DSpace at VNU: Geranyl Dihydrochalcones from Artocarpus altilis and Their Antiausteric Activity

Original Papers 193 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 Affiliations Key words " antiausterity agent l " Artocarpus altilis l " Moraceae l " pancreatic cancer l " PANC‑1 l Faculty of Chemistry, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam Vietnam Military Medical University, Ha Noi, Vietnam Frontier Research Core for Life Sciences, University of Toyama, Toyama, Japan Abstract ! Human pancreatic cancer cell lines have 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 is a novel antiausterity strategy in anticancer drug discovery In this study, the methanolic extract of the leaves of Artocarpus altilis showed 100 % preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions at a concen- Introduction ! received revised accepted July 23, 2013 Nov 13, 2013 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 Pancreatic cancer is one of the most deadly forms of cancer associated with the lowest 5-year survival rates known for cancers [1] It shows resistance to conventional chemotherapeutic agents such as 5-fluorouracil, paclitaxel, doxorubicin, and cisplatin [2] Pancreatic cancers are hypovascular [3] in nature resulting in an inadequate supply of nutrition and oxygen to aggressively proliferating cells However, pancreatic cancer cells show an extraordinary tolerance [4] to starvation enabling them to survive in hypovascular (austerity) 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 antiausterity agents, using the PANC-1 human pancreatic cancer cell line [5–15] Artocarpus altilis (Parkinson ex F A Zorn) Forsberg (Moraceae) is a medicinal plant locally known as “Sa ke” in Vietnam, and a decoction of its leaves has been used traditionally for the treatment of gout, hepatitis, hypertension, and diabetes [16] Previous work on this plant species re- tration of 50 µg/mL Further investigation of this extract led to the isolation of eight new geranylated dihydrochalcones named sakenins A–H (1– 8) together with four known compounds (9–12) Among them, sakenins F (6) and H (8) were identified as potent preferentially cytotoxic candidates with PC50 values of 8.0 µM and 11.1 µM, respectively Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica ported a number of geranyl dihydrochalcones [17] having cytotoxic activities and aurones having tyrosinase and α-glucosidase inhibitory activity [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 dihy" Fig 1) and four known comdrochalcones (1–8, l pounds (9–12) We herein report the structures of 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 HR‑ESI‑MS to be C25H32O6 [m/z 451.2091 (M + " Table 1) exNa)+] The 1H NMR spectrum of (l hibited signals due to a pair of ortho-coupled aromatic protons at δH 6.42 (1H, d, J = 8.1 Hz) and δH 6.48 (1H, d, J = 8.1 Hz), an aromatic ABX spin system (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 olefinic proton (δH 5.04, td, J = 6.5, 1.2 Hz), six methylenes (δH 2.99, t, J = 7.4 Hz; δH 2.78, t, J = 7.4 Hz; Nguyen MTT et al Geranyl Dihydrochalcones from … Planta Med 2014; 80: 193–200 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited Geranyl Dihydrochalcones from Artocarpus altilis and Their Antiausteric Activity Original Papers Fig Chemical structure of new compounds (1–8) isolated from Vietnamese Artocarpus altilis Table 1 H and 13C NMR data (500 and 125 MHz) for compounds 1–4 Position (in CD3OD) δH 1′ 2′ 3′ 4′ 5′ 6′ β α C=O 1′′ 6.15, d (2.4) 6.22, dd (8.9, 2.4) 7.53, d (8.9) 2.99, t (7.4) 2.78, t (7.4) 6.48, d (8.1) 6.42,d (8.1) 3.30, d (6.5) (in CD3OD) δC 112.6 164.8 102.3 164.8 107.7 132.3 39.3 27.6 204.5 130.9 126.6 142.9 143.1 112.3 121.0 24.6 2′′ 3′′ 4′′ 5.04, td (6.5, 1.2) 5′′ 1.33, m 22.2 6′′ 7′′ 8′′ 9′′ 10′′ 1.27, m 42.9 70.0 27.7 27.7 14.9 1.84, t (7.4) 1.01, s 1.01, s 1.62, d (1.2) 123.5 134.2 39.7 δH 6.26, d (2.4) 6.34, dd (8.8, 2.4) 7.64, d (8.8) 3.10, m 2.88, m 6.59, d (8.1) 6.53, d (8.1) 3.40, d (6.6) 5.18, td (6.6, 1.1) 1.99, m 2.21, m 1.61, m 1.32, m 3.35, m 1.08, s 1.03, s 1.74, s (in DMSO-d6) δC 114.0 166.4 103.8 166.4 109.3 133.7 40.7 29.0 205.9 132.2 128.1 144.5 144.3 113.8 121.1 26.1 125.2 135.6 37.7 6.25, d (2.3) 6.35, dd (8.8, 2.3) 7.78, d (8.8) 3.18, m 2.75, m 6.54, d (8.1) 6.52, d (8.1) 2.95, m 3.35, m 5.19, t (9.0) (in CD3OD) δC 112.4 165.0 102.4 164.2 108.2 132.9 38.0 26.9 203.7 127.6 126.4 146.2 139.2 115.5 120.3 33.8 2.06, m 84.5 148.1 30.6 30.5 2.16, m 25.8 76.6 78.7 21.2 20.7 16.5 5.12, td (7.0, 1.4) 2′-OH Nguyen MTT et al Geranyl Dihydrochalcones from … δH Planta Med 2014; 80: 193–200 1.65, s 1.57, s 4.90, s 5.14, s 12.62, s 124.7 131.1 25.4 17.5 109.9 δH 6.24, d (2.4) 6.30, dd (8.9, 2.4) 7.63, d (8.9) 3.12, t (7.4) 2.90, m 6.59, d (8.1) 6.57, d (8.1) 2.69, dd (16.5, 5.0) 2.48, dd (16.5, 13.0) 1.60, dd (13.0, 5.0) 2.00, m 1.74, m 1.77, m 1.64, m 3.32, dd (11.2, 4.0) 0.85, s 1.12, s 1.07, s δC 114.1 166.5 103.8 166.5 109.2 133.9 39.9 29.0 206.2 131.6 122.3 142.2 145.2 113.7 121.0 22.0 48.5 77.1 38.8 28.5 78.8 39.5 14.7 20.0 27.8 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited 194 Fig Connectivities (bold lines) deduced by the COSY and HSQC spectra and significant HMBC correlations (solid arrows) observed for new compounds 1–8 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, on the other hand, displayed signals of 25 carbons, which could be classified as a carbonyl carbon, fourteen sp2 carbons, a quaternary oxygenated carbon, six methylene carbons, and three methyl car" Table 1) Part of these data closely resembled those for bons (l compound AC-5-1 (2-geranyl-2′,4′,3,4-tetrahydroxydehydrochalcone) previously isolated from A altilis from Micronesia [19] and indicated the presence of a 2′,4′,3,4-tetrahydroxydihydrochalcone unit However, showed signals due to a quaternary oxygenated 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 geranyl unit in compound AC-5-1 Therefore, the presence of a hydroxygeranyl unit instead of a geranyl unit was assumed In the HMBC " Fig 2), two tertiary methyl groups, H -8′′ and spectrum of (l H3-9′′, at δH 1.01 showed correlations with the quaternary oxygenated 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-dimethyl2-octenyl group Furthermore, the HMBC correlations from methylene proton H2-1′′ (δH 3.30) in the geranyl unit to two aromatic carbons, C-2 (δC 126.6) and C-3 (δC 142.9), indicated the lo" Fig 2) Finally, the double bond cation of the side chain at C-2 (l geometry at C-2′′ was determined to be E based on the high-field shifted 13C chemical shift at the vinylmethyl carbon C-10′′ (δC 14.9) along with NOESY correlations between H-2′′/H2-4′′ and " Fig 3) Therefore, the structure of was conH3-10′′/H2-1′′ (l cluded as 2-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]-2′,4′,3,4-tetrahydroxydihydrochalcone Sakenin B (2) was isolated as a yellow amorphous solid The HR‑ESI‑MS of indicated its molecular formula to be C25H32O7 " Table 1), Its 1H and 13C NMR data closely resembled those of (l except for the appearance of a signal due to an oxymethine group (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 Hence, the presence of a hydroxyl group at C-6′′ in was evident, which was con" Fig 2) firmed by COSY, HSQC, and HMBC spectral analysis (l Therefore, the structure of was concluded as 2-[6,7-dihydroxy3,7-dimethyl-2(E)-octenyl]-2′,4′,3,4-tetrahydroxydihydrochalcone Sakenin C (3) was isolated as a yellow amorphous solid having the molecular formula C25H30O6 as determined by HR‑ESI‑MS The 1H NMR and 13C NMR data of resembled quite closely those " Table 1) However, they differ in the signals due to the gerof (l anyl side chain The DEPT and HSQC spectra of showed the signals 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, C2′′), an exomethylene group (δH 4.90, 5.14; δC 109.9; C-10′′), two quaternary 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 three methylene 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 by " Fig 2), and from them, the partial COSY and HSQC spectra (l structures C(1′′)H2–C(2′′)H–O and C(4′′)H2–C(5′′)H2–C(6′′)H were deduced In the HMBC spectrum, the two tertiary methyl groups H3-8′′ and H3-9′′ gave significant correlations to the quaternary olefinic carbon C-7′′ and the olefinic methine carbon C-6′′ suggesting the linkage of C-8′′ and C-9′′ with C-6′′ via the quaternary olefinic carbon C-7′′ Similarly, significant long-range correlations were observed from exomethylene protons (H2-10′′) to C-3′′, C2′′, and C-4′′, suggesting the connectivity between C-2′′–C3′′– " Fig 2) These data suggested that contains a geranyl side C4′′ (l chain in the form of 7-methyl-3-methyleneoct-6-enyl-2-ol Finally, the location of this side chain was determined to be at C-2 based on the HMBC correlations from H2-1′′ to C-2 and C-3 Nguyen MTT et al Geranyl Dihydrochalcones from … Planta Med 2014; 80: 193–200 195 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited Original Papers Original Papers Fig Key NOESY correlations (dashed arrow) observed for new compounds 1, 4, and Therefore, the structure of sakenin C was concluded as 2-[2-hydroxy-7-methyl-3-methyleneoct-6-enyl]-2′,4′,3,4-tetrahydroxydihydrochalcone Sakenin D (4) was isolated as a yellow amorphous solid Its molecular formula was determined as C25H30O6 based on HRESI‑MS The 1H and 13C NMR data of showed similar resonances indicative of the presence of a 2′,4′,3,4-tetrahydroxydihydrochal" Table 1) Excluding this, the HSQC revealed cone unit as in 1–3 (l the remaining signals in as three tertiary methyl groups, an oxymethine, a quaternary oxygenated carbon, an aliphatic methine, an aliphatic quaternary carbon, and three aliphatic methylene groups This data together with COSY analysis showed two partial connectivities between C(1′′)H2–C(2′′)H and C(4′′)H2– " Fig 2), the two terC(5′′)H2–C(6′′)H–O In the HMBC spectrum (l tiary methyl protons at δH 0.85 (C-8′′) and δH 1.12 (C-9′′) showed correlations with a methine carbon C-2′′, a quaternary carbon at δC 39.5 (C-7′′), and an oxymethine carbon at δC 78.8 (C-6′′), suggesting the linkage of C-2′′, C-6′′, C-8′′, and C-9′′ with C-7′′ Similarly, a linkage of C-10′′, C-2′′, and C-4′′ with C-3′′ was established from the HMBC correlations of the tertiary methyl proton at δH 1.07 (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 cyclized geranyl group in The relative stereochemistry of was established by coupling constant data and the NOESY spectral analysis The large coupling constant of H-6′′ (J5,6ax = 11.2) indicated that it should be axially oriented Furthermore, NOESY correlations between H-6′′/H-2′′, H-2′′/Hβ-1′′, and Hβ-1′′/H‑10′′ indicated that these groups are oriented in the same direction " Fig 3) and the ring bearing hydroxyl group adopts a chair con(l formation Therefore, the structure of sakenin E was concluded as shown The 1H and 13C NMR data of sakenin E (5) showed the signals of a dihydrochalcone unit as in 1–4 together with the signals ascribable for a cyclized geranyl group that could be classified into three tertiary methyl groups, two methylene groups, three meth" Table 2) ine groups, and two quaternary oxygenated carbons (l The COSY and HSQC analysis showed two partial connectivities from a methylene C-4′′ (δC 32.8, δH 1.95, 1.64) via a methylene C-5′′, (δC 20.9, δH 1.57, 1.25), and two methine groups C-6′′ (δC 38.7, 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-range 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 " Fig Its relative stereochemistry was determined by NOESY l spectral analysis NOESY correlations were observed between H4β“/H-10′′ and H-10′′/H2′′, and between H4α”/H-6′′ and H" Fig 6′′/H‑1′′ suggesting their spatial orientation as shown in l Therefore, the structure of was concluded as shown Nguyen MTT et al Geranyl Dihydrochalcones from … Sakenin F (6) was isolated as a yellow amorphous solid Its molecular 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" Table 2) hydroxydihydrochalcone unit similar to that of (l 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 The partial connectivity 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 confirmed " Fig 2) In the HMBC spectrum, correlations based on HMBC (l from methylene protons H2-1′′ (δH 3.28, 3.19) to an aromatic carbon 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 oxymethine proton H-2′′ to C-3, C-1′′, C-3′′, and a tertiary methyl carbon C-10′′ (δC 17.9, δH 1.61), and from the tertiary methyl proton H3-10′′ to C-2′′, C-3′′, and a methylene carbon C-4′′ (δC 39.2, δH 1.55) indicated the presence of a hydroxyl group at C-3′′ and the 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 molecular formula of C17H14O5 as determined from HR‑ESI‑MS The 1H and 13C NMR data showed signals of a dihydrochalcone " Table 2) The major difference obunit as in other compounds (l served was the occurrence of signals due to the presence of a benzofuran unit instead of a geranyl unit, which was confirmed " Fig 2) Therefore, the structure of by the HMBC spectrum (l was concluded as 1-(2,4-dihydroxyphenyl)-3-(7-hydroxybenzofuran-4-yl)propan-1-one The 1H and 13C NMR data of sakenin H (8) were similar to except for the presence of a methoxyl group with the disappearance of " Table 2) This was also signals due to protons in a furan unit (l supported by its molecular formula C18H18O6 as determined by HR‑ESI‑MS Therefore, the presence of a methoxydihydrofuran " Fig 2) group was assumed, which was confirmed by HMBC (l As a result, the structure of sakenin H (8) was concluded as 1(2,4-dihydroxyphenyl)-3-(7-hydroxy-2-methoxy-2,3-dihydrobenzofuran-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-methyl2-(4-methylpent-3-enyl)-2H-1-benzopyran-5-yl]propan-1-one (9) [17], 1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4methylpent-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 activity against a PANC-1 human pancreatic cancer cell line in normal Planta Med 2014; 80: 193–200 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited 196 Original Papers H and 13C NMR data (500 and 125 MHz) for compounds 5–8 Position (in CDCl3) δH 1′ 2′ 3′ 4′ 5′ 6′ β α C=O 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 7′′ 8′′ 9′′ 10′′ 2′-OH 4-OH 2′′-OMe 3′′-OH 6.38, d (2.5) 6.36, dd (8.3, 2.5) 7.64, d (8.3) 3.17, m 2.95, m 2.20, m 3.93, d (6.0) 1.95, m 1.64, m 1.57, m 1.25, m 3.83, m 1.39, s 1.42, s 1.39, s 12.65, s (in CD3 COCD3) δC 113.7 165.4 103.6 163.0 108.3 132.5 39.3 25.5 203.6 46.3 δH 6.32, d (2.4) 6.41, dd (8.8, 2.4) 114.1 166.6 103.7 165.9 109.0 (in CD3 COCD3) δH δC 6.25, d (2.4) 112.7 164.8 108.3 164.0 102.3 6.35, dd (8.8, 2.4) 134.0 39.2 28.3 205.2 30.1 7.77, d (8.8) 3.25, m 3.05, m 7.00, d (2.1) 132.8 38.6 26.7 203.5 105.3 7.90, d (2.1) 146.1 1.55, m 90.0 73.6 39.2 20.9 2.17, m 22.8 38.7 82.3 23.8 30.3 26.0 5.13, tt (7.2, 1.4) 76.5 76.5 32.8 7.82, d (8.8) 3.23, m 2.87, m (in DMSO-d6) δC 3.28, m 3.19, m 4.67, dd (9.5, 8.5) 1.65, s 1.23, s 1.61, s 12.78, s 7.63, s δH δC 6.32, d (2.4) 6.43, dd (8.8, 2.4) 7.81, d (8.8) 3.21, m 2.86, m 3.38, dd (16.5, 6.5) 3.00, dd (16.5, 2.0) 5.67, dd (6.5, 2.0) 113.9 167.0 103.5 166.0 108.7 133.6 39.0 27.9 205.1 36.6 108.3 126.1 131.0 26.0 22.4 17.9 12.61, s 12.77, s 3.45, s 55.6 3.50, s DMEM and nutrient-deprived medium (NDM), utilizing an antiausterity strategy All the tested geranyl dihydrochalcones showed preferential cytotoxicity in a nutrient-deprived condition without apparent toxicity in a nutrient-rich condition (Supporting Information, 2S) Their PC50 values, which means the 50 % preferential cell death in nutrition-deprived medium (NDM) without cytotoxicity in normal nutrient-rich medium (DMEM), " Table Arctigenin, an antiausterity strategyare depicted in l based anticancer agent which was used as a positive control in this study, showed a PC50 value of 0.83 µM Paclitaxel, a wellknown anticancer agent, was virtually inactive Among them, and displayed the most potent preferential cytotoxic activity " Table 3) with PC50 values of 8.0 µM and 11.1 µM, respectively (l Sakenin F (6) was further evaluated for its effect on the cell morphology 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 fluores" Fig a, the cells in control were alive and cence As shown in l stained with AO giving a green fluorescence However, when treated with 6, the morphology of PANC-1 cells was distinctly al" Fig b–d) with an increasing population of dead cells tered (l (red) The phase contrast image of PANC-1 cells treated with showed rounding of the cell membrane, rupture, and disintegra" Fig d) These results tion of cellular contents to the medium (l 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 polarimeter UV spectra were obtained on a Shimadzu UV-160A spectrophotometer IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3 solutions NMR spectra were taken on a Bruker Avance III 500 spectrometer (Bruker Biospin) with tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in δ values HR‑ESI‑MS was performed on a Micro O-QIITOF mass spectrometer (Bruker Daltonics) Analytical and preparative TLC were carried out on precoated Merck silica gel 60F254 or RP-18F254 plate (0.25 or 0.5 mm thickness) 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 (AN2964) has been deposited at the Department of Analytical Chemistry 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, h × 3) to yield a MeOH extract (350 g) The MeOH extract was suspended in H2O (1.5 L) and partitioned successively with 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 Nguyen MTT et al Geranyl Dihydrochalcones from … Planta Med 2014; 80: 193–200 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited Table 197 198 Original Papers a Compounds PC50, µMa Compounds 41.2 ± 1.3 19.9 ± 2.0 –d 58.7 ± 4.3 –d 8.0 ± 0.9 –d 10 11 12 Arctigeninb Paclitaxelc PC50, µM Table Preferential cytotoxicity of compounds isolated from Artocarpus altilis against the PANC-1 human pancreatic cancer cell line in nutrient-deprived medium (NDM) 11.1 ± 2.4 18.8 ± 1.6 75.2 ± 0.8 –d –d 0.8 ± 0.4 > 100 Concentration at which 50% cells were killed preferentially in NDM b Positive control; c negative control; d not tested due to meager Fig Morphology of PANC-1 cells in NDM: a control, b treated with µM of 6, c treated with 10 µM of 6, d treated with 10 µM of 6, phase contrast (Live cells were stained with AO giving green fluorescence; dead cells were stained with EB giving red fluorescence) (Color figure available online only.) The CHCl3 fraction (90 g) was subjected to silica gel column (12 × 80 cm) chromatography eluted with EtOAc–petroleum ether (0 : 100, : 95, 10 : 90, 20 : 80, 40 : 60, 60 : 40, 80 : 20, and 100 : 0; each L) to give fractions: fr (5.7 g), fr (18.5 g), fr (11.2 g), fr (15.7 g), fr (12.5 g), and fr (25.2 g) Fraction was rechromatographed 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 rechromatographed on silica gel column (2.5 × 60 cm) with 20% EtOAc–petroleum ether (1.5 L), followed by preparative TLC with EtOAc– hexane (25 %), to give (16.7 mg) and (1.3 mg) Subfraction 2– was rechromatographed on silica gel column (3.5 × 60 cm) with 25 % EtOAc–petroleum ether (2.5 L), followed by preparative TLC with MeOH–CHCl3 (2 : 98), to give (5.3 mg) and (14.2 mg) Subfraction 2–3 was separated by preparative TLC with % MeOH–CHCl3 to give 10 (14.3 mg) and 11 (1.6 mg) Fraction was further separated by silica gel column chromatography (5 × 60 cm) with % 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) Subfraction 5–2 was rechromatographed on silica gel column (2.5 × 60 cm) with % MeOH–CHCl3 (1.5 L), followed by preparative TLC with % MeOH–CHCl3, to give (9.3 mg), (8.9 mg), and (1.7 mg) Subfraction 5–3 was rechromatographed on silica gel column (3.5 × 60 cm) with % MeOH–CHCl3 (2.5 L), followed by preparative TLC with 5% MeOH–CHCl3, to give (12.4 mg) and Nguyen MTT et al Geranyl Dihydrochalcones from … (1.3 mg) Fraction was further separated by silica gel column chromatography (8 × 60 cm) with 10% MeOH–CHCl3 (5.5 L), followed by preparative TLC with 15% MeOH–CHCl3, to give (4.3 mg) and 12 (1.3 mg) Sakenin A (1): Yellow amorphous solid; UV (EtOH) λmax nm (log ε) 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, see " Table 1; HR‑ESI‑MS m/z 451.2091 [calcd for C l 25H32O6Na (M + Na)+, 451.2097] Sakenin B (2): Yellow amorphous solid; [α]25 D − 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, " Table 1; HR‑ESI‑MS m/z 1440 cm−1; 1H and 13C NMR, see l 449.1903 [calcd for C25H30O6Na (M – H2O + Na)+, 449.1940] Sakenin C (3): Yellow amorphous solid; [α]25 D − 8.1 (c 1.0, CHCl3); UV (EtOH) λmax nm (log ε) 342 (3.27), 311 (3.43), 280 (3.69); IR νmax (CHCl3) 3400, 2940, 1670, 1605, 1450, 1375 cm−1; 1H and 13 " Table 1; HR‑ESI‑MS m/z 431.1839 [calcd for C NMR, see l C25H28O5Na (M – H2O + Na)+, 431.1834] Sakenin D (4): Yellow amorphous solid; [α]25 D − 114 (c 1.0, CHCl3); UV (EtOH) λmax nm (log ε) 340 (3.61), 318 (3.85), 277 (4.17); IR νmax (CHCl3) 3420, 2975, 1610, 1505, 1485, 1450, 1370 cm−1; 1H " Table 1; HR‑ESI‑MS m/z 449.1933 [calcd for and 13C NMR, see l C25H30O6Na (M + Na)+, 449.1940] Planta Med 2014; 80: 193–200 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited isolated amount PC50 values represent the mean ± SD of three replicate experiments Original Papers Morphological assessment of cancer cells Cells (1.2 × 105/chamber) for the morphological change study were seeded in a chamber slide (Lab-Teke®) and incubated in a humidified CO2 incubator for 24 h for the cell attachment The cells were then washed twice with D‑PSB and treated with 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 Biological 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 ! Biological material Dulbeccoʼs phosphate-buffered saline (D‑PBS) was purchased from Nissui Pharmaceutical Dulbeccoʼs modified Eagleʼs medium (DMEM) was purchased from Wako Pure Chemical Sodium bicarbonate, potassium chloride, magnesium sulfate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and phenol red were purchased from Wako Pure Chemical HEPES was purchased from Dojindo Fetal bovine serum (FBS) was from Nichirei Biosciences Antibiotic antimycotic solution was from Sigma-Aldrich, Inc WST-8 cell counting kit was from Dojindo Cell culture flasks and 96-well plates were obtained 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 crystalline 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 standard 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 in 96-well plates (1.5 × 104/well) and incubated in fresh DMEM at 37 °C under % CO2 and 95 % air for 24 h After the cells were washed with D‑PBS, the medium was changed to serially diluted test samples in either DMEM or NDM with the control and blank in each well After 24 h incubation, cells were washed twice with D‑PBS, and 100 µL of DMEM containing 10% WST-8 cell counting kit solution was added to each well After h 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 (%) = This work was supported by a grant from the University of Science, 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 References Asuthkar S, Rao JS, Gondi CS Drugs in preclinical and early-stage clinical development for pancreatic cancer Expert Opin Investig Drugs 2012; 21: 143–152 Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL, Gallick GE, Logsdon CD, McConkey DJ, Choi W Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer Cancer Res 2009; 69: 5820–5828 Sakamoto H, Kitano M, Suetomi Y, Maekawa K, Takeyama Y, Kudo M Utility of contrast-enhanced endoscopic ultrasonography for diagnosis of small pancreatic carcinomas Ultrasound Med Biol 2008; 34: 525– 532 Izuishi K, Kato K, Ogura T, Kinoshita T, Esumi H Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy Cancer Res 2000; 60: 6201–6207 Awale S, Lu J, Kalauni SK, Kurashima Y, Tezuka Y, Kadota S, Esumi H Identification of arctigenin as an antitumor agent having the ability to eliminate the tolerance of cancer cells to nutrient starvation Cancer Res 2006; 66: 1751–1757 Awale S, Nakashima EMN, Kalauni SK, Tezuka Y, Kurashima Y, Lu J, Esumi H, Kadota S Angelmarin, a novel anti-cancer agent able to eliminate the tolerance of cancer cells to nutrient starvation Bioorg Med Chem Lett 2006; 16: 581–583 Awale S, Li F, Onozuka H, Esumi H, Tezuka Y, Kadota S Constituents of Brazilian red propolis and their preferential cytotoxic activity against human pancreatic PANC‑1 cancer cell line in nutrient-deprived condition Bioorg Med Chem 2008; 16: 181–189 Awale S, Linn TZ, Li F, Tezuka Y, Myint A, Tomida A, Yamori T, Esumi H, Kadota S Identification of chrysoplenetin from Vitex negundo as a potential cytotoxic agent against PANC′ and a panel of 39 human cancer cell lines (JFCR 39) Phytother Res 2011; 25: 1770–1775 Awale S, Ueda J, Athikomkulchai S, Abdelhamed S, Yokoyama S, Saiki I, Miyatake R Antiausterity agents from Uvaria dac and their preferential cytotoxic activity against human pancreatic cancer cell lines in a nutrient-deprived condition J Nat Prod 2012; 75: 1177–1183 [Abs(test sample) − Abs(blank)/Abs(Control) − Abs(blank)] × 100 Nguyen MTT et al Geranyl Dihydrochalcones from … Planta Med 2014; 80: 193–200 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited Sakenin E (5): Yellow amorphous solid; [α]25 D − 11.3 (c 1.0, CHCl3); UV (EtOH) λmax nm (log ε) 341 (3.55), 321 (3.78), 279 (4.23); IR νmax (CHCl3) 3410, 2970, 1615, 1510, 1480, 1445, 1370 cm−1; 1H " Table 2; HR‑ESI‑MS m/z 447.1775 [calcd for and 13C NMR, see l C25H28O6Na (M – H2O + Na)+, 447.1784] Sakenin F (6): Yellow amorphous solid; [α]25 D − 100 (c 1.0, CHCl3); UV (EtOH) λmax nm (log ε) 340 (3.40), 316 (3.74), 280 (3.97); IR νmax (CHCl3) 3420, 2970, 1645, 1515, 1495, 1455, 1375 cm−1; 1H " Table 2; HR‑ESI‑MS m/z 449.1963 [calcd for and 13C NMR, see l C25H30O6Na (M + Na)+, 449.1940] Sakenin G (7): Yellow amorphous solid; UV (EtOH) λmax nm (log ε) 370 (3.15); IR νmax (CHCl3) 3400, 3050, 1655, 1510, 1460, 1420, " Table 2; HR‑ESI‑MS m/z 1365 cm−1; 1H and 13C NMR, see l 321.0703 [calcd for C17H14O5Na (M + Na)+, 321.0739] Sakenin H (8): Yellow amorphous solid; [α]25 D − 116 (c 1.0, CHCl3); UV (EtOH) λmax nm (log ε) 295 (2.98); IR νmax (CHCl3) 3400, 3055, " Table 1725, 1505, 1460, 1420, 1360 cm−1; 1H and 13C NMR, see l 2; HR‑ESI‑MS m/z 353.0983 [calcd for C18H18O6Na (M + Na)+, 353.1001] 199 Original Papers 10 Awale S, Ueda J, Athikomkulchai S, Dibwe DF, Abdelhamed S, Yokoyama S, Saiki I, Miyatake R Uvaridacols E–H, highly oxygenated antiausterity agents from Uvaria dac J Nat Prod 2012; 75: 1999–2002 11 Li F, Awale S, Zhang H, Tezuka Y, Esumi H, Kadota S Chemical constituents of propolis from Myanmar and their preferential cytotoxicity against a human pancreatic cancer cell line J Nat Prod 2009; 72: 1283–1287 12 Win NN, Awale S, Esumi H, Tezuka Y, Kadota S Bioactive secondary metabolites from Boesenbergia pandurata of Myanmar and their preferential cytotoxicity against human pancreatic cancer PANC‑1 cell line in nutrient-deprived medium J Nat Prod 2007; 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37: 2161–2165 20 Lotulung PDN, Fajriah S, Hanafi M, Filaila E Identification of cytotoxic compound from Artocarpus communis leaves against P‑388 cells Pak J Biol Sci 2008; 11: 2517–2520 21 Shimizu K, Kondo R, Sakai K, Buabarn S, Dilokkunanant UJ 5-Reductase inhibitory component from leaves of Artocarpus altilis J Wood Sci 2000; 46: 385–389 22 Patil AD, Freyer AJ, Killmer L, Offen P, Taylor PB, Votta BJ, Johnson RK A new dimeric dihydrochalcone and a new prenylated flavone from the bud covers of Artocarpus altilis: potent inhibitors of cathepsin K J Nat Prod 2002; 65: 624–627 Planta Med 2014; 80: 193–200 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited 200 ... established from the HMBC correlations of the tertiary methyl proton at δH 1.07 (C-10′′) with the quaternary oxygenated carbon at δC 77.1 (C-3′′), C-2′′, and C-4′′ These data indicated the presence... methyl groups, an oxymethine, a quaternary oxygenated carbon, an aliphatic methine, an aliphatic quaternary carbon, and three aliphatic methylene groups This data together with COSY analysis showed... 7-methyl-3-methyleneoct-6-enyl-2-ol Finally, the location of this side chain was determined to be at C-2 based on the HMBC correlations from H2-1′′ to C-2 and C-3 Nguyen MTT et al Geranyl Dihydrochalcones from … Planta Med 2014;