Biosci Biotechnol Biochem., 77 (12), 2378–2382, 2013 CytotoxicActivityofNewPhenolicCompoundsfromVietnameseCaesalpiniasappan Tran Manh H UNG,1;2; y Nguyen Xuan H AI,1 Nguyen Trung N HAN,1 Ton That Q UANG,1 Tran Le Q UAN,1 To Dao C UONG,2 Nguyen Hai D ANG,3 and Nguyen Tien D AT3 Faculty of Chemistry, University of Science, Vietnam National University, 227 Nguyen Van Cu Street, District 5, HoChiMinh city, Vietnam College of Pharmacy, Catholic University of Daegu, Kyeongbuk 712-702, Korea Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay District, Hanoi, Vietnam Received June 19, 2013; Accepted September 11, 2013; Online Publication, December 7, 2013 [doi:10.1271/bbb.130493] Two newphenolic compounds, caesalpiniaphenols G– H (1 and 2), were isolated fromVietnameseCaesalpiniasappan heartwood The chemical structures were established mainly by extensive spectroscopic studies and chemical evidence Compounds and showed potent inhibitory activity against HL-60 cancer cell lines with respective IC50 values of 16.7 and 22.5 g/mL Treating HL-60 cells with various concentrations of resulted in growth inhibition and the induction of apoptosis Key words: Caesalpinia sappan; Leguminosae; caesalpiniaphenols; HL-60; apoptosis Caesalpiniasappan L (Leguminosae) is distributed in Southeast Asia It has been used as a herbal medicine for treating inflammation and to improve blood circulation,1) as well as for its anti-influenza, anti-allergic and neuroprotective activities.2–4) This plant is called Toˆ Mo:^ c in Vietnam and is scattered in low mountainous areas, being light-demanding and drought-tolerant Toˆ Mo:^ c has been used in traditional Vietnamese medicine for treating menstrual and post-partum haematometra, trauma blood static, dizziness and post-partum blood loss It has also been employed in the therapy of bloody dysentery, enteralgia, intestinal haemorrhage and infectious diarrhea, and for cleansing wounds.5) Many reports have shown that the main components in C sappan are phenolics with four structural sub-types: brazilin, chalcone, protosappanin and homoisoflavonoids.6–11) An ethanolic extract of C sappan has ameliorated hypercholesterolemia in C57BL/6 mice and suppressed the inflammatory response in human umbilical vein endothelial cells (HUVECs) through an anti-oxidizing mechanism.7) The compounds with a sappanchalcone skeleton have shown anti-inflammatory, anti-bacterial and antiinfluenza activities.8–13) Nguyen et al have reported that a methanolic extract and the active compoundsfrom the heartwood of C sappan collected in Vietnam exhibited significant XO inhibitory activity.14,15) However, limited information is available concerning the cytotoxicactivity principles of C sappan origining in Vietnam To y OH HO 8a O 4a 4' OH 2' 1' 6' OH OH O 3a O 11 10 HO 10b 10a 5a 6a OH Fig Chemical Structures of and from C sappan further study the phytochemical and biological activity available from this plant, fractionation of the ethyl acetate-soluble fraction resulted in the isolation of two newphenoliccompounds (1 and 2) This study describes the isolation and structural elucidation of these compounds, as well as an evaluation of their in vitro cytotoxicity and apoptosis induction toward HL-60, human premyelocytic leukemia cancer cell lines Materials and Methods General experimental procedures Optical rotation was measured with a Jasco DIP 370 digital polarimeter UV spectra were obtained in MeOH by using a Thermo 9423AQA2200E UV spectrometer, and IR spectra were obtained with a Bruker Equinox 55 FT-IR spectrometer The nuclear magnetic resonance (NMR) spectra were obtained with a Varian Unity Inova 400 MHz spectrometer Silica gel (Merck, 63– 200 mm particle size) and RP-18 silica gel (Merck, 75 mm particle size) were used for column chromatography TLC was carried out by using Merck silica gel 60 F254 and RP-18 F254 plates HPLC was performed with a Waters 600 Controller system equipped with a UV detector and a YMC Pak ODS-A column (20  250 mm, mm particle size, YMC Co., Japan), and HPLC solvents were obtained from Burdick & Jackson, USA Plant materials The heartwood ofCaesalpiniasappan L was collected in An Giang province of Vietnam in September 2010 Professor Tran Cong Luan at Vietnam National Institute of Medicinal Material botanically authenticated the plant, where voucher specimen number TCL-00120 describing the plant is deposited Extraction and isolation C sappan (1.5 kg) was extracted three times (3 h  L) with refluxing methanol After the solvent had been removed under reduced pressure, the residue was suspended in H2 O and then successively partitioned with n-hexane, EtOAc, and n-BuOH To whom correspondence should be addressed Tel: +84-8-3832-4457; Fax: +84-8-3835-0096; e-mail: tmhung801018@gmail.com Abbreviations: C sappan, Caesalpinia sappan; NMR, nuclear magnetic resonance; HL-60, human promyelocytic leukemia; HeLa, human cervical adenocarcinoma; MCF-7, human breast adenocarcinoma; LLC, Lewis lung carcinoma cells; ATCC, American Type Culture Collection Cytotoxic ActivityofPhenolicCompoundsfrom C sappan The EtOAc-soluble fraction (16 g) was chromatographed in a silica gel column, using a stepwise gradient of CHCl3 :MeOH (80:1 to 0:1), to yield sixteen sub-fractions (E.1–E.16) according to their TLC profiles Fraction E10 (1.12 g) was subjected to reversed-phase (ODS-A) column chromatography, eluting with MeOH/H2 O (from 3:1 to 1:0, L for each step) to afford six sub-fractions (E.10-1–E.10-6) Further purification of E10-5 (127 mg) by a semi-preparative Waters HPLC system [using an isocratic solvent system of 30% MeOH in H2 O ỵ 0:1% triuoroacetic acid at a ow rate of mL/min over 90 min; UV detection at 210 nm; a YMC Pak ODS-A column (20  250 mm, mm particle size] resulted in the isolation of compound (12.7 mg; tR ¼ 26:5 min) Further purification of E10-6 (68.5 mg) by the semi-preparative Waters HPLC system [using an isocratic solvent system of 25% MeOH in H2 O ỵ 0:1% triuoroacetic acid at a ow rate of mL/min over 90 min; UV detection at 210 nm; a YMC Pak ODS-A column (20  250 mm, mm particle size] resulted in the isolation of compound (5.7 mg; tR ¼ 32:5 min) Caesalpiniaphenol G (1): yellowish needles (MeOH); mp 176– 178 C; IR max (KBr) cmÀ1 : 3464 (hydroxy group), 1725 (carbonyl group), 1440 (aromatic absorption); H-NMR (400 MHz, methanol-d4 ) and 13 C-NMR (100 MHz, methanol-d4 ): see Table for the spectroscopic data; HR-EI-MS m=z: 316.0596 [M]ỵ , calcd for C16 H12 O7 , 316.0595 Caesalpiniaphenol H (2): colorless amorphous powder; ½ 25 D À165.2 (c 0.05, MeOH); IR max (KBr) cmÀ1 : 3380 (hydroxy group), 1452 (aromatic absorption); UV (MeOH) max (log ") nm: 225 (4.33), 260 (3.75); H-NMR (400 MHz, methanol-d4 ) and 13 C-NMR (100 MHz, methanol-d4 ): see Table for the spectroscopic data; HREI-MS m=z: 242.0968 [M]ỵ , calcd for C15 H14 O3 , 242.0969 Cell lines and culture HL-60 (human promyelocytic leukemia), HeLa (human cervical adenocarcinoma), MCF-7 (human breast adenocarcinoma) and LLC (Lewis lung carcinoma) cells were obtained from the American Type Culture Collection (ATCC) The cells were maintained in RPMI or IMDM (Gibco BRL, NY, USA) with 10% fetal bovine serum (FBS) supplemented with 2% penicillin and 100 mg/mL of streptomycin at 37 C in a humidified atmosphere containing 5% CO2 Cytotoxicactivity assay The cancer cell lines were maintained in RPMI 1640 or IMDM that included L-glutamine (Gibco) with 10% FBS (Gibco) and 2% penicillin-streptomycin (Gibco) The cells were cultured at 37 C in a 5% CO2 incubator The cytotoxicactivity was measured by using a modified MTT assay.16) Viable cells were seeded in the growth medium into 96-well microtiter plates (1  104 cells/ well) and then incubated at 37 C in a 5% CO2 incubator The test sample was dissolved in DMSO and adjusted to a final sample concentration ranging from to 100 mg/mL by diluting with the growth medium Each sample was prepared in triplicate, the final DMSO concentration being adjusted to 100 >100 2:4 Ỉ 0:2 >100 42:5 Æ 5:1 2:8 Æ 0:3 a The inhibitory effect is presented as giving 50% inhibition (IC50 ) relative to the vehicle control These data represent the average values of three repeated experiments (mean Ỉ SD) b Positive control C (100 MHz) 108.1 129.7 152.9 182.0 78.9 33.6 35.7 126.2 113.9 144.8 146.6 117.1 128.9 44.3 30.3 with the chromen-4-one skeleton with ten quaternary carbons, the chemical shifts of these quaternary carbons indicating that six of them were oxygen-bearing carbons with one carbonyl functionality evident at C 184.4 (C-4) (Table 1) By comparison, it was found that the H- and 13 C-NMR spectroscopic data for were closely related to those of sappanone A12) and 30 -deoxysappanone A,12) except for the additional hydroxyl groups at C-30 , C-50 and C-60 of ring B The complete NMR assignment and connectivity of were further determined by analyzing the COSY, HMQC and HMBC spectroscopic data (Fig 2) Based on the this analysis, the structure of compound was elucidated as (E)-3(2,3,4,5-tetrahydroxybenzylidene)-2,3-dihydro-7-hydroxychromen-4-one, trivially named caesalpiniaphenol G Compound was obtained as a colorless amorphous powder with the molecular formula C15 H14 O3 , as established by a molecular ion peak [M]ỵ at m=z 242.0968 in the HR-EI-MS data IR absorptions at 3380 cmÀ1 and 1452 cmÀ1 respectively implied the presence of hydroxyl and aromatic absorptions In the H-NMR spectrum, two aromatic proton singlet signals at H 6.41 (1H, s, H-7) and 6.64 (1H, s, H-10) were observed The resonances were ascribed at H 4.39 (d, J ¼ 11:2 Hz, H-5ax), 4.24 (dd, J ¼ 3:0, 11.2 Hz, H-5eq), 2.46 (1H, m, H-5a), 2.24 (d, J ¼ 12:8 Hz, H-6ax), 2.09 (dd, J ¼ 3:0, 12.8 Hz, H-6eq), 2.95 (d, J ¼ 17:2 Hz, H-11ax) and 3.27 (dd, J ¼ 5:6, 17.2 Hz, H-11eq), respectively accounting for the presence of one oxygenated methylene, a methine proton, and two methylene groups In the 13 C-NMR (DEPT) spectra (Table 2), 15 signals were recognized, viz  C,  CH,  CH2 , suggesting that the structural skeleton of should be close to that of 10,11- Fig Induction of DNA Fragmentation by in HL-60 Cells in Vitro HL-60 cells were treated with for 24 h (5, 10, 20 and 50 mg/mL) Total genomic DNA was extracted and resolved on 1% agarose gel Apoptotic DNA fragmentation was visualized by ethidium bromide staining M, size marker; , mature cell; ỵ, positive control treated with 2.5 mg/mL of camptothecin dihydroxydracaenone C,18) except that the signals for a cyclohexa-2,5-dienone group at C-4a and 11a of 10,11dihydroxydracaenone were absent in Instead, a set of olefinic proton signals at H 6.84 (d, J ¼ 10:0 Hz, H-1), 6.42 (brd, J ¼ 10:0 Hz, H-2) and 5.54 (brs, H-3) in the H-NMR spectrum and three carbon signals at C 108.1 (C-1), 129.7 (C-2) and 152.9 (C-3) in the 13 C-NMR spectrum, which are characteristic of a cyclopenta-1,3diene, were present (Table 2) The H–1 H COSY spectrum, coupled with the HMQC analysis, allowed the establishment of three H-atom spin systems H-1/ H-2/H-3, corresponding to structural fragments of C-1/ C-2/C-3 (Fig 2) The HMBC correlations of H-1, H-2 and H-3 with C-3a and C-10b indicated the direct linkage of cyclopenta-1,3-diene located at C-3a and C-10b The stereochemistry of was deduced from the negative optical rotation data, ½ 25 D À165.2 (c 0.05, MeOH), compared with that of 10,11-dihydroxydracae18) This evidence none C (½ 25 D À411.3, c 0.10, MeOH) was used to deduce the stereochemistry of as shown in Fig 1, with the trivial name of caesalpiniaphenol H Cytotoxic effects Cancer cells were seeded in 96-well plates, then incubated for h, and were treated with the caesalpiniaphenol G (1) and caesalpiniaphenol H (2) at various concentrations (5, 10, 20, 50 and 100 mg/mL) The inhibitory process was assessed by using an MTT assay.16) As shown in Table 3, both of and showed effective inhibition against HL-60 and HeLa cells with the IC50 values of 16:7 Ỉ 2:2, 22:5 Ỉ 5:1, 28:1 Ỉ 3:6 and 39:2 Ỉ 2:0 mg/mL, respectively Caesalpiniaphenol H (2) also exhibited significant inhibition against LLC cancer cells with the IC50 value as 42:5 Ỉ 5:1 mg/mL Cytotoxic ActivityofPhenolicCompoundsfrom C sappan 2381 Fig Increase of Caspase-3 Activity by in HL-60 Cells in Vitro After 12 h, 24 h and 48 h of incubation with the 1-treated HL-60 cells, the cell lysates were incubated for h at 37 C with the caspase-3 substrate (Ac-DEVD-AFC) The fluorescence intensity of the cell lysates was measured to determine the caspase-3 activity The blank group was used as 0.1% DMSO-treated cells Camptothecin (2.5 mg/mL) was used as a positive control Data are presented as the mean Ỉ SD of the results from three independent experiments However, in the case of MCF-7 cell lines, both of two compounds showed very weak inhibitory activity with the IC50 values over 100 mg/mL (Table 3) Apoptosis effects To evaluate whether the growth inhibition of HL-60 cells by caesalpiniaphenol G (1) was mediated through the apoptotic process, we performed a DNA laddering assay.16) HL-60 cells (5  105 cell/mL) were treated with 5, 10, 20 and 50 mg/mL of for 24 h, and the DNA bands were photographed under ultraviolet illumination The chromosomal DNA of the treated cells showed internucleosomal DNA fragmentation consisting of multiples of approximately 180–200 base pairs in the agarose gel electrophoresis (Fig 3) Caspase-3 is a cytosolic protein that normally exists as a 32 kDa inactive precursor, and is proteolytically cleaved into a heterodimer when the cell undergoes apoptosis The activityof caspase-3 was measured by the proteolytic cleavage of Ac-Asp-Glu-Val-Asp-8-amino-4-trifluoromethylcoumarin in the HL-60 cells for 12, 24, and 48 h (Av-DEVD-AFC)17) after treating with compound (5, 10, 20, 50 and 100 mg/mL) The result was the caspase-3 activity being increased 1–5-fold in a dose- and timedependent manner when compared to the control (Fig 4) proliferator-activated receptor (PPAR ).21) The toxic effects of brazilein have been evaluated in terms of the cell viability, induction of apoptosis, and activityof caspase-3 in BCC cells.22) Brazilin has also shown dosedependent inhibition of the cell proliferation and induction of apoptosis in glioma cells by increasing the ratio of cleaved poly-(ADP)-ribose polymerase and decreasing the expression of caspase-3 and caspase7.21) This in now the first report on the chemical constituents ofVietnamese C sappan and its cytotoxicactivity Treatment with one of the isolated compounds, caesalpiniaphenol G (1), induced apoptosis, thereby inhibiting the growth of HL-60 cancer cells The activation of caspase-3 is required for several typical hallmarks of apoptosis and is indispensable for apoptotic chromatin condensation and DNA fragmentation in the HL-60 cell type examined The apoptotic bodies induced by DNA fragmentation were clearly observed in HL-60 cells treated with caesalpiniaphenol G via electrophoresis Apoptosis is an important way to maintain cellular homeostasis between cell division and cell death The induction of apoptosis in cancer cells is therefore one of the most useful strategies for anticancer drug development According to these results, it is suggested that the active constituents fromVietnamese C sappan may be an important source for developing anti-cancer drugs Discussion Acknowledgments The heartwood of C sappan L is a common remedy in traditional medicine and possesses diverse biological activities Among the compounds isolated from C sappan, isoliquiritigenin 20 -methyl ether has inhibited the growth of oral cancer cells via a pathway involving MAP kinases, NF-B, and Nrf2.19) Sappanchalcone, a flavonoid, has suppressed oral cancer cell growth and induced apoptosis through the activation of p53dependent mitochondrial, p38, ERK, JNK, and NF-B signaling.20) In another report, brazilein, the main phenolic compound from C sappan, has exhibited an antioxidative effect, inhibited the intracellular lipid accumulation during adipocyte differentiation in 3T3L1 cells, and suppressed the induction of peroxisome This study was funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.05-2011.52 References 1) 2) 3) 4) Nagai M, Nagumo S, Eguchi I, Lee SM, and Suzuki T, Yakugaku Zasshi, 104, 935–938 (1984) Liu AL, Shu SH, Qin HL, Lee SM, Wang YT, and Du GH, Planta Med., 75, 337–339 (2009) Yodsaoue O, Cheenpracha S, Karalai C, Ponglimanont C, and Tewtrakul S, Phytother Res., 23, 1028–1031 (2009) Shen J, Zhang H, Lin H, Su H, Xing D, and Du L, Eur J Pharmacol., 558, 88–95 (2007) 2382 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) T M HUNG et al Bich DH, ‘‘Selected Medicinal Plants in Vietnam,’’ Science and Technology Publishing House, Hanoi, 1, pp 151–154 (1999) Shimokawa T, Kinjo J, Yamahara J, Yamasaki M, and Nohara T, Chem Pharm Bull., 33, 3545–3547 (1985) Choi BM, Lee JA, Gao SS, Eun SY, Kim YS, Ryu SY, Choi YH, Park R, Kwon DY, and Kim BR, Biofactors, 30, 149–157 (2007) Namikoshi M, Nakata H, and Saitoh T, Chem Pharm Bull., 35, 3615–3619 (1987) Kim B, Kim SH, Jeong SJ, Sohn EJ, Jung JH, Lee MH, and Kim SH, J Agric Food Chem., 60, 9882–9889 (2012) Lee MJ, Lee HS, Jung HJ, Lee CS, Kim JE, Moon HI, and break>Park WH, Immunopharmacol Immunotoxicol., 32, 671– 679 (2010) Washiyama M, Sasaki Y, Hosokawa T, and Nagumo S, Biol Pharm Bull., 32, 941–944 (2009) Cuong TD, Hung TM, Kim JC, Kim EH, Woo MH, Choi JS, Lee JH, and Min BS, J Nat Prod., 75, 2069–2075 (2012) Min BS, Cuong TD, Hung TM, Min BK, Shin BS, and Woo MH, Bioorg Med Chem Lett., 22, 7436–7439 (2012) Nguyen MT, Awale S, Tezuka Y, Tran QL, Watanabe H, and 15) 16) 17) 18) 19) 20) 21) 22) Kadota S, Biol Pharm Bull., 27, 1414–1421 (2004) Nguyen MT, Awale S, Tezuka Y, Tran QL, and Kadota S, Tetrahedron Lett., 45, 8519–8522 (2004) Lee MK, Hung TM, Cuong TD, Na M, Kim JC, Kim EJ, Park HS, Choi JS, Lee I, Bae K, Hattori M, and Min BS, Phytother Res., 25, 1579–1585 (2011) Zhao P, Iwamoto Y, Kouno I, Egami Y, and Yamamoto H, Phytochemistry, 65, 2455–2461 (2004) Zheng QA, Zhang YJ, and Yang CR, J Asian Nat Prod Res., 8, 571–577 (2006) Lee YM, Jeong GS, Lim HD, An RB, Kim YC, and Kim EC, Toxicol In Vitro, 24, 776–782 (2010) Lee YM, Kim YC, Choi BJ, Lee DW, Yoon JH, and Kim EC, Toxicol In Vitro, 25, 1782–1788 (2011) Yen CT, Nakagawa-Goto K, Hwang TL, Wu PC, Morris-Natschke SL, Lai WC, Bastow KF, Chang FR, Wu YC, and Lee KH, Bioorg Med Chem Lett., 20, 1037–1039 (2010) Liang CH, Chan LP, Chou TH, Chiang FY, Yen CM, Chen PJ, Ding HY, and Lin RJ, Evid Based Complement Alternat Med., 2013, 864–892 (2013) ... mg/mL Cytotoxic Activity of Phenolic Compounds from C sappan 2381 Fig Increase of Caspase-3 Activity by in HL-60 Cells in Vitro After 12 h, 24 h and 48 h of incubation with the 1-treated HL-60... constituents of Vietnamese C sappan and its cytotoxic activity Treatment with one of the isolated compounds, caesalpiniaphenol G (1), induced apoptosis, thereby inhibiting the growth of HL-60 cancer... linkage of cyclopenta-1,3-diene located at C-3a and C-10b The stereochemistry of was deduced from the negative optical rotation data, ½ 25 D À165.2 (c 0.05, MeOH), compared with that of 10,11-dihydroxydracae18)