Chemical Constituents from Flueggea Virosa and the Structural Revision of Dehydrochebulic Acid Trimethyl Ester

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Article Chemical Constituents from Flueggea virosa and the Structural Revision of Dehydrochebulic Acid Trimethyl Ester

Chih-Hua Chao 1,2,*, Ying-Ju Lin 3,4, Ju-Chien Cheng 5, Hui-Chi Huang 6, Yung-Ju Yeh 5, Tian-Shung Wu 7,8, Syh-Yuan Hwang 9 and Yang-Chang Wu 1,2,10,11,*

1 School of Pharmacy, China Medical University, Taichung 40402, Taiwan 2 Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 40447, Taiwan 3 School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan; [email protected] 4 Genetic Center, Department of Medical Research, China Medical University Hospital, Taichung 40447, Taiwan 5 Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 40402, Taiwan; [email protected] (J.-C.C.); [email protected] (Y.-J.Y.) 6 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan; [email protected] 7 Department of Pharmacy, National Cheng Kung University, Tainan 70101, Taiwan; [email protected] 8 Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 90741, Taiwan 9 Endemic Species Research Institute, Council of Agriculture, Nantou 55244, Taiwan; [email protected] 10 Center for Molecular Medicine, China Medical University Hospital, Taichung 40447, Taiwan 11 Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan * Correspondence: [email protected] (C.-H.C.); [email protected] (Y.-C.W.); Tel.: +886-4-2205-3366 (ext. 5157) (C.-H.C.)

Academic Editor: Derek J. McPhee Received: 9 August 2016; Accepted: 12 September 2016; Published: 16 September 2016

Abstract: In an attempt to study the chemical constituents from the twigs and leaves of Flueggea virosa, a new terpenoid, 9(10→20)-abeo-ent-podocarpane, 3β,10α-dihydroxy-12-methoxy-13- methyl-9(10→20)-abeo-ent-podocarpa-6,8,11,13-tetraene (1), as well as ﬁve known compounds were characterized. Their structures were elucidated on the basis of spectroscopic analysis. In addition, the structure of dehydrochebulic acid trimethyl ester was revised as (2S,3R)-4E-dehydrochebulic acid trimethyl ester based on a single-crystal X-ray diffraction study. The in vitro anti-hepatitis C virus (anti-HCV) activity and cytotoxicity against Huh7.5 cells for the isolated compounds were evaluated.

Keywords: Flueggea virosa; dehydrochebulic acid trimethyl ester; ent-podocarpane; anti-HCV; HCVcc

1. Introduction Flueggea virosa (Roxb. ex Wild) Baill, belonging to the Euphorbiaceae family, is a common medicinal plant in Africa and China [1,2]. In previous reports, Securinega alkaloids have been widely regarded as a representative group of the genus Flueggea [3], formerly classiﬁed as Securinega genus. However, our prior studies have demonstrated that the roots of F. virosa contained a series of non-alkaloids, including ent-podocarpanes [4,5], 9(10→20)-abeo-ent-podocarpanes [6], 3,4-seco-ent-podocarpanes [7], and 3,4-seco-30-nor-friedelanes [6], some of which were endowed with anti-HCV activities and weak toxicities. Among them, 4-hydroxy-12-methoxy-13-methyl-3,4-seco- ent-podocarpa-6,8,11,13-tetraen-3-oic acid was found to be a brand new type anti-HCV

Molecules 2016, 21, 1239; doi:10.3390/molecules21091239 www.mdpi.com/journal/molecules Molecules 2016, 21, 1239 2 of 7 MoleculesMolecules 2016 2016, ,21 21, ,1239 1239 22 of of 7 7 podocarpanesMolecules 2016, 21 ,[7], 1239 and 3,4-seco-30-nor-friedelanes [6], some of which were endowed with anti-HCV2 of 7 activitiespodocarpanespodocarpanes and [7], [7],weak andand 3,4-3,4toxicities.‐secoseco-30-‐30 ‐nornorAmong-friedelanes‐friedelanes them, [6],[6], 4-hydroxy-12-methoxy-13-methyl-3,4-somesome ofof whicwhichh werewere endowedendowed withwith anti-HCVantiseco‐HCV-ent- podocarpa-6,8,11,13-tetraen-3-oicactivitiesactivities andand weakweak toxicities.toxicities. acid AmongAmong was found them,them, to be4-hydroxy-12-methoxy-13-methyl-3,4-4‐ hydroxya brand ‐new12‐methoxy type anti-HCV‐13‐methyl agent‐3,4 ‐seco[7].seco -The‐entent-‐ non-alkaloidsagentpodocarpa-6,8,11,13-tetraen-3-oicpodocarpa [7].‐6,8,11,13 Thein the non-alkaloids other‐tetraen parts‐3‐oic of inacidacidthis the plantwaswas other found foundstill partsreceived toto bebe of aa less brand thisbrand attention. plant newnew type stilltype Thus, anti-HCV receivedanti the‐HCV isolation less agentagent attention. of [7].[7]. active TheThe constituentsThus,non-alkaloidsnon‐alkaloids the from isolation inin the the the otherother twigs of parts activeparts and ofleavesof constituents thisthis plantofplant F. virosa stillstill fromreceived receivedwas carried the less less twigs attention. out,attention. which and Thus, Thus, leavesresulted the the of isolation inisolation theF. virosaisolation of of active activewas of acarriedconstituentsconstituents new 9(10 out,→ from 20)-from whichabeo the the- resulted ent twigstwigs-podocarpane, andand in leaves leaves the isolation of namelyof F. F. virosa virosa of3β ,10was awasα new -dihydroxy-12-methoxy-13-methyl-9(10carried carried 9(10 out, →out,20)- which whichabeo- entresulted resulted-podocarpane, inin thethe isolationisolation namely→20)- ofof abeo3aaβ newnew,10-entα 9(10-dihydroxy-12-methoxy-13-methyl-9(109(10-podocarpa-6,8,11,13-tetraene→→20)-20)‐abeoabeo-‐entent-podocarpane,‐podocarpane, ( 1 namelynamely), alone→ 33 β20)-βwith,10,10abeoαα‐ -dihydroxy-12-methoxy-13-methyl-9(10fivedihydroxy-ent -podocarpa-6,8,11,13-tetraeneknown‐12 co‐methoxympounds.‐13 ‐methylIn addition,‐9(10 (1),→ alone→ 20)-the20)‐ withstructuralabeoabeo-‐ent ﬁveent-podocarpa-6,8,11,13-tetraene‐podocarpaknown revision compounds. of‐6,8,11,13 dehydrochebulic‐tetraene In addition, (ac(11),),id thealone alonetrimethyl structural withwith ester fivefive revision (Chart knownknown of1, dehydrochebulic originalcocompounds.mpounds. 2) was In Inalso acidaddition,addition, reported trimethyl the thein thisesterstructuralstructural study. (Chart revision revision1, original ofof dehydrochebulicdehydrochebulic2) was also reported acacidid in trimethyltrimethyl this study. esterester (Chart(Chart 1,1, originaloriginal 22)) waswas alsoalso reportedreported inin thisthis study. study.

Chart 1. Structures of compounds 1–6. ChartChart 1. 1. Structures Structures of of compounds compounds 1 1––66. . 2. Results Results and and Discussion Discussion 2.2. Results Results and and Discussion Discussion The MeOH MeOH extract extract from from the twigs the twigsand leaves and of leaves F. virosa of wasF. concentrated virosa was concentrated and the alkaloids and were the removedalkaloidsTheThe MeOH byMeOH were partition removedextract extract with from from byacidic the the partition twigs twigswater. and and The with leaves leaves result acidic of ingof F. F. water. nonalkaloidvirosa virosa was was The concentrated concentrated layer resulting was separated nonalkaloid and and the the alkaloids alkaloidsrepeatedly layer were were was by → columnseparatedremovedremoved chromatography byby repeatedly partitionpartition with bywith to column afford acidicacidic a water. chromatography water.new 9(10 TheThe result20)-resultingabeoing to- ent nonalkaloid affordnonalkaloid-podocarpane a new layerlayer 9(10 (1 ) was→ wasand20)- separatedfiveseparatedabeo known-ent -podocarpane repeatedlyrepeatedly compounds, byby → → which(columncolumn1) and were chromatography chromatography ﬁve identified known as compounds, 4 to Eto -dehydrochebulicafford afford a a new new which 9(10 9(10→ wereacid20)-20) trimethyl‐abeo identiﬁedabeo-‐entent-podocarpane‐ podocarpaneester as (2 4)E [8,9],-dehydrochebulic ( (112-hydroxy-20(101)) and and five five known known acid compounds, compounds,5)- trimethylabeo-4,5- → secoesterwhichwhich-podocarpa-5(10),6,8,11,13-pentaen-3-one ( 2were were)[8, 9identified identified], 12-hydroxy-20(10 as as 4 4EE-dehydrochebulic‐dehydrochebulic→5)-abeo-4,5- acid acid(3seco) trimethyl [10],trimethyl-podocarpa-5(10),6,8,11,13-pentaen-3-one 3β,12-dihydroxy-13-methylpodocarpa-6,8,11,13- ester ester ( (22)) [8,9], [8,9], 12-hydroxy-20(10 12‐hydroxy‐20(10→5)-5)‐abeo (abeo3)[-4,5-‐104,5],‐ tetraene3secosecoβ,12-dihydroxy-13-methylpodocarpa-6,8,11,13-tetraene-podocarpa-5(10),6,8,11,13-pentaen-3-one‐podocarpa (4) [10],‐ betulinic5(10),6,8,11,13 acid ‐3pentaenβ-calfeate‐3‐ (one5) [11], ((33)) and[10],[10], (+) 3 3ampelosinββ,12-dihydroxy-13-methylpodocarpa-6,8,11,13-,12 (4‐)[dihydroxy10], E betulinic (6) [12].‐13‐methylpodocarpa acid 3β-calfeate‐6,8,11,13 (5)[11],‐ andtetraenetetraene (+) ampelosin( (44)) [10], [10], betulinic betulinic E (6)[ 12acid acid]. 3 3ββ‐-calfeatecalfeate ( (55)) [11], [11], and and (+) (+) ampelosin ampelosin E E ( (66)) [12]. [12].

Figure 1. Selected 1H−1H COSY (▬) and HMBC (→) correlations of 1. 1 1 FigureFigureFigure 1.1. 1.Selected Selected Selected 1 H1HH−−11HH COSY COSY ( (▬▬)) and and HMBC HMBC ( ( →→)) )correlations correlations correlations of of 1 1. . Compound 1 was obtained as a colorless oil and a molecular formula of C19H26O3 was deduced for thisCompound compound 1 basedwas obtained on the molecular as a colorless ion peakoil and [M a − molecular H]− at m/z formula 301.1805 of in C the19H (26−O)-HR-APCI-MS3 was deduced Compound 1 was obtainedobtained asas aa colorlesscolorless oiloil andand aamolecular molecular formula formula of of C C1919HH2626O3 was deduced 1 − 13 (calcdforfor this this for compoundcompound compound C19H25O3 based,based based301.1809). on on on the the the Inspection molecular molecular molecular ionof ion ion the peak peak peak overall [M [M [M− − − H] H-H]H]− −and atat at mm m ///zC-NMRz 301.1805 301.1805 data in in thethe therevealed (( −(−−)-HR-APCI-MS)-HR-APCI-MS)‐HR that‐APCI it is‐MS a member(calcd for of C9(1019H→25O20)-3, 301.1809).abeo-ent-podocarpanes Inspection of [6], the with overall slight 111H chemical‐ and 131313C ‐shiftNMR differences data revealed for resonances that it is a (calcd forfor CC1919H25OO33, ,301.1809). 301.1809). Inspection Inspection of of the the overall overall H-H- and and C-NMRC-NMR data data revealed revealed that that it it is a → ofmembermember substituent ofof of 9(109(10 9(10 patterns.→→20)-20)-20)abeo‐abeoabeo The--ent‐entent 12-methoxy-podocarpanes-podocarpanes‐podocarpanes group [ 6[6], [6],], was with with with re slight adilyslight slight chemicalassigned chemical chemical shiftfrom shift shift differences itsdifferences differences proton forchemical for for resonances resonances resonances shift at of δsubstituentofofH substituent 3.82substituent (s), while patterns. patterns. patterns. the 6,7-double The The The 12-methoxy 12-methoxy 12‐ methoxybond was groupgroup group deduced was was reaccording readilyreadilyadily assigned assigned to the protonfrom from from its its itschemical proton proton proton chemical chemicalshifts chemical at δshift shiftH shift6.58 at at (1H,δH 3.82 dd, (s), J = while12.1, 2.6the Hz) 6,7‐ doubleand 5.78 bond (1H, was dd, deducedJ = 12.1, 4.4according Hz) (Table to the 1) proton[6]. The chemical above assignment shifts at δH was 6.58 atδH δ3.82H 3.82 (s), while (s), while the 6,7-double the 6,7-double bond was bond deduced was deduced according according to the proton to the chemical proton chemicalshifts at δH shifts 6.58 confirmed(1H, dd, J by= 12.1, the interpretation2.6 Hz) and 5.78 of 1(1H,H–1H dd, COSY J = 12.1, and 4.4 HMBC Hz) (Tablecorrelations 1) [6]. (FigureThe above 1). Theassignment 3β-OH was was at(1H,δH dd,6.58 J = (1H, 12.1, dd, 2.6 JHz)= 12.1, and 5.78 2.6Hz) (1H, and dd, 5.78J = 12.1, (1H, 4.4 dd, Hz)J = (Table 12.1, 1) 4.4 [6]. Hz) The (Table above1)[ assignment6]. The above was 1 1 determinedassignmentconfirmedconfirmed by by wasbased thethe confirmed interpretation oninterpretation the chemical by the ofof interpretationshift 1HH––1 HandH COSYCOSY coupling and ofand1 H– HMBCpatternsHMBC1H COSY correlationscorrelations of H-3 and [δ HMBCH 3.51 (Figure(Figure (1H, correlations 1).1). t, JTheThe = 2.8 33β (Figureβ‐Hz)].-OHOH wasThewas1). relativedetermined configuration based on ofthe 1 chemicalwas further shift confirmed and coupling by the patterns analysis of of H NOE‐3 [δH correlations3.51 (1H, t, J (Figure = 2.8 Hz)]. 2). The The Thedetermined 3β-OH based was determined on the chemical based shift on and the coupling chemical patterns shift and of couplingH-3 [δH 3.51 patterns (1H, t, ofJ = H-32.8 Hz)]. [δH 3.51The NOE(1H,relativerelative t,correlationsJ configuration =configuration 2.8 Hz)]. of The H-5/H-20a,of of 1 relative1 was was further further configurationH-5/H-1a, confirmed confirmed and of by H-5/Hby1 wasthe the 3analysis analysis-18 further confirmed of confirmedof NOENOE that correlationscorrelationsby 1 thepossessed analysis (Figure (Figure the of 2). 2).same NOE The The NOE correlations of H‐5/H‐20a, H‐5/H‐1a, and H‐5/H3‐18 confirmed that 1 possessed the same correlationsNOE correlations (Figure 2of). TheH-5/H-20a, NOE correlations H-5/H-1a, of and H-5/H-20a, H-5/H3-18 H-5/H-1a, confirmed and that H-5/H 1 possessed3-18 confirmed the thatsame1

Molecules 2016, 21, 1239 3 of 7 relative configurations (10α-OH, 5β-H) as those of 9(10→20)-abeo-ent-podocarpanes isolated previouslypossessed the from same the relative roots of conﬁgurations F. virosa [6] Accordingly, (10α-OH, 5β-H) the as structure those of 9(10of 1→ was20)- determinedabeo-ent-podocarpanes as 3β,10α- dihydroxy-12-methoxy-13-methyl-9(10isolated previously from the roots of F.→ virosa20)-abeo[6]- Accordingly,ent-podocarpa-6,8,11,13-tetraene. the structure of 1 was determined as 3β,10α-dihydroxy-12-methoxy-13-methyl-9(10→20)-abeo-ent-podocarpa-6,8,11,13-tetraene.

Table 1. 1H- and 13C-NMR Spectroscopic Data (400 and 100 MHz, resp.; CDCl3) of 1. 1 13 Table 1. H- and C-NMR Spectroscopic Data (400 and 100 MHz, resp.; CDCl3) of 1. 1

δH (J in Hz) δC (Mult.)1 HMBC 1 2.05 td (14.1, 4.4) 33.0 (CH2) 2, 3, 20 δH (J in Hz) δC (Mult.) HMBC 1.61 m 12 2.05 td (14.1, 2.27 4.4)m 33.0 25.5 (CH (CH2)2) 1 2, 3, 20 1.611.62 m m 2 2.27 m 25.5 (CH ) 1 3 3.51 t (2.8) 75.6 (CH)2 1, 5 1.62 m 4 38.2 (C) 3 3.51 t (2.8) 75.6 (CH) 1, 5 45 2.45 dd (4.4, 2.6) 38.2 49.6 (C)(CH) 4, 6, 7, 10, 18, 19 56 2.45 5.78 dd (4.4,dd (12.1, 2.6) 4.4) 49.6 128.0 (CH) (CH) 4, 6,5, 7,8, 10,10 18, 19 67 5.78 6.58 dd (12.1,dd (12.1, 4.4) 2.6) 128.0 131.3 (CH) (CH) 5, 4,9, 5,14 8, 10 78 6.58 dd (12.1, 2.6) 131.3128.4 (CH) (C) 5, 9, 14 89 128.4135.2 (C) (C) 910 135.276.4 (C)(C) 1011 6.56 s 112.276.4 (C) (CH) 8, 12, 13, 20 1112 6.56 s 112.2156.9 (CH) (C) 8, 12, 13, 20 12 156.9 (C) 13 124.4 (C) 13 124.4 (C) 1414 6.95 6.95 s s 132.4 132.4 (CH) (CH) 7, 9, 7, 12 9, 12 15 2.17 s 15.6 (CH3) 12, 13, 14 15 2.17 s 15.6 (CH3) 12, 13, 14 3 1818 1.00 1.00 s s 27.2 27.2 (CH (CH3)) 3, 4, 3, 5, 4, 19 5, 19 1919 1.09 1.09 s s 22.4 22.4 (CH (CH3)3) 3, 4, 3, 5, 4, 18 5, 18 2020 2.96 2.96 d (14.2) d (14.2) 51.6 51.6 (CH (CH2)2) 1, 5, 1, 8, 5, 9, 8, 10, 9, 10,11 11 2.68 2.68 d (14.2) d (14.2) 12-OMe12-OMe 3.82 3.82 s s 55.3 55.3 (CH (CH3)3) 12 12

Figure 2 2.. Selected NOE correlations of 1.

The structure of dehydrochebulic acid trimethyl ester (original 2, Chart 1) with a cis- The structure of dehydrochebulic acid trimethyl ester (original 2, Chart1) with a methylbutenedioate scaffold was reported from Phyllanthus urinaria by Yao et al. in 1993 [9]. cis-methylbutenedioate scaffold was reported from Phyllanthus urinaria by Yao et al. in 1993 [9]. However, the authors proposed the wrong geometry for the methylbutenedioate moiety because of However, the authors proposed the wrong geometry for the methylbutenedioate moiety because failure to compare the NMR data for this moiety with those reported in the literature. The 1H- and of failure to compare the NMR data for this moiety with those reported in the literature. The 1H- 13C-NMR data of 2 measured in acetone-d6 and pyridine-d6 were in good agreement with those and 13C-NMR data of 2 measured in acetone-d and pyridine-d were in good agreement with those reported in the literature [8,9]. In the selective 1D6 NOESY experiment6 (Figure 3), irradiation of olefinic reported in the literature [8,9]. In the selective 1D NOESY experiment (Figure3), irradiation of H-5 did not show enhancements with other signals, suggesting that the 4,5-double bond in 2 has a olefinic H-5 did not show enhancements with other signals, suggesting that the 4,5-double bond in trans geometry. A single-crystal X-ray analysis was performed on 2 (Figure 4), which led the absolute 2 has a trans geometry. A single-crystal X-ray analysis was performed on 2 (Figure4), which led the configurations to be defined as 2S,3R according to the value of Flack parameter 0.09 (14), and absolute configurations to be defined as 2S,3R according to the value of Flack parameter 0.09 (14), confirmed the geometry of a 4E-double bond. Accordingly, the structure of 2 was revised as (2S,3R)- 4E-dehydrochebulic acid trimethyl ester (Chart 1, revised 2).

Molecules 2016, 21, 1239 4 of 7 Molecules 2016, 21, 1239 4 of 7

and conﬁrmed the geometry of a 4E-double bond. Accordingly, the structure of 2 was revised as Molecules(2S,3 2016R)-4, E21-dehydrochebulic, 1239 acid trimethyl ester (Chart1, revised 2). 4 of 7

Figure 3. (a) 1H-NMR spectrum of 2 measured in acetone-d6 and (b) selective 1D NOESY spectrum of 1 1 FigureFigure 3. (a 3.) (H-NMRa) H-NMR spectrum spectrum of of 2 2measuredmeasured in acetone-acetone-d6d6and and (b ()b selective) selective 1D 1D NOESY NOESY spectrum spectrum of of 2: selective irradiation of H-5 (δH = 6.83 ppm) using selnogp.3 Bruker program (parameter set: ns 128, 2: selective2: selective irradiation irradiation of ofH-5 H-5 (δ (Hδ H= =6.83 6.83 ppm) ppm) using using selnogp.3selnogp.3Bruker Bruker program program (parameter (parameter set: nsset: 128, ns 128, p12 = 80 ms, and d8 = 400 ms). p12 p12= 80 = ms, 80 ms,and and d8 d8= 400 = 400 ms). ms).

Figure 4. X-ray ORTEP drawing of 2.

In order to continue our FigureexplorationFigure 4. X-ray 4. X-rayfor ORTEPanti-HCV ORTEP drawing drawing agents of 2 .fromof 2. a natural source, the isolated compounds were subjected to anti-HCV and cytotoxic evaluation. As shown in Table 2, the anti-HCV In orderactivity to ofIn continue ordercompounds to continue our 1– 6exploration ourwas exploration assayed forusing for anti-HCV anti-HCV the cell-based agents agents fromHCV from a cell natural aculture natural source, (HCVcc) thesource, isolated infection the isolated system,compounds while the were toxicity subjected toward to anti-HCVhuman hepatoma and cytotoxic Huh7.5 evaluation. cells was Asalso shown measured in Table using2, thea 3-(4,5- compounds were subjected to anti-HCV and cytotoxic evaluation. As shown in Table 2, the anti-HCV dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2anti-HCV activity of compounds 1–6 was assayed using the cell-based HCVH-tetrazolium cell culture (HCVcc)salt (MTS) activity of compoundsinfection system, 1 while–6 was the toxicityassayed toward using human the hepatoma cell-based Huh7.5 HCV cells was cell also culture measured (HCVcc) using a infection assay. In addition, the therapeutic indices (TI = IC50/EC50) were also calculated to estimate their 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) system, potencywhile the as anti-HCV toxicity agents.toward The human result showedhepatoma that compoundsHuh7.5 cells 3–5 waspossessed also bettermeasured potencies using than a 3-(4,5- assay. In addition, the therapeutic indices (TI = IC50/EC50) were also calculated to estimate their dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2honokiolpotency (TI as = anti-HCV2.1) [13], agents.a natural The anti-HCV result showed agent that from compounds Magnolia3 –officinalis5 possessed [14],H better-tetrazolium with potencies TI values than ofsalt 6.8, (MTS) assay. In9.2, addition, andhonokiol 2.9, respectively. (TIthe = 2.1)therapeutic [13], a natural indices anti-HCV (TI agent = fromIC50/ECMagnolia50) were ofﬁcinalis also[14 ],calculated with TI values to of estimate 6.8, their potency as anti-HCV9.2, and 2.9, respectively.agents. The result showed that compounds 3–5 possessed better potencies than Table 2. Anti-HCV activities (EC50) and cytotoxicity (IC50) of 1–6. honokiol (TI = 2.1) [13], a natural anti-HCV agent from Magnolia officinalis [14], with TI values of 6.8, a b c 9.2, and 2.9, respectively. Compound EC50 (μM) IC50 (μM) (TI) 1 27.4 ± 1.4 >100 - d 2 98.4 ± 2.1 >100 - d Table 2. Anti-HCV3 activities12.8 (EC ± 3.150 ) and 87.1 cytotoxicity ± 5.1 6.8 (IC 50) of 1–6. 4 7.7 ± 2.7 70.5 ± 4.2 9.2 a b c Compound5 EC20.850 (μ ±M) 2.0 IC 60.750 ±(μ 2.0M) 2.9(TI) 1 6 27.466.7 ± ± 1.4 1.8 >100 >100 - d - d honokiol2 98.4 22.4 ± ± 2.1 0.5 48.8 >100 ± 0.8 2.1 - d a EC50: concentration that inhibi3 ts HCVcc12.8 infection ± 3.1 by 50%. 87.1 b± IC 5.150: concentration 6.8 that inhibits cell growth by 50%. c Therapeutic4 index (TI) =7.7 IC50 ±/EC 2.750 . d not calculated. 70.5 ± 4.2 9.2 5 20.8 ± 2.0 60.7 ± 2.0 2.9 6 66.7 ± 1.8 >100 - d honokiol 22.4 ± 0.5 48.8 ± 0.8 2.1

a EC50: concentration that inhibits HCVcc infection by 50%. b IC50: concentration that inhibits cell

growth by 50%. c Therapeutic index (TI) = IC50/EC50. d not calculated.

Molecules 2016, 21, 1239 5 of 7

Table 2. Anti-HCV activities (EC50) and cytotoxicity (IC50) of 1–6.

a b c Compound EC50 (µM) IC50 (µM) (TI) 1 27.4 ± 1.4 >100 - d 2 98.4 ± 2.1 >100 - d 3 12.8 ± 3.1 87.1 ± 5.1 6.8 4 7.7 ± 2.7 70.5 ± 4.2 9.2 5 20.8 ± 2.0 60.7 ± 2.0 2.9 6 66.7 ± 1.8 >100 - d honokiol 22.4 ± 0.5 48.8 ± 0.8 2.1 a b EC50: concentration that inhibits HCVcc infection by 50%. IC50: concentration that inhibits cell growth by c d 50%. Therapeutic index (TI) = IC50/EC50. not calculated.

3. Experimental Section

3.1. General Experimental Procedures Melting point and optical rotation data were recorded on a Yanako MP-500P micro melting point apparatus (Yanaco, Kyoto, Japan) and a JASCO P2000 digital polarimeter (JASCO Corporation, Tokyo, Japan), respectively. IR spectra were recorded as a thin ﬁlm on a KBr plate or using a conventional KBr pellet on a Shimadzu IR Prestige21 FT-IR spectrometer (Shimadzu, Milan, Italy). UV data were obtained using a Shimadzu UV-1700 UV/Vis spectrometer (Shimadzu). NMR spectra (400 MHz) were recorded using a Bruker Avance 400 spectrometer equipped with a 5-mm Dual z-gradient probe 1H/13C (Bruker, Rheinstetten, Germany). HR-APCI-MS and HR-ESI-MS were measured with an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientiﬁc Corp., Waltham, MA, USA). Column chromatography (CC) was performed on silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany) and RP-18 gel (230–400 mesh, SiliaBond C18, SiliCycle, Quebec City, QC, Canada). Silica gel plates (Kieselgel 60 F254, 0.25 mm, Merck) were used for thin-layer chromatography (TLC) analysis, and spots were visualized by spraying with 10% H2SO4 solution followed by heating.

3.2. Plant Material Twigs and leaves of F. virosa were collected in North Pingtung, Taiwan, in August 2013. Botanical identiﬁcation was performed by Dr. S.-Y. Hwang. A voucher specimen (chao-002) was deposited in the School of Pharmacy, China Medical University.

3.3. Extraction and Isolation The twigs and leaves of F. virosa (35 kg) were crushed and extracted exhaustively with MeOH (5 × 40 L). The organic extract was concentrated to an aqueous suspension and was further partitioned between CHCl3 and H2O. The CHCl3 extract was washed with 3% aqueous tartaric acid three times to remove alkaloids. The resulting nonalkaloid extract (420 g) was fractionated by open column chromatography on silica gel using hexane/EtOAc and EtOAc/MeOH mixtures of increasing polarity to yield 14 fractions. Fraction 9, eluted with EtOAc/MeOH (9:1), was repeatedly separated by RP-18 column chromatography with gradient elution (MeOH/H2O, 5% to 40%) to yield 2 (200.2 mg) and 6 (12.1 mg). Fraction 7 was fractionated by silica gel column chromatography using gradient elution (hexane/EtOAc, 100:0 to 53:47) to afford 20 subfractions (7A to 7T). Compounds 1 (100.2 mg), 4 (266.1 mg), 3 (19.1 mg), and 5 (80.0 mg) were obtained from fraction 7L by repeated column chromatography over silica gel with (hexane/EtOAc, 100:0 to 60:40). 3β,10α-Dihydroxy-12-methoxy-13-methyl-9(10→20)-abeo-ent-podocarpa-6,8,11,13-tetraene (1): pale yellow 25 oil; [α]D +139 (c 1.65, CHCl3); UV (MeOH) λmax (log ε) 214 (4.23), 261 (3.94) nm; IR (neat) vmax 3444, 3003, 2953, 2931, 2870, 1610, 1568, 1504, 1446, 1435, 1384, 1336, 1315, 1257, 1217, 1114, 1097, 1045, 1020, Molecules 2016, 21, 1239 6 of 7

−1 1 13 − and 981 cm ; for H- and C-NMR data in CDCl3, see Table2;( −)-APCI-MS m/z 301 [M − H] ; − (−)-HR-APCI-MS m/z 301.1805 [M − H] (calcd for C19H25O3, 301.1809). ◦ 25 (2S,3R)-4E-Dehydrochebulic acid trimethyl ester (2): colorless crystal; mp 216–218 C (MeOH), [α]D +15 (c 28.45, MeOH); UV (MeOH) λmax (log ε) 226 (4.47), 287 (3.97) nm; IR (KBr) vmax 3394, 3257, 1761, 1752, 1730, 1708, 1649 1622, 1608, 1537, 1494, 1431, 1390, 1369, 1352, 1309, 1278, 1244, 1209, 1180, 1114, 1066, and 1006 cm−1.

3.4. Crystallographic Data of 2 A colorless crystal of 2 with sizes of 0.62 × 0.50 × 0.47 mm3 was obtained at room temperature by slow evaporation in MeOH solution. Diffraction intensity data were acquired with a CCD area detector with graphite-monochromated CuKα radiation (λ = 1.54178 Å). Crystal data for 2:C17H16O11, M = 396.30, orthorhombic, a = 7.1567(2) Å, b = 9.7147(3) Å, c = 25.1934(7) Å, α = 90.00◦, β = 90.00◦, ◦ 3 −1 γ = 90.00 , V = 1751.58(9) Å , T = 150(2) K, space group P212121, Z = 4, µ(CuKα) = 1.115 mm , 6642 reflections collected, 3372 independent reflections (Rint = 0.0199). The final R1 values were 2 0.0298 (I > 2σ(I)). The final wR(F ) values were 0.0787 (I > 2σ(I)). The final R1 values were 0.0315 (all data). The final wR(F2) values were 0.0800 (all data). The goodness of fit on F2 was 1.028. Flack parameter = 0.09(14). Crystallographic data for this compound have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 1484756) (Supplementary Materials). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44(0)-1223-336033 or e-mail: [email protected]].

3.5. Infection Inhibition Assay The JC1-Luc2A HCV reporter viral particles were prepared as described previously [15,16]. In brief, in vitro-transcribed genomic JC1-Luc2A RNA was delivered into Huh7.5.1 cells by electroporation. The virus-containing supernatant was collected for 4 days, clarified by low-speed centrifugation, passed through a filter with a pore size of 0.45 µm, and quantitated by fluorescent focus assay. The MTS assay was used to determine the cell viability, while HCV infection inhibition assay was performed according to the published protocol in [6,15]. Huh7.5.1 cells were seeded into the 96-well culture plates at a cell density of 1 × 104 cells/well for 24 h followed by incubation with HCV reporter virus (with 0.1 MOI) for an additional 4 h. After removing the virus containing supernatant, the cells were washed 3 times with PBS and then replaced with medium containing indicated compound for 72 h. The luciferase activity assay was performed by the collected cell lysates. Honokiol, a known natural product with anti-HCV activity, was used as an inhibition positive control.

4. Conclusions A new 9(10→20)-abeo-ent-podocarpane (1) and five known compounds were isolated from the twigs and leaves of F. virosa. The structures of the isolates were identified on the basis of NMR, MS, and IR spectroscopic data. The structure 4E-dehydrochebulic acid trimethyl ester was revised and its absolute configuration was determined for the first time. Comparing the present study with our prior investigation [4–7], it was found that the diversity in chemical constitutions of the roots were quite different from those of the twigs and leaves of F. virosa. Furthermore, 3β,12-dihydroxy-13-methylpodocarpa-6,8,11,13-tetraene (4) was found to be abundant in the twigs and leaves; however, the 12-O-methyl analogue of 4 was isolated as the main constituent in the roots [4]. Although the 9(10→20)-abeo-ent-podocarpane skeleton had been reported from the roots of this plant [6], it was still rare in a natural source. The present anti-HCV evaluation suggested that the ent-podocarpane derivatives might be the active ingredients in this plant.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 9/1239/s1. Molecules 2016, 21, 1239 7 of 7

Acknowledgments: The authors are grateful for financial support from the Ministry of Science and Technology (MOST102-2113-M-039-001, MOST103-2320-B-039-004-MY3, and MOST104-2911-I-002-302). This study was also supported in part from the grant of Chinese Medicine Research Center, China Medical University (the Ministry of Education, the Aim for the Top University Plan). Author Contributions: Chih-Hua Chao and Yang-Chang Wu conceived and designed the experiments; Ying-Ju Lin, Yung-Ju Yeh, and Ju-Chien Cheng performed the biological assay; Hui-Chi Huang and Tian-Shung Wu analyzed the data; Syh-Yuan Hwang contributed to the collection and species identification. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1 and 2 are available from the authors.

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