Fitoterapia133(2019)175-179.Contents lists available at ScienceDirectFitoterapia?ELSEVIERjournalhomepage:www.elsevier.com/locate/fitoteMelosuavine I, an apoptosis-inducing bisindole alkaloid from MelodinussuaveolensupdatesZhong-Ying Fangab,1, Yi-Dan Ren'l, Si-Yu Du', Miao Zhang, Yun-Shan Wang, Lei FangabHua Zhanga,'Schoolof Biological Sciences and Technology,University of Jinan, Jinan 250022, PR ChinaKey laboratory of Natural Pharmaceutical Chemisty, Shandong University of Traditional Chinese Medicine, Jinan 250200, China Intenational Biotechnology R&D Center, Schoolt of Ocean, Shandong University, Weihai 264209, ChinaARTICLEINFOABSTRACTKeywards.A new bisindole alkaloid, melosuavine I (1) possessing an aspidosperma-aspidosperma dimeric skeleton, wasMelodinus suaveolensisolated from the leaves of Melodirus suaveolens.The structure with absolute configuration of1 was elucidated byBisindole alkaloida combination of MS, NMR and computational methods. MTT assays indicated that 1 exhibited significant cy-Anticancertotoxicity on human breast cancer BT549 cells with an ICso value of 0.89 μM. Further study showed that 1Apoptosisinhibited BT549 cell proliferation by inducing apoptosis through activation of caspase 3 and p53, and down-regulation of Bcl-2.1.Introduction2. ExperimentalBisindole alkaloids are one important group of plant-derived alka-2.1. General experimental proceduresloids that have attracted significant interest in anticancer drug dis-covery due to their established therapeutic importance and structuralOptical rotations were acquired on a Rudolph VI digital polarimeterdiversity [1]. Plants of the genus Melodinus (Apocynaceae), which(Rudolph Research Analytical, USA). IR spectra were obtained on amainly occurinthetropical and subtropicalareas of Asia,haveprovenNicolet 5700FT-IR spectrometer.ECD spectra weretaken on ato be a good source of bisindole alkaloids [2-10]. To date, > 40 bi-Chirascan circular dichroism spectrometer (Applied Photophysics Ltd.,sindole alkaloids have been isolated and characterized from the genus,UK). The 1D and 2D NMR spectra were recorded on a Bruker AVANCEsome of which exhibit significant cytotoxic effects against a variety ofDRX600 NMR spectrometer (Bruker BioSpin Corporation, Switzerland)human cancer cell lines [9,11]. M. suaveolens is used for the treatmentwith trimethylsilane (TMS) as internal standard. HRESIMS experimentsof hernia, infantile malnutrition and dyspepsia in Chinese folk medicinewere conducted on an Agilent 6250 Accurate-Mass Q-TOF LC/MS[12]. As part of our ongoing effort to discover anticancer alkaloids from(Agilent Technologies, USA). Semipreparative HPLC separations werethe Melodinus genus [13,14], the leaves of M. suaveolens were phyto-performed on an Agilent 1260 series LC instrument (Agilentchemically investigated, which led to the isolation of melosuavine I (1),Technologies, USA) using an Agilent SB-Cis (9.4 × 250 mm) column.All solvents used for column chromatography were of analytical gradea new bisindole alkaloid possessing an aspidosperma-aspidosperma(Tianjin Fuyu Fine Chemical Co. Ltd., China) and solvents used forskeleton (Fig.1). Herein, we report the isolation and structural eluci-dation of the new alkaloid, as well as its antiproliferative activity byHPLC were of HPLC grade (Oceanpak Alexative Chemical Ltd.,MTT assay. In addition, the mechanism of apoptosis induced by 1 wasSweden). Normal phase column chromatography was performed onsilica gel (200-300 mesh, Qingdao Marine Chemical Inc., China).also investigated.Cancer cell lines (A549, K562, PC3 and BT549) were purchased fromthe Cell Bank of the Chinese Academy of Science (Shanghai, China).? Corresponding authors at: School of Biological Sciences and Technology, University of Jinan, Jinan 250022, PR ChinaE-mail addresses: bio_fangl@ujn.edu.cn (L. Fang), bio_zhangh@ujn.edu.cn (H. Zhang). These authors contributed equally to this work.https://doi.org/10.1016/j.fitote.2018.12.026Received 3 December 2018; Received in revised form 30 December 2018; Accepted 30 December 2018Available online 18 January 20190367-326x/ 2019 Elsevier B.V. All rights reserved
Contents lists available at ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Melosuavine I, an apoptosis-inducing bisindole alkaloid from Melodinus suaveolens Zhong-Ying Fanga,b,1 , Yi-Dan Renc,1 , Si-Yu Dub , Miao Zhanga , Yun-Shan Wangc , Lei Fanga,b,⁎ , Hua Zhanga,⁎ a School of Biological Sciences and Technology, University of Jinan, Jinan 250022, PR China b Key laboratory of Natural Pharmaceutical Chemistry, Shandong University of Traditional Chinese Medicine, Jinan 250200, China c International Biotechnology R&D Center, School of Ocean, Shandong University, Weihai 264209, China ARTICLE INFO Keywords: Melodinus suaveolens Bisindole alkaloid Anticancer Apoptosis ABSTRACT A new bisindole alkaloid, melosuavine I (1) possessing an aspidosperma-aspidosperma dimeric skeleton, was isolated from the leaves of Melodinus suaveolens. The structure with absolute configuration of 1 was elucidated by a combination of MS, NMR and computational methods. MTT assays indicated that 1 exhibited significant cytotoxicity on human breast cancer BT549 cells with an IC50 value of 0.89 μM. Further study showed that 1 inhibited BT549 cell proliferation by inducing apoptosis through activation of caspase 3 and p53, and downregulation of Bcl-2. 1. Introduction Bisindole alkaloids are one important group of plant-derived alkaloids that have attracted significant interest in anticancer drug discovery due to their established therapeutic importance and structural diversity [1]. Plants of the genus Melodinus (Apocynaceae), which mainly occur in the tropical and subtropical areas of Asia, have proven to be a good source of bisindole alkaloids [2–10]. To date, > 40 bisindole alkaloids have been isolated and characterized from the genus, some of which exhibit significant cytotoxic effects against a variety of human cancer cell lines [9,11]. M. suaveolens is used for the treatment of hernia, infantile malnutrition and dyspepsia in Chinese folk medicine [12]. As part of our ongoing effort to discover anticancer alkaloids from the Melodinus genus [13,14], the leaves of M. suaveolens were phytochemically investigated, which led to the isolation of melosuavine I (1), a new bisindole alkaloid possessing an aspidosperma-aspidosperma skeleton (Fig. 1). Herein, we report the isolation and structural elucidation of the new alkaloid, as well as its antiproliferative activity by MTT assay. In addition, the mechanism of apoptosis induced by 1 was also investigated. 2. Experimental 2.1. General experimental procedures Optical rotations were acquired on a Rudolph VI digital polarimeter (Rudolph Research Analytical, USA). IR spectra were obtained on a Nicolet 5700 FT-IR spectrometer. ECD spectra were taken on a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., UK). The 1D and 2D NMR spectra were recorded on a Bruker AVANCE DRX600 NMR spectrometer (Bruker BioSpin Corporation, Switzerland) with trimethylsilane (TMS) as internal standard. HRESIMS experiments were conducted on an Agilent 6250 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, USA). Semipreparative HPLC separations were performed on an Agilent 1260 series LC instrument (Agilent Technologies, USA) using an Agilent SB-C18 (9.4 × 250 mm) column. All solvents used for column chromatography were of analytical grade (Tianjin Fuyu Fine Chemical Co. Ltd., China) and solvents used for HPLC were of HPLC grade (Oceanpak Alexative Chemical Ltd., Sweden). Normal phase column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., China). Cancer cell lines (A549, K562, PC3 and BT549) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). https://doi.org/10.1016/j.fitote.2018.12.026 Received 3 December 2018; Received in revised form 30 December 2018; Accepted 30 December 2018 ⁎ Corresponding authors at: School of Biological Sciences and Technology, University of Jinan, Jinan 250022, PR China. E-mail addresses: bio_fangl@ujn.edu.cn (L. Fang), bio_zhangh@ujn.edu.cn (H. Zhang). 1 These authors contributed equally to this work. Fitoterapia 133 (2019) 175–179 Available online 18 January 2019 0367-326X/ © 2019 Elsevier B.V. All rights reserved. T
Z.-Y. Fang et a.Fitoterapia 133 (2019) 175-17918'Table 119'H and 13C NMR Data of melosuavine I in CDClg.15'1716COOCH38cNo.8cNo.8k(J in Hz)4&(J in Hz)20'21123rz38.97 (s)8.96 (s)NH167.0166.66113'52.33.73 (d, 15.3)3.91 (d, 4.6)59.26'3.22 (d, 15.3)121001955'51.551.53.11 (m)3.00 (m)111111'g'l2.79 (m)3.03 (m)13NH3CO18OCH36644.42.05 (m)43.02.08 (m)161012H1.86 (m)1.98 (m)COOCH37897'54.955.58131.0131.7Fig. 1. Structure of compound 1.9122.27.20 (d, 8.8)122.87.27 (d, 8.1)10105.310°105.66.43 (m)6.43 (m)1111'160.1160.12.2. Plant material1店96.56.43 (m)12'96.76.43 (m)13144.5144.6The leaves of M. suaveolens were collected from Yunnan Province,1414'135.5125.75.78 (dd, 10.2, 4.6)China, in October 2016, and authenticated by Prof.Yongqing Zhang15130.515'133.45.63 (s)5.88 (d, 10.2)16°1692.291.1(College of Pharmacy, Shandong University of Traditional Chinese1717'30.22.50 (d, 15.0)31.02.61 (d, 15.2)Medicine). A voucher specimen (No. 201610TM01) was deposited at2.32 (d, 15.0)2.18 (d, 15.2)the Herbarium of School of Biological Science and Technology,1818'8.27.90.63 (t, 7.4)0.70 (t, 7.4)University of Jinan, China.191928.21.01 (m)29.81.05 (m)0.89 (m)0.93 (m)202040.338.02.3.Extractionandisolation212170.565.42.70 (s)3.09 (s)OCHs55.7OCHs55.73.75 (s)3.77 (s)The air-dried and powdered leaves of M suaveolens (16.0 kg) wereCO2CHs51.13.79 (s)CO2CH351.13.79 (s)extractedwith95%EtOH(3×50L)atroomtemperaturetoafforda169.1CO,CHa169.1CO,CHscrudeextract (600g).The extractwas dissolved in2%HCl solution andextracted with ethyl acetate to give a non-alkaloid extract. The acidicdifferent concentrations of 1 for 48h. Then, the cells were fixed forwater-solubleportionwasbasifiedtopH10witha10%ammoniaso-30 min with 4% paraformaldehyde and stained with Hoechst 33342 forlution and extracted with CHClg to give an alkaloidal extract (149 g).1Omin at room temperature in the dark.Finally,the samples wereThe alkaloidal extract was separated on a silica gel column eluted withwashed with phosphate-buffered saline (PBS) and mounted on a slidea gradient of CH2Cl2-MeOH (from 200:1 to 10:1) to give seven frac-forfluorescence microscopy.tions, A-G. Fraction G (23 g) was chromatographed on silica gel elutingwith a gradient of petroleum ether-acetone to give 11 subfractions (Br-Bu) (374 mg). Subfraction Bg was further purified by Sephadex LH-202.6.PEactive caspase3assay(CHClg-MeOH,1:1)andpreparativeHPLCusing85%MeOH/H2Otoyield 1 (4mg, tr = 22.80 min).To detect apoptotic cells, PE active caspase 3 assay was appliedaccording to the manufacturer's protocol [14]. Briefly, BT549 cells were2.3.1. Melosuavine1(1)exposedtodifferentconcentrationsof1for24h,washedbyPBStwice,White amorphous powder; [α]5-152.3 (c 0.05, MeOH); UVand re-suspended in 400 μL cytofix buffer. After incubation in ice bath(MeOH) 入max (log e)210,230, 308 nm; ECD (c =0.25mg/mL, MeOH)for 20min, the cells were collected and washed by perm bufferforZmax (Ae)228 (+6.17),257(+15.02),328 (11.06)nm; IR (KBr) Vmaxtwice.Then, 20μLPE-conjugated active caspase3 antibody was added.3440,2956,2935,2855,1737,1676,1618,1496,1446,1262,789 cm-1;*H and 13C NMR data, see Table 1; HRESIMS m/z 731.3805Thesampleswereanalyzedbyflowcytometry(BDFACSAriaIIl,USA)afterincubationfor30minatroomtemperature.[M + H]+ (calcd for C44HsiN,O6, 731.3803).2.4. Cell viability assay2.7. Western blot analysisCell proliferation was evaluated by MTT assay [14]. Cells wereWestern blotting for p53, caspase 3, Bcl-2 and β-actin was doneseeded into 96-well plates at a density of 5000 per well. After overnightessentially as described [14].BT549 cells treated with various con-incubation, cells were treated with 1 using a series of concentrationscentrations of 1for24h were harvested inlysisbuffer[20mMTris-HClranging from 0.16 to 100μM, while control cells were treated with(pH7.5), 1% NP-40, 1 mM sodium vanadate, 1 mM EDTA, 1 mM EGTA,equal volume DMSO. After 48 h treatment, 0.5% MTT (Amresco, USA)50mMNaF,1mM phenylmethyl sulfonylfluoride(PMSF)J,and cen-solution was added to each well, and incubated for 4h. Then the MTTtrifuged at 14,000 rpm for 15min at 4°C. The supernatant was thesolution was removed and150μL DMSO added to dissolve the for-whole-cell extracts. Total protein extracts (30 μg per lane) were re-mazan. After mixing for 5 min, optical density was detected at 570 nmsolvedby12%SDS-PAGEandtransferredontoPVDFmembranes(Cat.on a microplate reader (Perkin Elmer corporation Enspire, USA). TheIPVH00010, Millipore, USA). Membrane was blocked with 5% non-fatOrigin 7.5 software was used to analyze the data and give the ICsodry milk for1h at room temperature and then incubated with primaryvalue. Assays were repeated at least three times.antibody overnight at 4°C including p53 (Cell Signaling, USA), Bcl.2(Cell signaling, USA), Caspase 3 (Cell Signaling, USA), and β-actin2.5. Hoechst staining(Sigma, USA). After three washes, anti-mouse or anti-rabbit goat-HRP.conjugated secondary antibodies were incubated for 2h at room tem-The nuclear morphological changes of BT549 cells exposed to 1perature and visualized by enhanced chemiluminescence (ECL, Cat.were evaluated using Hoechst 33342 staining [15]. Briefly, cells wereWBKLS0050, Millipore, USA).seeded into 6-well plates at the density of 1 x 105/cell and treated with176
2.2. Plant material The leaves of M. suaveolens were collected from Yunnan Province, China, in October 2016, and authenticated by Prof. Yongqing Zhang (College of Pharmacy, Shandong University of Traditional Chinese Medicine). A voucher specimen (No. 201610TM01) was deposited at the Herbarium of School of Biological Science and Technology, University of Jinan, China. 2.3. Extraction and isolation The air-dried and powdered leaves of M. suaveolens (16.0 kg) were extracted with 95% EtOH (3 × 50 L) at room temperature to afford a crude extract (600 g). The extract was dissolved in 2% HCl solution and extracted with ethyl acetate to give a non-alkaloid extract. The acidic water-soluble portion was basified to pH 10 with a 10% ammonia solution and extracted with CHCl3 to give an alkaloidal extract (149 g). The alkaloidal extract was separated on a silica gel column eluted with a gradient of CH2Cl2-MeOH (from 200:1 to 10:1) to give seven fractions, A–G. Fraction G (23 g) was chromatographed on silica gel eluting with a gradient of petroleum ether-acetone to give 11 subfractions (B1- B11) (374 mg). Subfraction B8 was further purified by Sephadex LH-20 (CHCl3-MeOH, 1:1) and preparative HPLC using 85% MeOH/H2O to yield 1 (4 mg, tR = 22.80 min). 2.3.1. Melosuavine I (1) White amorphous powder; [α] 25 D − 152.3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 210, 230, 308 nm; ECD (c = 0.25 mg/mL, MeOH) λmax (Δε) 228 (+6.17), 257 (+15.02), 328 (−11.06) nm; IR (KBr) νmax 3440, 2956, 2935, 2855, 1737, 1676, 1618, 1496, 1446, 1262, 789 cm−1 ; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 731.3805 [M + H]+ (calcd for C44H51N4O6, 731.3803). 2.4. Cell viability assay Cell proliferation was evaluated by MTT assay [14]. Cells were seeded into 96-well plates at a density of 5000 per well. After overnight incubation, cells were treated with 1 using a series of concentrations ranging from 0.16 to 100 μM, while control cells were treated with equal volume DMSO. After 48 h treatment, 0.5% MTT (Amresco, USA) solution was added to each well, and incubated for 4 h. Then the MTT solution was removed and 150 μL DMSO added to dissolve the formazan. After mixing for 5 min, optical density was detected at 570 nm on a microplate reader (Perkin Elmer corporation Enspire, USA). The Origin 7.5 software was used to analyze the data and give the IC50 value. Assays were repeated at least three times. 2.5. Hoechst staining The nuclear morphological changes of BT549 cells exposed to 1 were evaluated using Hoechst 33342 staining [15]. Briefly, cells were seeded into 6-well plates at the density of 1 × 105 /cell and treated with different concentrations of 1 for 48 h. Then, the cells were fixed for 30 min with 4% paraformaldehyde and stained with Hoechst 33342 for 10 min at room temperature in the dark. Finally, the samples were washed with phosphate-buffered saline (PBS) and mounted on a slide for fluorescence microscopy. 2.6. PE active caspase 3 assay To detect apoptotic cells, PE active caspase 3 assay was applied according to the manufacturer's protocol [14]. Briefly, BT549 cells were exposed to different concentrations of 1 for 24 h, washed by PBS twice, and re-suspended in 400 μL cytofix buffer. After incubation in ice bath for 20 min, the cells were collected and washed by perm buffer for twice. Then, 20 μL PE-conjugated active caspase 3 antibody was added. The samples were analyzed by flow cytometry (BD FACSAria III, USA) after incubation for 30 min at room temperature. 2.7. Western blot analysis Western blotting for p53, caspase 3, Bcl-2 and β-actin was done essentially as described [14]. BT549 cells treated with various concentrations of 1 for 24 h were harvested in lysis buffer [20 mM Tris-HCl (pH 7.5), 1% NP-40, 1 mM sodium vanadate, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM phenylmethyl sulfonylfluoride (PMSF)], and centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was the whole-cell extracts. Total protein extracts (30 μg per lane) were resolved by 12% SDS-PAGE and transferred onto PVDF membranes (Cat. IPVH00010, Millipore, USA). Membrane was blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibody overnight at 4 °C including p53 (Cell Signaling, USA), Bcl- 2(Cell signaling, USA), Caspase 3 (Cell Signaling, USA), and β-actin (Sigma, USA). After three washes, anti-mouse or anti-rabbit goat-HRPconjugated secondary antibodies were incubated for 2 h at room temperature and visualized by enhanced chemiluminescence (ECL, Cat. WBKLS0050, Millipore, USA). Fig. 1. Structure of compound 1. Table 1 1 H and 13C NMR Data of melosuavine I in CDCl3. No. δC δH(J in Hz) No. δC δH(J in Hz) 1 8.96 (s) 1′ 8.97 (s) 2 167.0 2′ 166.6 3 52.3 3.73 (d, 15.3) 3′ 59.2 3.91 (d, 4.6) 3.22 (d, 15.3) 5 51.5 3.00 (m) 5′ 51.5 3.11 (m) 2.79 (m) 3.03 (m) 6 44.4 2.05 (m) 6′ 43.0 2.08 (m) 1.86 (m) 1.98 (m) 7 54.9 7′ 55.5 8 131.0 8′ 131.7 9 122.2 7.20 (d, 8.8) 9′ 122.8 7.27 (d, 8.1) 10 105.3 6.43 (m) 10′ 105.6 6.43 (m) 11 160.1 11′ 160.1 12 96.5 6.43 (m) 12′ 96.7 6.43 (m) 13 144.5 13′ 144.6 14 135.5 14′ 125.7 5.78 (dd, 10.2, 4.6) 15 130.5 5.63 (s) 15′ 133.4 5.88 (d, 10.2) 16 92.2 16′ 91.1 17 30.2 2.50 (d, 15.0) 17′ 31.0 2.61 (d, 15.2) 2.32 (d, 15.0) 2.18 (d, 15.2) 18 7.9 0.63 (t, 7.4) 18′ 8.2 0.70 (t, 7.4) 19 28.2 1.01 (m) 19′ 29.8 1.05 (m) 0.89 (m) 0.93 (m) 20 40.3 20′ 38.0 21 70.5 2.70 (s) 21′ 65.4 3.09 (s) OCH3 55.7 3.75 (s) OCH3 55.7 3.77 (s) CO2CH3 51.1 3.79 (s) CO2CH3 51.1 3.79 (s) CO2CH3 169.1 CO2CH3 169.1 Z.-Y. Fang et al. Fitoterapia 133 (2019) 175–179 176
Z.-Y. Fang et alFitoterapia133(2019)175-1792.8.ECD calculationsThe conformational analyses of 1 were performed by using theOPLS3 molecular mechanics force field via the Macro Model panel ofMaestro 10.2.The conformers were further optimized with the softwarepackage Gaussian 09 at the B3LYP/6-311+ +G (2d, p) level, and theharmonic vibrational frequencies were also calculated to confirm theirstability.The stable conformers weresubject to ECD calculationbytime-dependent density functional theory (TD-DFT)method at theFig.3.KeyROESYcorrelations Oof1.B3LYP/6-311++G (2d,p)level in MeOH.TheECD spectra ofdifferentconformers were simulated using a Gaussian function with a half-bandwidth of 0.36 eV, and the final ECD spectra were obtained according to25Exptlof1the Boltzmann distribution of each conformer. The calculated ECDCalcdof120spectrum of 1was comparedwith theexperimental one15-3.Results and discussion10-Melosuavine I (1) was obtained as white amorphous powder. Its5molecular formula, C4gHsoN.O6,corresponding to 22 degrees of un-saturation, was assigned based on the HRESIMS peak at m/z 731.38050[M + H]+. The UV spectrum showed absorption maxima at 308, 230,and 210 nm, characteristic of an indole chromophore [16].The IR ab--5sorption at 3440 and 1737 cm-1 suggested the presence of NH and-10 -ester carbonyl functions,respectively..The H NMR data (Table 1)showed signals of two NH groups at 8 8.97 (1H, s) and 8.96 (1H, s),-15together with signals of four O-methyl groups at 8 3.79 (6H, s), 3.77200250300350400(3H,s),and3.75(3H, s).Tripletsat8u0.63(3H,t,J=7.4Hz,Me-18)Wavelength (nm)and0.70(3H,t,J=7.4Hz,Me-18wereassignedtoprotonsoftwomethyl groups connected to methylenes. The 13c (Table 1) and HSQCFig. 4. Experimental and calculated ECD spectra of 1 (in MeOH)NMR spectra of 1 displayed a total of 44 signals including six methyl,six methylene, 14 methine, and 18 quaternary carbons.Of them,12 arylindicated that therelative configurations ofunits A and Bwere identicalcarbons, six quaternary carbons (8c 91.1, 92.2, 166.6, 167.0, 169.1 andto those of 11-methoxytabersonine [18]. The ROESY correlation of H-169.1), and two methoxy signals (8c 51.1, 51.1) were characteristic3'/H-21' suggested the α-orientation for H-3' (Fig. 3). The absoluteresonances for two β-anilinoacrylate moieties conjugated with twoconfiguration of 1 was established by comparing the experimental ECDmethyl ester units [9]. Based on the analysis of 1D and 2D NMR spectra,spectrum with the calculated one (Fig. 4), and the good agreement1wasestablished as abisindolealkaloid assembled from aunion of twobetweenthecalculated and experimental ECD curves allowed theas-aspidosperma-typeunits (Table1) [17].signmentof7S,20R,21S3'R,7'S,20'R,21'Sconfigurationsfor1.Thus1In the unit A (Fig.2), the H and 13c NMR data resembled those ofwas established as shownand named melosuavineI11-methoxytabersonine exceptfor the replacement of one olefinicAspidosperma-type alkaloids are cytotoxic to cancer cell lines, somethine carbon (8c133.2, C-14) by one olefinic quaternary carbon (8cthe cytotoxicity of melosuavine I against four cancer cell lines (A549,135.5),whichwassupportedbytheHMBCcorrelationsfromH-3andK562,PC3andBT549)wasevaluatedbyMTTassay[19,20].Melo-H-15toC-14.ComparisontheNMRdataof unitBwith those of11-suavine I exhibited cytotoxic activities against all cell lines with ICsomethoxytabersonine revealed identical structural features, except forvaluesof11.38±0.11,10.73±0.23，3.44±0.07,andthe absence of a methylene carbon (8c 50.8) and the presence of a0.89 ± 0.03 μM, respectively. According to the results above, melo-methine resonance (8c 59.2), as supported by the 'H-'H COSY corre-suavine I showed the strongest cytotoxicity against BT549 cells andlation between H-3′ and H-14' [18]. Finally, the linkage of units A and Bthus intensive mechanism study was carried out in BT549 cells.between C-14 and C-3'was established by the HMBC correlations fromInduction of apoptosis of cancer cells is a promising anticancerH-3to C-3,C-14and C-15.Hence,theplanarstructureof 1 was es-strategy. To investigate whether induction of apoptosis contributed totablished as shown. The relative configuration of 1 was determined bythe significant cytotoxicity against BT549 cells, Hoechst staining wasanalysis of the ROESY spectrum (Fig- 2). In the ROESY spectrum, theapplied in our study. As shown in Fig. 5, BT549 cells exhibited chro-correlationsof H-9/H-21,H-21/H-18,H-9'/H-21',andH-21/H-18matin condensation and cell shrinkage after treatment of melosuavine Iat different concentrations.The above morphological changes of BT549UnitAcells indicated the proapoptotic effect of melosuavine I. PE active cas-COOCHpase 3 assay was further conducted to confirm the apoptosis-inducingeffect of melosuavine I. As shown in Fig.6, the percentage of activecaspase 3 gradually increased to 2.68%, 4.29% and 40.8% after treat-NHment with 0.1,1.0, and 10μM of 1 when compared withthecontrol(0.48%). These results demonstrated that melosuavine I induced cas-pases-dependent cell apoptosis in BT549 cell. Accordingly, the effects ofmelosuavineIontheexpression level ofapoptotic-related proteins,Bcl-V0CF2, caspase 3, and p53 were further investigated by western blottingHCOOCH3UnitBassay (Fig. 7). The results suggested that melosuavine I decreased theexpression level of antiapoptotic Bcl-2 and increased the level of cas-pase 3 compared with control in a dose-dependent manner. Meanwhile,Fig. 2. Selected COSY O and HMBC O correlations of of 1.177
2.8. ECD calculations The conformational analyses of 1 were performed by using the OPLS3 molecular mechanics force field via the Macro Model panel of Maestro 10.2. The conformers were further optimized with the software package Gaussian 09 at the B3LYP/6–311++G (2d, p) level, and the harmonic vibrational frequencies were also calculated to confirm their stability. The stable conformers were subject to ECD calculation by time-dependent density functional theory (TD-DFT) method at the B3LYP/6–311++G (2d, p) level in MeOH. The ECD spectra of different conformers were simulated using a Gaussian function with a half-band width of 0.36 eV, and the final ECD spectra were obtained according to the Boltzmann distribution of each conformer. The calculated ECD spectrum of 1 was compared with the experimental one. 3. Results and discussion Melosuavine I (1) was obtained as white amorphous powder. Its molecular formula, C44H50N4O6, corresponding to 22 degrees of unsaturation, was assigned based on the HRESIMS peak at m/z 731.3805 [M + H]+. The UV spectrum showed absorption maxima at 308, 230, and 210 nm, characteristic of an indole chromophore [16]. The IR absorption at 3440 and 1737 cm−1 suggested the presence of NH and ester carbonyl functions, respectively. The 1 H NMR data (Table 1) showed signals of two NH groups at δH 8.97 (1H, s) and 8.96 (1H, s), together with signals of four O-methyl groups at δH 3.79 (6H, s), 3.77 (3H, s), and 3.75 (3H, s). Triplets at δH 0.63 (3H, t, J = 7.4 Hz, Me-18) and 0.70 (3H, t, J = 7.4 Hz, Me-18′) were assigned to protons of two methyl groups connected to methylenes. The 13C (Table 1) and HSQC NMR spectra of 1 displayed a total of 44 signals including six methyl, six methylene, 14 methine, and 18 quaternary carbons. Of them, 12 aryl carbons, six quaternary carbons (δC 91.1, 92.2, 166.6, 167.0, 169.1 and 169.1), and two methoxy signals (δC 51.1, 51.1) were characteristic resonances for two β-anilinoacrylate moieties conjugated with two methyl ester units [9]. Based on the analysis of 1D and 2D NMR spectra, 1 was established as a bisindole alkaloid assembled from a union of two aspidosperma-type units (Table 1) [17]. In the unit A (Fig. 2), the 1 H and 13C NMR data resembled those of 11-methoxytabersonine except for the replacement of one olefinic methine carbon (δC 133.2, C-14) by one olefinic quaternary carbon (δC 135.5), which was supported by the HMBC correlations from H-3 and H-15 to C-14. Comparison the NMR data of unit B with those of 11- methoxytabersonine revealed identical structural features, except for the absence of a methylene carbon (δC 50.8) and the presence of a methine resonance (δC 59.2), as supported by the 1 H-1 H COSY correlation between H-3′ and H-14′ [18]. Finally, the linkage of units A and B between C-14 and C-3′ was established by the HMBC correlations from H-3′ to C-3, C-14 and C-15. Hence, the planar structure of 1 was established as shown. The relative configuration of 1 was determined by analysis of the ROESY spectrum (Fig. 2). In the ROESY spectrum, the correlations of H-9/H-21, H-21/H-18, H-9′/H-21′, and H-21′/H-18′ indicated that the relative configurations of units A and B were identical to those of 11-methoxytabersonine [18]. The ROESY correlation of H- 3′/H-21′ suggested the α-orientation for H-3′ (Fig. 3). The absolute configuration of 1 was established by comparing the experimental ECD spectrum with the calculated one (Fig. 4), and the good agreement between the calculated and experimental ECD curves allowed the assignment of 7S,20R,21S,3′R,7′S,20′R,21′S configurations for 1. Thus, 1 was established as shown and named melosuavine I. Aspidosperma-type alkaloids are cytotoxic to cancer cell lines, so the cytotoxicity of melosuavine I against four cancer cell lines (A549, K562, PC3 and BT549) was evaluated by MTT assay [19,20]. Melosuavine I exhibited cytotoxic activities against all cell lines with IC50 values of 11.38 ± 0.11, 10.73 ± 0.23, 3.44 ± 0.07, and 0.89 ± 0.03 μM, respectively. According to the results above, melosuavine I showed the strongest cytotoxicity against BT549 cells and thus intensive mechanism study was carried out in BT549 cells. Induction of apoptosis of cancer cells is a promising anticancer strategy. To investigate whether induction of apoptosis contributed to the significant cytotoxicity against BT549 cells, Hoechst staining was applied in our study. As shown in Fig. 5, BT549 cells exhibited chromatin condensation and cell shrinkage after treatment of melosuavine I at different concentrations. The above morphological changes of BT549 cells indicated the proapoptotic effect of melosuavine I. PE active caspase 3 assay was further conducted to confirm the apoptosis-inducing effect of melosuavine I. As shown in Fig. 6, the percentage of active caspase 3 gradually increased to 2.68%, 4.29% and 40.8% after treatment with 0.1, 1.0, and 10 μM of 1 when compared with the control (0.48%). These results demonstrated that melosuavine I induced caspases-dependent cell apoptosis in BT549 cell. Accordingly, the effects of melosuavine I on the expression level of apoptotic-related proteins, Bcl- 2, caspase 3, and p53 were further investigated by western blotting assay (Fig. 7). The results suggested that melosuavine I decreased the expression level of antiapoptotic Bcl-2 and increased the level of caspase 3 compared with control in a dose-dependent manner. Meanwhile, Fig. 2. Selected COSY () and HMBC () correlations of of 1. Fig. 3. Key ROESY correlations () of 1. Fig. 4. Experimental and calculated ECD spectra of 1 (in MeOH). Z.-Y. Fang et al. Fitoterapia 133 (2019) 175–179 177
Z.-Y. Fang et al.Fitoterapia 133 (2019) 175-179DMSO0.1uM10μMFig.5. Treatment with melosuavine I in48 h for determination of apoptotic changes analyzed under a fluorescence microscope.theexpressionlevelofp53inBT549cellsaftertreatmentwithmelo-Conflictof interestsuavine I was increased. These results indicated that melosuavine Icould induced the apoptosis of BT549 cells which involved activation ofThe authors declare that there are no conflicts of interest with thiscaspase 3 and p53 and down-regulation of Bcl-2.work.Acknowledgments4.ConclusionsThe chemical investigation of the leaves of M. suaveolens led to theThis work was financially supported by Natural Science Foundationisolation of a new bisindole alkaloid, melosuavine I (1). The structureof China (21772065, 81673527), Shandong Provincial Natural Sciencewith absoluteconfigurationsof 1was elucidated on thebasis of MS,Foundation(ZR2017MH019,JQ201721),NationalKeyResearchNMR and computational methods.The cytotoxicity of melosuavine IProject (2017YFC1702702), Shandong Provincial Key Research Projectagainstfourcancer cell lines(A549,K562,PC3andBT549)was eval-(2017GSF19115,2018GSF118158),andShandongTalentsTeamuated. The results showed that melosuavine I exhibited significant cy-Cultivation Plan of University Preponderant Discipline (No. 10027).totoxicity on BT549 cells with an ICso value of 0.89μM. Intensivemechanismstudyindicated thatmelosuavineIinhibited cell prolifera-tion by inducing apoptosis in BT549 cells through activation of caspaseAppendixA.Supplementary data3 and p53, and down-regulation Bcl-2.Supplementary datato this articlecanbe found online at https://doi.org/10.1016/j.fitote.2018.12.026.melosuavinel0.1μM1.0 μM10 μMControlCaspase3-A+Caspase3-A+Caspase3-A+Caspase3-A-Caspase3-A-Caspase3-A-Caspase3-A+Capase3-A-2K2.0ke7.32.0k95.789.22.684.290.4840.899.5MN0001.5KunIan8200nk1.0K3103Caspase3Fig. 6. Melosuavine I induced apoptosis in BT549 cell marked with PE-Caspase 3. BT549 cell were treated with DMSO alone or melosuavine I at 0.1, 1.0 and 10 μMfor 24h, and analyzed by flow cytometry.178
the expression level of p53 in BT549 cells after treatment with melosuavine I was increased. These results indicated that melosuavine I could induced the apoptosis of BT549 cells which involved activation of caspase 3 and p53 and down-regulation of Bcl-2. 4. Conclusions The chemical investigation of the leaves of M. suaveolens led to the isolation of a new bisindole alkaloid, melosuavine I (1). The structure with absolute configurations of 1 was elucidated on the basis of MS, NMR and computational methods. The cytotoxicity of melosuavine I against four cancer cell lines (A549, K562, PC3 and BT549) was evaluated. The results showed that melosuavine I exhibited significant cytotoxicity on BT549 cells with an IC50 value of 0.89 μM. Intensive mechanism study indicated that melosuavine I inhibited cell proliferation by inducing apoptosis in BT549 cells through activation of caspase 3 and p53, and down-regulation Bcl-2. Conflict of interest The authors declare that there are no conflicts of interest with this work. Acknowledgments This work was financially supported by Natural Science Foundation of China (21772065, 81673527), Shandong Provincial Natural Science Foundation (ZR2017MH019, JQ201721), National Key Research Project (2017YFC1702702), Shandong Provincial Key Research Project (2017GSF19115, 2018GSF118158), and Shandong Talents Team Cultivation Plan of University Preponderant Discipline (No. 10027). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2018.12.026. Fig. 5. Treatment with melosuavine I in48 h for determination of apoptotic changes analyzed under a fluorescence microscope. Fig. 6. Melosuavine I induced apoptosis in BT549 cell marked with PE-Caspase 3. BT549 cell were treated with DMSO alone or melosuavine I at 0.1, 1.0 and 10 μM for 24 h, and analyzed by flow cytometry. Z.-Y. Fang et al. Fitoterapia 133 (2019) 175–179 178
Z.-Y.Fang et al.Fitoterapia 133 (2019) 175-179(A)Fig. 7. Melosuavine I modulated apoptosis regulatory proteins in BT549 cellline. (A) Bcl-2 was inhibited by Melosuavine I after 48 h treatment in BT549cells and exhibited a dose dependent manner versus control (B) Melosuavine I0.1μM1μMControl10μMinduced the expression of Caspase 3 and exhibited a dose dependent mannerversus control. (C) Melosuavine I significantly induced the expression of p53,Caspase3.and also exhibited a dose dependent manner versus control. Data were shownas means ± SD of the triplicate measurements and illustrated in histograms.β-actinSamplegroups were significantly different from the control group (*p < 0.01),and significant difference were existed between different treatment con-0centration of khasuanine Aon cells (#p < 0.05).a7.*福References54[1] S.E OConnor, J.J. Maresh, Chemistry and biology of monoterpene indole alkaloidbiosynthesis, Nat.Prod.Rep.23 (2006)532547.[2]Delectis Florae Reipublicae Popularis Sinicae Edita, Florae Reipubicae PopularisSinicae, Science Press, Beijing,1977.[3] Y.L.Zhou, J.H,Ye,ZM.L, Z.H.Huang, Study on the Alkaloids ofMelodinus tenuicaudatus,Planta Med.54 (1988)315-317.0[4] H.Mehri,M.Plat, The structure of scaandomelidine,bisindole alkaloid fromcontrol 0.1μM1μM10μMMelodinus scandens, J.Nat, Prod. 55 (1992) 241-244.[5] Y.L. He, W.M, Chen, X.Z.Feng, Melomorsine, a new dimeric indoline alkaloids frommelosuavinelMelodinus morsei,J.Nat.Prod.57 (1994) 411-414.[6] K.X. Yan, XZ. Feng, Melaxillarinine, a new bisindole alkaloid from Melodinus ax-illaris,Chin.Chem.Lett.8(1997)313-314.(B)[7] F. Tao, Y. L, Y.Y. Wang, X.H. Cai, Y.P. Liu, X.D. Luo, Cytotoxic indole alkaloidsfromMelodinustenuicauatus,J.Nat.Prod.73(2010)1075-1079.[8] XH.Cai,Y.i, Y.P.Liu, .N.LI, M.F.Bao,.D. uo, Alkaloids fromMelodinus0.1μM1uM10μMControlyunnanensis, Phtytochemistry 116 (2012)116-124.[9] Y.P.Liu,Y.L.Zhao,I.Feng,G.G.Cheng,B.H.Zhang,Y.Li,X.H.Cai,.D.uoBcl-2Melosuavines A-H, cytotoxic bisindole alkaloid derivatives from Melodinus suaveo-lens,J.Nat.Prod.76(2013)2322-2329.[1o] s.Shao,H.Zhang,C.M.Yuan,Y.Zhang,MM.Cao,H.Y.Zhang.Y.Feng,X.Dingβ-actinQ.Zhou, Q.Zhao, H.P.He,X.J.Hao, Cytotoxic indole alkaloids from thefruits ofMelodinus cochinchinensis, Phtytochemistry 116 (2015) 367-373.1.24[11] J.H. Jiang, W.D. Zhang, Y.G. Chen, Phytochemical and pharmacological propertiesof the genus Melodinus, Trop.J.Pharm.Res.14 (2015)2325-2344.a1[12] J.H.Ye, Y.L. Zhou,Z.H.Huang,F.Picot, Akaloids from Melodinus suaveolenPhytochemistry30 (1991)3168-3170.[13] L.Fang,s.M.Tian, J.Zhou, Y.L.Lin, Z.W.Wang.X.Wang,Melaxillines a and Bmonoterpenoid indole alkaloids from Melodinus axilaris, Fitoterapia 115 (2016)0.6 173-176.[14] J. Zhou, J.H,.Feng,Fang,A novel monoterpenoid indole alkaloid with anticancer0.4activity fromMelodinuskhasianus,Bioorg.Med.Chem.Lett.27(2017)893-896.[15].Tang,L.Liu,Q.RHe,W.an,.Li,J.M.Gao,nsamycinswithanti0.2 proliferative and antineuroinflammatory activity from moss-soil-derivedStreptomycescacaoisubsp.asoensisH2S5,J.Nat.Prod.81(2018)1984-19910[] .heludo, Gerasmenko,Kolsh,J.Sckigt, New alkaoids ofthesarcontrol0.1μM1uM10μMgine group from Rawolfia serpentina hairy root culture, JNat.Prod,65 (2002)1006-1010.melosuavinel[17] Y.P. Liu, Y. Li, X.H Cai, X.Y. Li, L.M. Kong, G.G. Cheng, X.D. Luo, MelodininesM-U, cytotoxic alkaloids from Melodims suaveolens, J.Nat. Prod.75 (2012)220-224(C)[18] F.EZiegler, G.B. Benett, Claisen rearrangement in indole alkaloid synthesis, Totalsynthesis of(± )tabersonine, J.Am.Chem.Soc.95 (1973)7458-7464.[19] X.H.Cai, H.Jiang, Y.Li, G.G.Cheng,Y.P.Liu, T.Feng,X.D.Luo, Cytotoxic indoleControl0.1μM1μM10μMalkaloids from Melodinus fusformis and M.morsei,Chin.J.NatMed.9 (2011)259263..p53[2o] M.E.Qazzaz, V.J.Raja, K.H.Lim, T.S.Kam, J.B.Lee, P.Gershkovich,T.D. Bradshaw, in vitro anticancer properties and biological evaluation of novelnatural alkaloid jerantinine B, Cancer Lett. 370 (2016)185-197.β-actin20 1050control0.1uM1μM10μMmelosuavinel179
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