7.4 with 10% NaHCO 3 Arctigenin, the positive control in this study, was isolated from the seeds of Arctium lappa 2 After 24 h of incubation with each test compound in DMEM and NDM, the cells were washed twice with PBS and replaced with 100 μL of DMEM containing a 10% WST-8 cell counting kit solution After 3 h of incubation, the absorbance at 450 nm was measured (PerkinElmer EnSpire multilabel reader) Cell viability was calculated from the mean values of data from three wells by using the following equation:

Cell viability (%) [Abs Abs /Abs

Abs ] 100%

(test sample) (blank) (control)

(blank)

Morphological Assessment of Cancer Cells PANC-1 cells were seeded in 24-well plates (6 × 10 4 /well) and incubated in fresh DMEM at

37 °C under 5% CO 2 and 95% air for 24 h After the cells were washed twice with PBS, the medium was changed to NDM (control) or nicolaioidesin C (15, 1.5 μM) in NDM (treated) After a 24 h incubation, 8 μL of EB/AO reagent was added to the each test well and incubated for 5 min, and the morphology was captured using an EVOS

FL cell imaging system (20× objective) under ﬂuorescent and phase contrast mode.

■ ASSOCIATED CONTENT

*S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnat-prod.6b00784

Copies of spectroscopic data for 1−8 (PDF)

■ AUTHOR INFORMATION

Corresponding Authors

*E-mail (N T Nguyen):ntnhan@hcmus.edu.vn Tel: +84-907-426-331 Fax: +84-838-353-659

*E-mail (S Awale): suresh@inm.u-toyama.ac.jp Tel: +81-76-434-7640 Fax: +81-76-+81-76-434-7640

ORCID

Nhan Trung Nguyen:0000-0001-5142-4573

Notes

The authors declare no competingﬁnancial interest

■ ACKNOWLEDGMENTS

This research was supported by a grant from Vietnam’s National Foundation for Science and Technology Development (No 104.01-2013.72) to N.T.N and by a Grant in Aid for Scientiﬁc Research (16K08319) from the Japan Society for the Promotion

of Science (JSPS), Japan, to S.A

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