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 preferentiallyinhibit the survival of cancer cells under low nu-trient conditions is a novel antiausterity strategyin anticancer drug discovery. In this study, themethanolic extract of the leaves of Artocarpus al-tilis showed 100% preferential cytotoxicityagainst PANC-1 human pancreatic cancer cellsunder nutrient-deprived conditions at a concen-

tration of 50 µg/mL. Further investigation of thisextract led to the isolation of eight new gerany-lated dihydrochalcones named sakenins A–H (1–8) together with four known compounds (9–12).Among them, sakenins F (6) and H (8) were iden-tified as potent preferentially cytotoxic candi-dates with PC50 values of 8.0 µM and 11.1 µM, re-spectively.

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Geranyl Dihydrochalcones from Artocarpus altilis andTheir Antiausteric Activity

Authors Mai Thanh Thi Nguyen1, Nhan Trung Nguyen1, Khang Duy Huu Nguyen1, Hien Thu Thi Dau1, Hai Xuan Nguyen1,Phu Hoang Dang1, Tam Minh Le1, Trong Huu Nguyen Phan1, Anh Hai Tran2, Bac Duy Nguyen2, Jun-ya Ueda3,Suresh Awale3

Affiliations 1 Faculty of Chemistry, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam2 Vietnam Military Medical University, Ha Noi, Vietnam3 Frontier Research Core for Life Sciences, University of Toyama, Toyama, Japan

Key wordsl" antiausterity agentl" Artocarpus altilisl" Moraceael" pancreatic cancerl" PANC‑1

received July 23, 2013revised Nov. 13, 2013accepted Nov. 22, 2013

BibliographyDOI http://dx.doi.org/10.1055/s-0033-1360181Published online January 15,2014Planta Med 2014; 80: 193–200© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

CorrespondenceDr. Suresh AwaleFrontier Research Core for LifeSciencesUniversity of Toyama2630 SugitaniToyama 930–0194JapanPhone: + 81764347640Fax: + 81764347640suresh@inm.u-toyama.ac.jp

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Introduction!

Pancreatic cancer is one of the most deadly formsof cancer associated with the lowest 5-year sur-vival rates known for cancers [1]. It shows resis-tance to conventional chemotherapeutic agentssuch 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 cellsshow an extraordinary tolerance [4] to starvationenabling them to survive in hypovascular (auster-ity) conditions. Thus, development of drugsaimed at countering this tolerance to nutrientstarvation is a novel antiausterity approach [5] inanticancer drug discovery. Working under thishypothesis, we screened medicinal plants fromwidely different origins for the discovery of anti-austerity agents, using the PANC-1 human pan-creatic cancer cell line [5–15].Artocarpus altilis (Parkinson ex F.A. Zorn) Fors-berg (Moraceae) is a medicinal plant locallyknown as “Sa ke” in Vietnam, and a decoction ofits leaves has been used traditionally for the treat-ment of gout, hepatitis, hypertension, and diabe-tes [16]. Previous work on this plant species re-

Nguyen MTT et al. Gera

ported a number of geranyl dihydrochalcones[17] having cytotoxic activities and aurones hav-ing tyrosinase and α-glucosidase inhibitory activ-ity [18]. In our screening program, recently wefound that the methanolic extract of the leavesdisplayed 100% preferential cytotoxicity againstPANC-1 cells in nutrient-deprived condition at50 µg/ml. A further phytochemical study on thisextract furnished the isolation of eight new dihy-drochalcones (1–8,l" Fig. 1) and four known com-pounds (9–12). We herein report the structures ofthese compounds and their antiausterity activity.

Results and Discussion!

Sakenin A (1) was isolated as a yellow amorphoussolid. Its molecular formula was determined byHR‑ESI‑MS to be C25H32O6 [m/z 451.2091 (M +Na)+]. The 1H NMR spectrum of 1 (l" Table 1) ex-hibited signals due to a pair of ortho-coupled aro-matic protons at δH 6.42 (1H, d, J = 8.1 Hz) and δH6.48 (1H, d, J = 8.1 Hz), an aromatic ABX spin sys-tem (δH 7.53, d, J = 8.9 Hz; δH 6.22, dd, J = 8.9,2.4 Hz; δH 6.15, d, J = 2.4 Hz) together with an ole-finic proton (δH 5.04, td, J = 6.5, 1.2 Hz), six meth-ylenes (δH 2.99, t, J = 7.4 Hz; δH 2.78, t, J = 7.4 Hz;

nyl Dihydrochalcones from… Planta Med 2014; 80: 193–200

Table 1 1H and 13C NMR data (500 and 125MHz) for compounds 1–4.

Position 1 (in CD3OD) 2 (in CD3OD) 3 (in DMSO-d6) 4 (in CD3OD)

δH δC δH δC δH δC δH δC1′ 112.6 114.0 112.4 114.1

2′ 164.8 166.4 165.0 166.5

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

4′ 164.8 166.4 164.2 166.5

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

C=O 204.5 205.9 203.7 206.2

1 130.9 132.2 127.6 131.6

2 126.6 128.1 126.4 122.3

3 142.9 144.5 146.2 142.2

4 143.1 144.3 139.2 145.2

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

3′′ 134.2 135.6 148.1 77.1

4′′ 1.84, t (7.4) 39.7 1.99, m 37.7 2.06, m 30.6 2.00, m 38.8

2.21, m 1.74, m

5′′ 1.33, m 22.2 1.61, m 30.5 2.16, m 25.8 1.77, m 28.5

1.32, m 1.64, m

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

7′′ 70.0 78.7 131.1 39.5

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, s5.14, s

109.9 1.07, s 27.8

2′-OH 12.62, s

Fig. 1 Chemical structure of new compounds (1–8) isolated from Vietnamese Artocarpus altilis.

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Fig. 2 Connectivities (bold lines) deduced by the COSY and HSQC spectra and significant HMBC correlations (solid arrows) observed for new compounds 1–8.

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δH 3.30, d, J = 6.5 Hz; δH 1.84, t, J = 7.4 Hz; δH 1.33, m δH 1.27, m),and three methyl groups. The 13C NMR and DEPT spectra of 1, onthe other hand, displayed signals of 25 carbons, which could beclassified as a carbonyl carbon, fourteen sp2 carbons, a quaternaryoxygenated carbon, six methylene carbons, and three methyl car-bons (l" Table 1). Part of these data closely resembled those forcompound AC-5-1 (2-geranyl-2′,4′,3,4-tetrahydroxydehydro-chalcone) previously isolated from A. altilis from Micronesia [19]and indicated the presence of a 2′,4′,3,4-tetrahydroxydihydro-chalcone unit. However, 1 showed signals due to a quaternaryoxygenated carbon (δC 70.0) and a methylene (δH 1.27, δC 42.9)group instead of signals due to an olefin moiety as in the geranylunit in compound AC-5-1. Therefore, the presence of a hydroxy-geranyl unit instead of a geranyl unit was assumed. In the HMBCspectrum of 1 (l" Fig. 2), two tertiary methyl groups, H3-8′′ andH3-9′′, at δH 1.01 showed correlations with the quaternary oxy-genated carbon C-7′′ at δC 70.0 and the methylene carbon C-6′′(δC 42.9), suggesting the presence of a 7-hydroxy-3,7-dimethyl-2-octenyl group. Furthermore, the HMBC correlations frommethylene proton H2-1′′ (δH 3.30) in the geranyl unit to two aro-matic carbons, C-2 (δC 126.6) and C-3 (δC 142.9), indicated the lo-cation of the side chain at C-2 (l" Fig. 2). Finally, the double bondgeometry at C-2′′was determined to be E based on the high-fieldshifted 13C chemical shift at the vinylmethyl carbon C-10′′ (δC14.9) along with NOESY correlations between H-2′′/H2-4′′ andH3-10′′/H2-1′′ (l" Fig. 3). Therefore, the structure of 1 was con-cluded as 2-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]-2′,4′,3,4-tet-rahydroxydihydrochalcone.Sakenin B (2) was isolated as a yellow amorphous solid. TheHR‑ESI‑MS of 2 indicated its molecular formula to be C25H32O7.Its 1H and 13C NMR data closely resembled those of 1 (l" Table 1),except for the appearance of a signal due to an oxymethine group

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(δH 3.35, δC 76.6) instead of methylene signals assigned to H2-6′′(δH 1.27, δC 42.9) in the geranyl side chain of 1. Hence, the pres-ence of a hydroxyl group at C-6′′ in 2was evident, whichwas con-firmed by COSY, HSQC, and HMBC spectral analysis (l" Fig. 2).Therefore, the structure of 2 was concluded as 2-[6,7-dihydroxy-3,7-dimethyl-2(E)-octenyl]-2′,4′,3,4-tetrahydroxydihydrochal-cone.Sakenin C (3) was isolated as a yellow amorphous solid havingthe molecular formula C25H30O6 as determined by HR‑ESI‑MS.The 1H NMR and 13C NMR data of 3 resembled quite closely thoseof 1 (l" Table 1). However, they differ in the signals due to the ger-anyl side chain. The DEPT and HSQC spectra of 3 showed the sig-nals of only two tertiary methyl groups [δH 1.65, δC 25.4 (C-8′′)and δH 1.57, δC 17.5 (C-9′′)], an oxymethine (δH 5.19, δC 84.5, C-2′′), an exomethylene group (δH 4.90, 5.14; δC 109.9; C-10′′), twoquaternary olefinic carbons [δC 148.1 (C-3′′) and δC 131.1 (C-7′′)],an olefinic methine group (δH 5.12, δC 124.7, C-6′′), and threemethylene groups [δH 3.35, 2.95, δC 33.8 (C-1′′), δH 2.06, δC 30.6(C-4′′), and δH 2.16, δC 25.8 (C-5′′)]. These data were analyzed byCOSY and HSQC spectra (l" Fig. 2), and from them, the partialstructures C(1′′)H2–C(2′′)H–O and C(4′′)H2–C(5′′)H2–C(6′′)H werededuced. In the HMBC spectrum, the two tertiary methyl groupsH3-8′′ and H3-9′′ gave significant correlations to the quaternaryolefinic carbon C-7′′ and the olefinic methine carbon C-6′′ sug-gesting the linkage of C-8′′ and C-9′′with C-6′′ via the quaternaryolefinic carbon C-7′′. Similarly, significant long-range correlationswere observed from exomethylene protons (H2-10′′) to C-3′′, C-2′′, and C-4′′, suggesting the connectivity between C-2′′–C3′′–C4′′ (l" Fig. 2). These data suggested that 3 contains a geranyl sidechain 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-2based on the HMBC correlations from H2-1′′ to C-2 and C-3.

en MTT et al. Geranyl Dihydrochalcones from… Planta Med 2014; 80: 193–200

Fig. 3 Key NOESY correlations (dashed arrow) ob-served for new compounds 1, 4, and 5.

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Therefore, 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 mo-lecular formula was determined as C25H30O6 based on HR-ESI‑MS. The 1H and 13C NMR data of 4 showed similar resonancesindicative of the presence of a 2′,4′,3,4-tetrahydroxydihydrochal-cone unit as in 1–3 (l" Table 1). Excluding this, the HSQC revealedthe remaining signals in 4 as three tertiary methyl groups, anoxymethine, 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 twopartial connectivities between C(1′′)H2–C(2′′)H and C(4′′)H2–

C(5′′)H2–C(6′′)H–O. In the HMBC spectrum (l" Fig. 2), the two ter-tiary methyl protons at δH 0.85 (C-8′′) and δH 1.12 (C-9′′) showedcorrelations with a methine carbon C-2′′, a quaternary carbon atδC 39.5 (C-7′′), and an oxymethine carbon at δC 78.8 (C-6′′), sug-gesting the linkage of C-2′′, C-6′′, C-8′′, and C-9′′ with C-7′′. Simi-larly, a linkage of C-10′′, C-2′′, and C-4′′with C-3′′was establishedfrom the HMBC correlations of the tertiary methyl proton at δH1.07 (C-10′′) with the quaternary oxygenated carbon at δC 77.1(C-3′′), C-2′′, and C-4′′. These data indicated the presence of a cy-clized geranyl group in 4. The relative stereochemistry of 4 wasestablished by coupling constant data and the NOESY spectralanalysis. The large coupling constant of H-6′′ (J5,6ax = 11.2) indi-cated that it should be axially oriented. Furthermore, NOESY cor-relations between H-6′′/H-2′′, H-2′′/Hβ-1′′, and Hβ-1′′/H‑10′′ 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 Ewas concluded asshown.The 1H and 13C NMR data of sakenin E (5) showed the signals of adihydrochalcone unit as in 1–4 together with the signals ascrib-able for a cyclized geranyl group that could be classified intothree tertiary methyl groups, twomethylene groups, three meth-ine groups, and two quaternary oxygenated carbons (l" Table 2).The COSY and HSQC analysis showed two partial connectivitiesfrom a methylene C-4′′ (δC 32.8, δH 1.95, 1.64) via a methyleneC-5′′, (δC 20.9, δH 1.57, 1.25), and two methine groups C-6′′ (δC38.7, δH 3.83) and C-1′′ (δC 46.3, δH 2.20) to an oxymethine C-2′′(δC 76.5, δH 3.93), which were connected based on the long-rangecorrelations observed in HMBC so as to get a planar structurehaving a geranyl group in the form of a bicyclic ring as shown inl" Fig. 2. Its relative stereochemistry was determined by NOESYspectral analysis. NOESY correlations were observed betweenH4β“/H-10′′ and H-10′′/H2′′, and between H4α”/H-6′′ and H-6′′/H‑1′′ suggesting their spatial orientation as shown in l" Fig. 3.Therefore, the structure of 5 was concluded as shown.

Nguyen MTT et al. Geranyl Dihydrochalcones from… Planta Med 2014; 80: 193–20

Sakenin F (6) was isolated as a yellow amorphous solid. Its molec-ular formula was determined to be C25H30O6 from HR‑ESI‑MS.The 1H and 13C NMR data showed the presence of a 2′,4′,3,4-tetra-hydroxydihydrochalcone unit similar to that of 1 (l" Table 2).However, it showed a difference in signals due to the geranylgroup. Comparison of molecular formulae indicated that sakeninF (6) contains two hydrogen atoms less than 1, which suggeststhe presence of a cyclic form of the geranyl group in 6. The partialconnectivity between C(1′′)H2–C(2′′)H–O and C(4′′)H2–C(5′′)H2–

C(6′′)H was deduced from COSY and HSQC and were confirmedbased on HMBC (l" Fig. 2). In the HMBC spectrum, correlationsfrommethylene protons H2-1′′ (δH 3.28, 3.19) to an aromatic car-bon C-2′ (δC 166.6), an oxymethine carbon C-2′′ (δC 90.0, δH 4.67),and a quaternary oxygenated carbon C-3′′ (δC 73.6), from the oxy-methine proton H-2′′ to C-3, C-1′′, C-3′′, and a tertiary methyl car-bon C-10′′ (δC 17.9, δH 1.61), and from the tertiary methyl protonH3-10′′ to C-2′′, C-3′′, and a methylene carbon C-4′′ (δC 39.2, δH1.55) indicated the presence of a hydroxyl group at C-3′′ and thepresence of a cyclized geranyl group. Therefore, the structure ofsakenin F (6) was concluded as shown.Sakenin G (7) was isolated as a yellow amorphous solid having amolecular formula of C17H14O5 as determined from HR‑ESI‑MS.The 1H and 13C NMR data showed signals of a dihydrochalconeunit as in other compounds (l" Table 2). The major difference ob-served was the occurrence of signals due to the presence of abenzofuran unit instead of a geranyl unit, which was confirmedby the HMBC spectrum (l" Fig. 2). Therefore, the structure of 7was concluded as 1-(2,4-dihydroxyphenyl)-3-(7-hydroxybenzo-furan-4-yl)propan-1-one.The 1H and 13C NMR data of sakenin H (8) were similar to 7 exceptfor the presence of a methoxyl group with the disappearance ofsignals due to protons in a furan unit (l" Table 2). This was alsosupported by its molecular formula C18H18O6 as determined byHR‑ESI‑MS. Therefore, the presence of a methoxydihydrofurangroup was assumed, which was confirmed by HMBC (l" Fig. 2).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 theirspectroscopic 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], 1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methylpent-3-enyl)-2H-1-benzopyran-5-yl]propan-1-one (10)[20], 2-geranyl-2′,3,4,4′-tetrahydroxydihydrochalcone (11) [21],and cycloaltilisin (12) [22].The isolated compounds were tested for their cytotoxic activityagainst a PANC-1 human pancreatic cancer cell line in normal

0

Table 2 1H and 13C NMR data (500 and 125MHz) for compounds 5–8.

Position 5 (in CDCl3) 6 (in CD3 COCD3) 7 (in DMSO-d6) 8 (in CD3 COCD3)

δH δC δH δC δH δC δH δC1′ 113.7 114.1 112.7 113.9

2′ 165.4 166.6 164.8 167.0

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

4′ 163.0 165.9 164.0 166.0

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

C=O 203.6 205.2 203.5 205.1

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

3.19, m 3.00, dd (16.5, 2.0)

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

3′′ 76.5 73.6

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

7′′ 82.3 131.0

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

4-OH 7.63, s

2′′-OMe 3.45, s 55.6

3′′-OH 3.50, s

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DMEM and nutrient-deprived medium (NDM), utilizing an anti-austerity strategy. All the tested geranyl dihydrochalconesshowed preferential cytotoxicity in a nutrient-deprived condi-tion without apparent toxicity in a nutrient-rich condition (Sup-porting Information, 2S). Their PC50 values, whichmeans the 50%preferential cell death in nutrition-deprived medium (NDM)without cytotoxicity in normal nutrient-rich medium (DMEM),are depicted in l" Table 3. Arctigenin, an antiausterity strategy-based anticancer agent which was used as a positive control inthis study, showed a PC50 value of 0.83 µM. Paclitaxel, a well-known anticancer agent, was virtually inactive. Among them, 6and 8 displayed the most potent preferential cytotoxic activitywith PC50 values of 8.0 µM and 11.1 µM, respectively (l" Table 3).Sakenin F (6) was further evaluated for its effect on the cell mor-phology of PANC-1 cells in NDM. The microscopic images wereanalyzed under phase-contrast and fluorescence mode usingethidium bromide/acridine orange (EB/AO) reagent. AO is a cellpermeable dye and gives a green or orange fluorescence in livecells. EB is permeable to dead cells only and gives a red fluores-cence. As shown in l" Fig. 4a, the cells in control were alive andstained with AO giving a green fluorescence. However, whentreated with 6, the morphology of PANC-1 cells was distinctly al-tered (l" Fig. 4b–d) with an increasing population of dead cells(red). The phase contrast image of PANC-1 cells treated with 6showed rounding of the cell membrane, rupture, and disintegra-tion of cellular contents to the medium (l" Fig. 4d). These resultsindicated that A. altilis and its constituents could have a potentialutility for the development of drugs against pancreatic cancer.

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Materials and Methods!

General experimental proceduresOptical rotations were recorded on a PerkinElmer 241 digital po-larimeter. UV spectra were obtained on a Shimadzu UV-160Aspectrophotometer. IR spectra were measured with a ShimadzuIR-408 spectrophotometer in CHCl3 solutions. NMR spectra weretaken on a Bruker Avance III 500 spectrometer (Bruker Biospin)with tetramethylsilane (TMS) as an internal standard, and chem-ical shifts are expressed in δ values. HR‑ESI‑MSwas performed ona Micro O-QIITOF mass spectrometer (Bruker Daltonics). Analyti-cal and preparative TLC were carried out on precoated Merck sil-ica gel 60F254 or RP-18F254 plate (0.25 or 0.5mm thickness).

Plant materialThe leaves of cultivated Artocarpus altilis were collected in BinhDuong Province, Vietnam, in October 2009 and identified by Ms.Hoang Viet, Faculty of Biology, University of Science, NationalUniversity 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 ChiMinh City, Vietnam.

Extraction and isolationAir-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 MeOHextract was suspended in H2O (1.5 L) and partitioned successivelywith petroleum ether (3 × 0.3 L), CHCl3 (3 × 0.3 L), and EtOAc(3 × 0.3 L) to yield petroleum ether (70 g), CHCl3 (92 g), EtOAc(75 g), and H2O (110 g) fractions, respectively.

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Table 3 Preferential cytotoxicityof compounds isolated from Arto-carpus altilis against the PANC-1human pancreatic cancer cell linein nutrient-deprived medium(NDM).

Compounds PC50, µMa Compounds PC50, µM

1 41.2 ± 1.3 8 11.1 ± 2.4

2 19.9 ± 2.0 9 18.8 ± 1.6

3 –d 10 75.2 ± 0.8

4 58.7 ± 4.3 11 –d

5 –d 12 –d

6 8.0 ± 0.9 Arctigeninb 0.8 ± 0.4

7 –d Paclitaxelc > 100

a Concentration at which 50% cells were killed preferentially in NDM. b Positive control; c negative control; d not tested due to meager

isolated amount. PC50 values represent the mean ± SD of three replicate experiments

Fig. 4 Morphology of PANC-1 cells in NDM:a control, b treated with 1 µM of 6, c treated with10 µM of 6, d treated with 10 µM of 6, phase con-trast. (Live cells were stained with AO giving greenfluorescence; dead cells were stained with EB givingred fluorescence).(Color figure available online only.)

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The CHCl3 fraction (90 g) was subjected to silica gel column(12 × 80 cm) chromatography eluted with EtOAc–petroleumether (0 :100, 5 :95, 10:90, 20:80, 40:60, 60 :40, 80 :20, and100:0; 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 wasrechromatographed on silica gel column (6.5 × 80 cm) with 15%EtOAc–petroleum ether (4 L) to give three subfractions (fr. 2–1,2.8 g; fr. 2–2, 5.6 g; fr. 2–3, 3.5 g). Subfraction 2–1 was rechroma-tographed on silica gel column (2.5 × 60 cm) with 20% EtOAc–pe-troleum ether (1.5 L), followed by preparative TLC with EtOAc–hexane (25%), to give 8 (16.7mg) and 3 (1.3mg). Subfraction 2–2 was rechromatographed on silica gel column (3.5 × 60 cm) with25% EtOAc–petroleum ether (2.5 L), followed by preparative TLCwith MeOH–CHCl3 (2 :98), to give 4 (5.3mg) and 9 (14.2mg).Subfraction 2–3 was separated by preparative TLC with 5%MeOH–CHCl3 to give 10 (14.3mg) and 11 (1.6mg). Fraction 5was further separated by silica gel column chromatography(5 × 60 cm) with 5% MeOH–CHCl3 (3 L) to give four subfractions(fr. 5–1, 1.7 g; fr. 5–2, 2.4 g; fr. 5–3, 4.5 g; fr. 5–4, 2.8 g). Subfrac-tion 5–2 was rechromatographed on silica gel column(2.5 × 60 cm) with 5% MeOH–CHCl3 (1.5 L), followed by prepara-tive TLC with 3% MeOH–CHCl3, to give 1 (9.3mg), 2 (8.9mg), and5 (1.7mg). Subfraction 5–3 was rechromatographed on silica gelcolumn (3.5 × 60 cm) with 5% MeOH–CHCl3 (2.5 L), followed bypreparative TLC with 5% MeOH–CHCl3, to give 6 (12.4mg) and 7

Nguyen MTT et al. Geranyl Dihydrochalcones from… Planta Med 2014; 80: 193–20

(1.3mg). Fraction 6 was further separated by silica gel columnchromatography (8 × 60 cm) with 10% MeOH–CHCl3 (5.5 L), fol-lowed by preparative TLC with 15% MeOH–CHCl3, to give 2(4.3mg) and 12 (1.3mg).Sakenin A (1): Yellow amorphous solid; UV (EtOH) λmax nm (logε) 330 (0.51), 288 (1.50), 227.5 (1.88), 213 (2.35); IR νmax (CHCl3)3560, 3410, 3020, 1660, 1605, 1440 cm−1; 1H and 13C NMR, seel" Table 1; HR‑ESI‑MS m/z 451.2091 [calcd. for C25H32O6Na (M +Na)+, 451.2097].Sakenin B (2): Yellow amorphous solid; [α]D25 − 8.6 (c 1.0, CHCl3);UV (EtOH) λmax nm (log ε) 335 (0.50), 290 (1.55), 227.5 (1.90),213 (2.38); IR νmax (CHCl3) 3550, 3400, 3030, 1660, 1600,1440 cm−1; 1H and 13C NMR, see l" Table 1; HR‑ESI‑MS m/z449.1903 [calcd. for C25H30O6Na (M – H2O + Na)+, 449.1940].Sakenin C (3): Yellow amorphous solid; [α]D25 − 8.1 (c 1.0, CHCl3);UV (EtOH) λmax nm (log ε) 342 (3.27), 311 (3.43), 280 (3.69); IRνmax (CHCl3) 3400, 2940, 1670, 1605, 1450, 1375 cm−1; 1H and13C NMR, see l" Table 1; HR‑ESI‑MS m/z 431.1839 [calcd. forC25H28O5Na (M – H2O + Na)+, 431.1834].Sakenin D (4): Yellow amorphous solid; [α]D25 − 114 (c 1.0, CHCl3);UV (EtOH) λmax nm (log ε) 340 (3.61), 318 (3.85), 277 (4.17); IRνmax (CHCl3) 3420, 2975, 1610, 1505, 1485, 1450, 1370 cm−1; 1Hand 13C NMR, see l" Table 1; HR‑ESI‑MS m/z 449.1933 [calcd. forC25H30O6Na (M + Na)+, 449.1940].

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Sakenin E (5): Yellow amorphous solid; [α]D25 − 11.3 (c 1.0, CHCl3);UV (EtOH) λmax nm (log ε) 341 (3.55), 321 (3.78), 279 (4.23); IRνmax (CHCl3) 3410, 2970, 1615, 1510, 1480, 1445, 1370 cm−1; 1Hand 13C NMR, see l" Table 2; HR‑ESI‑MS m/z 447.1775 [calcd. forC25H28O6Na (M – H2O + Na)+, 447.1784].Sakenin F (6): Yellow amorphous solid; [α]D25 − 100 (c 1.0, CHCl3);UV (EtOH) λmax nm (log ε) 340 (3.40), 316 (3.74), 280 (3.97); IRνmax (CHCl3) 3420, 2970, 1645, 1515, 1495, 1455, 1375 cm−1; 1Hand 13C NMR, see l" Table 2; HR‑ESI‑MS m/z 449.1963 [calcd. forC25H30O6Na (M + Na)+, 449.1940].Sakenin G (7): Yellow amorphous solid; UV (EtOH) λmax nm (logε) 370 (3.15); IR νmax (CHCl3) 3400, 3050, 1655, 1510, 1460, 1420,1365 cm−1; 1H and 13C NMR, see l" Table 2; HR‑ESI‑MS m/z321.0703 [calcd. for C17H14O5Na (M + Na)+, 321.0739].Sakenin H (8): Yellow amorphous solid; [α]D25 − 116 (c 1.0, CHCl3);UV (EtOH) λmax nm (log ε) 295 (2.98); IR νmax (CHCl3) 3400, 3055,1725, 1505, 1460, 1420, 1360 cm−1; 1H and 13C NMR, see l" Table2; HR‑ESI‑MS m/z 353.0983 [calcd. for C18H18O6Na (M + Na)+,353.1001].

Biological materialDulbeccoʼs phosphate-buffered saline (D‑PBS) was purchasedfrom Nissui Pharmaceutical. Dulbeccoʼs modified Eagleʼs me-dium (DMEM) was purchased fromWako Pure Chemical. Sodiumbicarbonate, potassium chloride, magnesium sulfate, sodium di-hydrogen phosphate, potassium dihydrogen phosphate, sodiumchloride, and phenol red were purchased fromWako 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 wasfrom Dojindo. Cell culture flasks and 96-well plates were ob-tained from Falcon Becton Dickinson Labware (BD Biosciences).Paclitaxel (purity 97%) was purchased from Sigma-Aldrich. (−)Arctigenin was isolated from the seed of Arctium lappa in crystal-line form [5], and its purity was determined to be > 96% fromNMR and HPLC. Nutrient-deprived medium (NDM) was preparedaccording to a previously described protocol [5].

Cancer cell line and cell cultureThe PANC-1 (RBRC-RCB2095) human pancreatic cancer cell linewas purchased from Riken BRC cell bank and maintained in stan-dard DMEM with 10% FBS supplement, 100 U/mL penicillin G,0.1mg/mL streptomycin sulfate, and 0.25 µg/mL amphotericin B.

Preferential cytotoxic activity against PANC-1 cells innutrient-deprived medium (NDM)The in vitro preferential cytotoxicity of the crude extract and theisolated compounds was determined by a previously describedprocedure [6]. Briefly, human pancreatic cancer cells were seededin 96-well plates (1.5 × 104/well) and incubated in fresh DMEM at37°C under 5% CO2 and 95% air for 24 h. After the cells werewashed with D‑PBS, the medium was changed to serially dilutedtest samples in either DMEM or NDM with the control and blankin each well. After 24 h incubation, cells were washed twice withD‑PBS, and 100 µL of DMEM containing 10% WST-8 cell countingkit solution was added to each well. After 3 h incubation, the ab-sorbance at 450 nmwasmeasured (PerkinElmer EnSpire Multila-bel Reader). Cell viability was calculated from the mean values ofdata from three wells by using the following equation:

Cell viability (%) = [Abs(test sample) −Abs(blank)/Abs(Control) −Abs(blank)] × 100

Nguy

Morphological assessment of cancer cellsCells (1.2 × 105/chamber) for the morphological change studywere seeded in a chamber slide (Lab-Teke®) and incubated in ahumidified CO2 incubator for 24 h for the cell attachment. Thecells were then washed twice with D‑PSB and treated with 1 µMand 10 µM of sakenin F (6) in NDM. After 24 h incubation, cellswere treated with EB/AO, and morphology was observed usingan inverted Nikon Eclipse TS 100 microscope (40 × objective)with phase-contrast and fluorescent mode. Microscopic imageswere taken with a Nikon DS‑L‑2 camera directly attached to themicroscope.

Supporting informationBiological experimental details as well as 1H, 13C, DEPT, COSY,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 toMTTN, a Grant from ToyamaSupport Center for Young Principal Investigators in Advanced LifeSciences, 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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