Article Henrin A: A New Anti-HIV Ent-Kaurane Diterpene from henryi

Wan-Fei Li 1,†, Juan Wang 2,3,†, Jing-Jie Zhang 1, Xun Song 2, Chuen-Fai Ku 2, Juan Zou 1, Ji-Xin Li 1, Li-Jun Rong 4, Lu-Tai Pan 1,* and Hong-Jie Zhang 2,*

Received: 17 September 2015; Accepted: 13 November 2015; Published: 24 November 2015 Academic Editor: Ge Zhang

1 Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China; [email protected] (W.-F.L.); [email protected] (J.-J.Z.); [email protected] (J.Z.); [email protected] (J.-X.L.) 2 School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Kowloon, Hong Kong, China; [email protected] (J.W.); [email protected] (X.S.); [email protected] (C.-F.K.) 3 School of Public Health, Jilin University, Changchun 130021, China 4 Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, USA; [email protected] * Correspondences: [email protected] (L.-T.P.); [email protected] (H.-J.Z.); Tel./Fax: +86-852-8823-3016 (L.-T.P.); Tel.: +852-3411-2956 (H.-J.Z.); Fax: +852-3411-2461 (H.-J.Z.) † These authors contributed equally to this work.

Abstract: Henrin A (1), a new ent-kaurane diterpene, was isolated from the leaves of Pteris henryi. The chemical structure was elucidated by analysis of the spectroscopic data including one-dimensional (1D) and two-dimensional (2D) NMR spectra, and was further confirmed by X-ray crystallographic analysis. The compound was evaluated for its biological activities against a panel of cancer cell lines, dental bacterial biofilm formation, and HIV. It displayed anti-HIV potential with an IC50 value of 9.1 µM (SI = 12.2).

Keywords: Pteris henryi; ent-kaurane diterpene; henrin A; bioactivity evaluation; anti-HIV activity

1. Introduction Ent-kaurane compounds are members of a class of diterpenes with a four-membered ring system, which are richly found in the Isodon genus (Lamiaceae) [1–3]. They have been known for having a variety of biological activities, including anticancer and antibacterial activities [4–6]. Tetracyclic ent-kauranes have also been found in the of the genus Pteris ( family), and some of them have demonstrated biological activities [7–13]. Phytochemical and biological investigation of the plants in the genus Pteris may produce potentially novel bioactive diterpenes [14,15]. The genus Pteris comprises more than 300 species, 66 of which are distributed in China [16]. Few phytochemical studies have been reported for the chemical constituents of the plants in this genus [17]. Our present study focused on the species P. henryi Chirst, a perennial herb that has been used as an herbal medicine for the treatment of burns and scalds, lyssodexis, traumatic hemorrhages, leucorrhea, and difficulty and pain in micturition [18]. The plant is mainly distributed in the Guizhou and Yunnan provinces, People’s Republic of China [19]. In this study, we report the isolation, structural determination, and biological activity evaluation of henrin A (1), a new ent-kaurane diterpene (Figure1).

Int. J. Mol. Sci. 2015, 16, 27978–27987; doi:10.3390/ijms161126071 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2015, 16, 27978–27987 Int. J. Mol. Sci. 2015, 16, page–page

OH 12 OH 20 11 13 HO 9 14 16 1 17 2 10 H 8 3 5 7 15 4 6 H 18 19 Figure 1. Structure of compound henrin A (1).

2. Results Results and and Discussion Discussion

2.1. Compound IdentificationIdentification The dried leaves of P. henryi were extracted with MeOH to affordafford a MeOH extract, which was separated through silica gelgel chromatographychromatography toto yieldyield henrinhenrin AA ((11).). Henrin A (1) was obtained as colorless crystals with UV (MeOH) λmax (log ε) at 204 (1.60) nm (Figure S1). In the IR spectrum,Table 1. 1H and1 showed 13C NMR absorption data of compound of hydroxyl 1 a. groups (3386, 1099, and 1049 cm´1) (Figure S2). Its molecular formula was determined to be C H O by means of analyzing No. δH (mult, J in Hz) b δC (mult) c No. δH (mult,20 J in34 Hz)3 b δC (mult) c its NMR1α spectroscopic 2.15 (brddd, data 12.0, (Table 3.8,1 1.9)), and further 50.3 t verified 11α by the 1.65 HR-EIMS (overlap) data with 20.7m/z t 345.2405 + [M + Na]1β (calcd 345.2400)0.67 (brt, (Figure11.7) S3). The - molecule11β of 1 has1.82 four(overlap) double-bond equivalences. - 1 However,2α no carbonyl 3.87 (brtt, absorption 11.5, 4.3) was observed 65.4 d in the 12 IRα spectrum. 1.68 (overlap) As evidenced from 34.6 thet H and 13 C NMR3α spectral 1.72 data(brddd, (Table 12.5,1) (Figures4.3, 1.9) S4–S6) 51.9 as t well as12β the HMQC1.64 (overlap) correlation data (Figure - S7), the δ 20 carbons3β of compound 1.07 (brt,1 were12.0) characterized - as four 13 methyl groups - ( H 0.87, 0.93,1.09, 77.4 s and 1.18 δ δ (each 3H,4 s); C 19.4 (q), 21.3- (q), 22.8 (q), and35.7 34.2s (q)),14α an oxy-methine 1.66 (overlap) group ( H 44.03.87 t (1H, brtt, δ δ J = 11.5,5 4.3β Hz); 0.82C 65.4 (brd, (d)), 11.5) two oxy-tertiary 57.0 d carbons 14β ( C 77.41.83 (s) (d, and 11.9) 81.1 (s)), eight - methylene δ carbons,6 twoα non-oxygenated 1.34 (brqd, 12.1, methine 1.9) carbons 21.2 (t H 0.82 15α (1H, 1.50 brd, (dd,J = 14.5, 11.5 1.3) Hz), and 56.7 0.96 t (1H, brd, δ J = 7.2 Hz);6β C 57.01.59 (d) (brd, and 57.112.3) (d)), and three - quaternary15β carbons.1.63 (d, No 14.7) signals were - observed in δ 1 δ 13 the range7α of H 5–7 1.60 ppm (overlap) of the H NMR spectrum 43.2 t and 16 in the range - of C 90–160ppm 81.1 sof the C NMR spectrum,7β 1.44 indicating (brtd, 11.9, that 3.4) there is no carbon-carbon - 17 double-bond 1.18 (s) in 1. The 21.3 lack q of olefinic signals in8 the molecule and - the calculation of 42.2 four s double-bond 18 equivalences 0.93 (s) determined 34.2 q that 1 has a tetracyclic-ring9β system. 0.96 (brd, Compound 7.2) 1 was 57.1 thus d suggested 19 to have 0.87 a saturated (s) tetracyclic 22.8 q diterpene having an10ent -kaurane skeleton. - 41.9 s 20 1.09 (s) 19.4 q Through analysis of the long-range correlation data observed in the HMBC (heteronuclear a Data were recorded in CD3OD; δ values are given in ppm with reference to the signal of CD3OD multiple-bond correlation spectroscopy) spectrum (Figure S8), together with the HMQC (δ 3.31 ppm) for 1H and to the center peak of the signal of CD3OD (δ 49.0 ppm) for 13C; b Multiplicities 1 1 (heteronuclearin parentheses multiple-quantum represent: s (singlet correlation), brs (broad spectroscopy) singlet), dd (doublet (Figure of S7) doublet), and H– brddH (broad COSY doublet (correlated spectroscopy)of doublet), (Figure brtd (broad S9) datatriplet (Figure of doublet),2), the brqd three (broad oxy-carbon quartet of groupsdoublet), (one ddd oxy-methine(doublet of doublet and two oxy-tertiaryof doublet), carbons) brddd (broad in 1 could doublet be of assigned doublet of accordingly. doublet), t (triplet), Starting brt (broad from triplet), the singlet and brtt signals (broad of the 13 methyltriplet protons of triplet); at C-18 c Multiplicities and C-19 represent: (δH 0.93 s (H (quaternary3-18), 0.87 carbon), (H3-19)), d (CH), the t (CHC NMR2), and atq (CHδC 351.9). (t) was assigned to C-3 due to the presence of its HMBC correlations to the two methyl protons, which in 1 1 turnHenrin suggested A ( the1) was oxy-methine obtained as group colorless at C-2 crystals due to with the presenceUV (MeOH) of the λmaxH– (logH ε COSY) at 204 correlations (1.60) nm between(Figure S1). H-2 (InδH the3.87) IR and spectrum, H2-3 (δH 11.07 showed and1.72). absorption The presence of hydrox of theyl HMBCgroups correlations(3386, 1099, of and the singlet1049 cm signals−1) (Figure of the S2). methyl Its molecular protons atformula C-17 (δ wasH 1.18) determined to both oxy-tertiary to be C20H34 carbonsO3 by means at δC 77.4of analyzing and 81.1 suggestedits NMR spectroscopic that both C-13 data and (Table C-16 were1), and subsitituted further verified with a hydroxyby the HR-EIMS group, respectively. data with m/z 345.2405 [M +In Na] the+ ROESY(calcd 345.2400) (rotating frame(Figure nuclear S3). The Overhauser molecule effectof 1 spectroscopy)has four double-bond spectrum equivalences. (Figure S10), theHowever, presence no ofcarbonyl the ROE absorption (rotating was frame observed Overhauser in the effect) IR spectrum. correlations As evidenced (Figure3) offrom H-2 the ( δ H1H3.87) and with13C NMR H2-20 spectral suggested data the(Table hydroxy 1) (Figures group S4–S6) of C-2 as to well be β as-oriented. the HMQC In addition,correlation the data presence (Figure of S7), the ROEthe 20 correlations carbons of ofcompound H-5 (δH 0.82)1 were to characterized H3-18 and the as lack four of methyl ROE cross-peaks groups (δH of 0.87, H-5 0.93,1.09, to H-2 and and H 31.18-19 suggested(each 3H, s); H-5 δC to19.4 be (q),β-oriented. 21.3 (q), 22.8 Furthermore, (q), and 34.2 the (q)),β-orientation an oxy-methine of H-9 group (δH 0.96) (δH 3.87 was (1H, deduced brtt, onJ = the11.5, basis 4.3 Hz); of the δC observation65.4 (d)), two of oxy-tertiary the ROE correlation carbons (δ ofC 77.4 H-9 with(s) and H-5 81.1 and (s)), the eight lack methylene of ROE correlation carbons, betweentwo non-oxygenated H-9 and H3-20. methine carbons (δH 0.82 (1H, brd, J = 11.5 Hz), and 0.96 (1H, brd, J = 7.2 Hz); δC 57.0 (d) and 57.1 (d)), and three quaternary carbons. No signals were observed in the range of δH 5–7 ppm of the 1H NMR spectrum and in the range of δC 90–160 ppm of the 13C NMR spectrum, indicating that there is no carbon-carbon double-bond in 1. The lack of olefinic signals in the 27979 2 Int.Int. J.J. Mol.Mol. Sci.Sci. 20152015,, 1616,, page–pagepage–page

Int. J. Mol. Sci. 2015 16 moleculemolecule andand ,thethe, 27978–27987 calculationcalculation ofof fourfour dodouble-bonduble-bond equivalencesequivalences determineddetermined thatthat 11 hashas aa tetracyclic-ringtetracyclic-ring system.system. CompoundCompound 11 waswas thusthus suggestedsuggested toto havehave aa saturatedsaturated tetracyclictetracyclic diterpenediterpene havinghaving anan entent-kaurane-kaurane skeleton.skeleton.Table 1. 1H and 13C NMR data of compound 1 a. ThroughThrough analysisanalysis ofof thethe long-rangelong-range correlationcorrelation datadata observedobserved inin thethe HMBCHMBC (heteronuclear(heteronuclear multiple-bond correlation spectroscopy)b spectrumc (Figure S8), togetherb with the cHMQC multiple-bondNo. correlationδH (mult, J inspectroscopy) Hz) δC (mult)spectrum No.(FigureδH (mult,S8), togetherJ in Hz) withδC (mult)the HMQC 1 1 (heteronuclear multiple-quantum correlation spectroscopy) (Figure S7) and 1 H–1 H COSY (correlated (heteronuclear1α 2.15multiple-quantum (brddd, 12.0, 3.8, 1.9)correlation 50.3 spectroscopy) t 11α (Figure1.65 S7) (overlap) and H– H COSY 20.7 (correlated t Int. J. Mol. Sci. 2015, 16, page–page spectroscopy)Int.spectroscopy)Int. J.J. Mol.Mol.1β Sci.Sci. 20152015 (Figure(Figure,, 1616,, page–pagepage–page0.67 S9)S9) (brt, datadata 11.7) (Figure(Figure 2),2), thethe - ththreeree oxy-carbonoxy-carbon 11β 1.82groupsgroups (overlap) (one(one oxy-methineoxy-methine - andand twotwo oxy-tertiaryoxy-tertiary2α carbons)carbons)3.87 (brtt,inin 11 11.5,couldcould 4.3) bebe assignedassigned 65.4 accordingly.accordingly. d 12α StartiStarti1.68ngng fromfrom (overlap) thethe singletsinglet signals 34.6signals t ofof thethe molecule and the calculation of four double-bond equivalences determined that 1 has a moleculemolecule3α andand1.72 thethe (brddd, calculationcalculation 12.5, 4.3, of 1.9)of fourHfour dodo 51.9uble-bonduble-bond3 t 12 βequivalencesequivalences3 1.64 (overlap) 13determineddetermined thatthatC - 11 hashas aa methylmethyl protonsprotons atat C-18C-18 andand C-19C-19 ((δδH 0.930.93 (H(H3-18),-18), 0.870.87 (H(H3-19)),-19)), thethe 13CC NMRNMR atat δδC 51.951.9 (t)(t) waswas tetracyclic-ringtetracyclic-ringassignedtetracyclic-ring3β to C-3 system. system.system.due1.07 to (brt,the CompoundCompoundCompound presence 12.0) 1 1of waswaswas its HMBCthusthusthus - suggestedsuggestedsuggested correlations 13 tototo havehavehave to the aaa saturatedsaturatedsaturatedtwo - methyl tetracyclictetracyclictetracyclic protons, 77.4 sditerpene diterpenediterpenewhich in assigned4 to C-3 due to the - presence of its HMBC 35.7 s correlations 14α to1.66 the (overlap)two methyl protons, 44.0 twhich in havinghaving anan entent-kaurane-kaurane-kaurane skeleton.skeleton.skeleton. 1 1 turnturn suggestedsuggested5β thethe 0.82oxy-methineoxy-methine (brd, 11.5) groupgroup atat C-2C-2 57.0 duedue d toto thethe 14β presencepresence1.83 ofof (d, thethe 11.9) 1H–H–1HH COSYCOSY -correlationscorrelations ThroughThrough analysisanalysisH ofof thethe 2 long-rangelong-rangeH correlationcorrelation datadatadata observedobservedobserved ininin thethethe HMBCHMBCHMBC (heteronuclear(heteronuclear(heteronuclear betweenbetween6α H-2H-2 ((δδH 1.34 3.87)3.87) (brqd, andand 12.1, HH2-3-3 1.9) ((δδH 1.071.07 andand 21.2 1.72).1.72). t TheThe 15 αpresencepresence1.50 ofof (dd, thethe 14.5, HMBCHMBC 1.3) correlationscorrelations 56.7 t ofof thethe multiple-bond correlation spectroscopy) spectrum (Figure S8), together with the HMQC multiple-bondmultiple-bond6β correlationcorrelation1.59 (brd, 12.3)spectroscopy)spectroscopy) spectrumspectrum -H 15(Figure(Figureβ 1.63S8),S8), (d, togethertogether 14.7) withwithC thethe - HMQCHMQC singletsinglet signalssignals ofof thethe methylmethyl protonsprotons atat C-17C-17 ((δδH 1.18)1.18) toto bothboth oxy-tertiaryoxy-tertiary carbonscarbons atat δδC 77.477.4 andand 81.181.1 (heteronuclear7α multiple-quantum1.60 (overlap) correlation 43.2 spectroscopy) t 16 (Figure S7) - and 111H–111H COSY 81.1 (correlated s suggested(heteronuclearsuggested(heteronuclear thatthat bothmultiple-quantumbothmultiple-quantum C-13C-13 andand C-16C-16 correlation correlationwerewere subsititutedsubsitituted spectroscopy)spectroscopy) withwith aa hydroxy hydroxy(Figure(Figure S7) S7) group,group, andand respectively.respectively.H–H– HH COSYCOSY (correlated(correlated spectroscopy)spectroscopy)7β (Figure(Figure1.44 (brtd, S9)S9) 11.9,datadata 3.4) (Figure(Figure 2),2), thethe - ththreeree oxy-carbonoxy-carbon 17 groupsgroups 1.18 (s) (one(one oxy-methineoxy-methine 21.3 q andand twotwo 8 - 42.2 s 18 0.93 (s) 34.2 q oxy-tertiaryoxy-tertiary carbons)carbons) inin 11 couldcould bebe assignedassigned accordingly.accordingly. StartiStartingng fromfrom thethe singletsinglet signalssignals ofof thethe oxy-tertiary9β carbons)0.96 in (brd, 1 could 7.2) be assigned 57.1 accordingly. d 19 StartiOHOH ng 0.87from (s) the singlet 22.8signals q of the methyl protons at C-18 and C-19 (δH 0.93 (H3-18), 0.87 (H3-19)), the 131313C NMR at δC 51.9 (t) was methylmethyl 10 protonsprotons atat C-18C-18 and -and C-19C-19 ((δδHH 0.930.93 41.9(H(H33-18), s-18), 0.870.87 20 (H(H33-19)),-19)),OHOH the 1.09the (s)CC NMRNMR atat δδ 19.4CC 51.951.9 q (t)(t) waswas assignedassigneda toto C-3C-3 duedue toto thethe presencepresence ofof itsits HMBCHMBC correlationscorrelations toto thethe twotwo methylmethyl protons,protons, whichwhich inin Data were recorded in CD3OD; δ values are given in ppm with reference to the signal of CD3OD (δ 3.31 ppm) 1 HO 13 b 111 111 turnturnturn suggestedsuggestedsuggestedfor H and the tothethe the oxy-methineoxy-methineoxy-methine center peakHO of groupgroupgroup the signal atat ofC-2C-2 CD due3dueOD (totoδ 49.0 thethe ppm) presencepresence for C; ofof theMultiplicitiesthe H–H–HH COSYCOSY in parentheses correlationscorrelations represent: s (singlet), brs (broad singlet), dd (doublet of doublet), brdd (broad doublet of doublet), brtd (broad betweenbetween H-2H-2 ((δδHH 3.87)3.87)3.87) andandand HHH222-3-3-3 (((δδHH 1.071.071.07 andandand 1.72).1.72).1.72).H TheTheThe presencepresencepresence ofofof thethethe HMBCHMBCHMBC correlationscorrelationscorrelations ofofof thethethe triplet of doublet), brqd (broad quartet of doublet), dddH (doublet of doublet of doublet), brddd (broad doublet singletsingletof signalssignals doublet of ofof doublet), thethe methylmethyl t (triplet), protonsprotons brt (broad atat C-17C-17 triplet), ((δδHH and 1.18)1.18)1.18) brtt tototo (broad bothbothboth triplet oxy-tertiaryoxy-tertiaryoxy-tertiary of triplet); carbonsccarbonscarbonsMultiplicities atatat δδC represent:C 77.477.477.4 andandand 81.181.181.1 suggestedsuggesteds (quaternary thatthat bothboth carbon), C-13C-13 d andand (CH), C-16C-16 t (CH were2were), and subsititutedsubsitituted q (CH3). withwith aa hydroxyhydroxy group,group, respectively.respectively. HH OHOH

OHOH FigureFigure 2.2. KeyKey COSYCOSY (marked(marked asas blueblue boldbold bondsbonds (H(H H)) H)) andand HMBCHMBC (the(the redred arrowsarrows (H(H C)) C)) correlationscorrelations forfor compoundcompound 11HO.HO. HH InIn thethe ROESYROESY (rotating(rotating frameframe nuclearnuclear OverhauserOverhauser effecteffect spectroscopy)spectroscopy) spectrumspectrum (Figure(Figure S10),S10), the presence of the ROE (rotating frame Overhauser effect) correlations (Figure 3) of H-2 (δH 3.87) the presence of the ROE (rotating frame OverhausH er effect) correlations (Figure 3) of H-2 (δH 3.87) 2 HH withwith HH2-20-20 suggestedsuggested thethe hydroxyhydroxy groupgroup ofof C-2C-2 toto bebe ββ-oriented.-oriented. InIn addition,addition, thethe presencepresence ofof thethe H 3 3 ROEROE correlationscorrelations ofof H-5H-5 ((δδH 0.82)0.82) toto HH3-18-18 andand thethe lacklack ofof ROEROE cross-peakscross-peaks ofof H-5H-5 toto H-2H-2 andand HH3-19-19

H suggestedsuggested H-5H-5 toto bebe ββ-oriented.-oriented. Furthermore,Furthermore, thethe ββ-orientation-orientation ofof H-9H-9 ((δδH 0.96)0.96) waswas deduceddeduced onon thethe basisbasisFigure Figureofof thethe 2. 2. observationobservation KeyKeyKey COSYCOSYCOSY (marked(marked (marked ofof thethe ROEasROEasas blueblueblue correlationcorrelation boldboldbold bondsbondsbonds of of (H(H(H H-9H-9 H)) H)) H))withwith andand and H-5H-5 HMBCHMBC HMBC andand (the (the (thethethe redred redlacklack arrowsarrows arrows ofof ROEROE (H(H (H correlationcorrelation C)) C)) C)) correlations for compound3 1. betweenbetweencorrelationscorrelations H-9H-9 andand forfor HH compoundcompound3-20.-20. 11..

InInIn thethethe ROESYROESYROESY (rotating(rotating(rotating frameframeframe nuclearnuclearnuclear OverhauserOverhauserOverhauser effecteffecteffect spectroscopy)spectroscopy)spectroscopy) spectrumspectrumspectrum (Figure(Figure(Figure S10),S10),S10), thethethe presencepresencepresence ofofof thethethe ROEROEROE (rotating(rotating(rotating frameframeframe OverhausOverhausOverhauserer effect)effect) correlationscorrelations (Figure(Figure 3)3) ofof H-2H-2 ((δδHH 3.87)3.87)3.87) withwith HH222-20-20-20 suggestedsuggestedsuggested thethethe hydroxyhydroxyhydroxy groupgroupgroup ofofof C-2C-2C-2 tototo bebebe ββ-oriented.-oriented.-oriented. InInIn addition,addition,addition, thethethe presencepresencepresence ofofof thethethe ROEROE correlationscorrelations ofof H-5H-5 ((δδHH 0.82)0.82)0.82) tototo HHH333-18-18-18 andandand thethethe lacklacklack ofofof ROEROEROE cross-peakscross-peakscross-peaks ofofof H-5H-5H-5 tototo H-2H-2H-2 andandand HHH333-19-19-19 suggestedsuggested H-5H-5 toto bebe ββ-oriented.-oriented.-oriented. Furthermore,Furthermore,Furthermore, thethethe ββ-orientation-orientation-orientation ofofof H-9H-9H-9 (((δδHH 0.96)0.96)0.96) waswaswas deduceddeduceddeduced ononon thethethe basisbasis ofof thethe observationobservation ofof thethe ROEROE correlationcorrelation ofof H-9H-9H-9 withwithwith H-5H-5H-5 andandand thethethe lacklacklack ofofof ROEROEROE correlationcorrelationcorrelation betweenbetween H-9H-9 andand HH333-20.-20.-20.

FigureFigure 3.3. KeyKey ROESYROESY correlationscorrelations (indic(indic (indicatedatedated asas magenta magenta arrows arrows (H (H H))H)) for for compound compound 11..

To further confirm the chemical structure, compound 1 was crystallized in MeOH to afford a To further confirm confirm the the chemical chemical structure, structure, compound compound 11 waswas crystallized crystallized in in MeOH MeOH to toafford afford a 1 1 1 colorlesscolorless crystalcrystal withwith thethe monoclinicmonoclinic spacespace groupgroup ofof P2P21221221,, whichwhich waswas analyzedanalyzed byby X-rayX-ray a colorless crystal with the monoclinic space group of P212121, which was analyzed by X-ray 33 27980

FigureFigure 3.3. KeyKeyKey ROESYROESYROESY correlationscorrelationscorrelations (indic(indic(indicatedated asas magentamagenta arrowsarrows (H(H H))H)) forfor compoundcompound 11...

ToTo furtherfurther confirmconfirm thethe chemicalchemical structure,structure, compoundcompound 11 waswaswas crystallizedcrystallizedcrystallized ininin MeOHMeOHMeOH tototo affordaffordafford aaa colorlesscolorless crystalcrystal withwith thethe monoclinicmonoclinic spacespace groupgroup ofof P2P21112211122111,,, whichwhichwhich waswaswas analyzedanalyzedanalyzed bybyby X-rayX-rayX-ray 33 Int. J. Mol. Sci. 2015, 16, 27978–27987

Int. J. Mol. Sci. 2015, 16, page–page crystallography.crystallography. Through Through structural structural refinementrefinement [20,21], [20,21 the], the chemical chemical structure structure of 1 was of confirmed1 was confirmed as as shownshownin in Figure 4.4. Like Like the the IsodonIsodon plants,plants, the diterpenes the diterpenes produced produced by the by in the genus ferns Pteris in the are genus Pterisallare ent all-kauraneent-kaurane diterpenes diterpenes [7–13]. [ 7Compound–13]. Compound 1 is also1 is an also ent an-kauraneent-kaurane diterpene diterpene due to due the to the 1111 ˝ observationobservation of the of the negative negative optical optical rotation rotation (([rααs]DD ´−5.66°)5.66 as) as well well as asthe the consideration consideration of the of similar the similar biogeneticbiogenetic pathways pathways used used by by the thePteris Pteris plantsplants for producing producing the the same same class class of congeners of congeners [22–24]. [22 –24]. Accordingly,Accordingly,1 was 1 was determined determined to to be beent ent-2-2ββ,, l3l3α, 16 16αα-trihydroxy-kaurane,-trihydroxy-kaurane, and and given given the trivial the trivial name name of henrinof henrin A. A.

C12 C11 C20 C13 C17

C1 C14 C9 C16 C2 C10 C8 C15 C18 C5 C3 C7 C4 C6

C19

Figure 4. 4. ORTEPORTEP (oak (oak ridge ridge thermal thermal ellipsoid ellipsoid plot program) plot program) drawing drawingof compound ofcompound 1 (Blue ball: 1carbon;(Blue grey ball: ball: hydrogen; red ball: oxygen). carbon; grey ball: hydrogen; red ball: oxygen). 2.2. Biological Activity 2.2. Biological Activity Henrin A (1) was evaluated for its cytotoxic activity against a panel of human cancer cell lines comprisingHenrin A (KB1) was(cervical), evaluated HCT116 for (colon), its cytotoxic A549 (l activityung), and against MCF-7 a(breast) panel cell of humanlines. No cancer inhibitory cell lines comprisingactivity KBagainst (cervical), these cell HCT116 lines was (colon), observed A549 for (lung), 1 at a andconcetration MCF-7 of (breast) 20 µg/mL. cell lines.Due to No the inhibitory low activitycytotoxicity against of these the compound, cell lines was1 was observed further evaluated for 1 at for a concetrationits antimicrobial of potential. 20 µg/mL. Due to the low cytotoxicityThe ofdental the compound, biofilm formation1 was furtherinhibitory evaluated as well foras the its antimicrobialantifungal activity potential. assays (Table 2) determinedThe dental that biofilm 1 had formation no antimicrobial inhibitory activity as wellagainst as the the two antifungal dental pathogens activity Streptococcus assays (Table 2) mutans and S. sobrinus at a concetration of 20 µg/mL. Compound 1 was also evaluated for its determined that 1 had no antimicrobial activity against the two dental pathogens Streptococcus mutans antifungal activity against the athelete’s foot fungus Trichophyton rubrum. No antifungal inhibitory µ and S.activity sobrinus was atobserved a concetration for this compound of 20 g/mL. at a concentration Compound of 120was µg/mL also (Table evaluated 2). for its antifungal activity against the athelete’s foot fungus Trichophyton rubrum. No antifungal inhibitory activity was observedTable for this 2. Antimicrobial compound atactivity a concentration of henrin A of(1) 20againstµg/mL biofilm (Table of 2two). dental bacteria and the athelete’s foot fungus. Table 2. Antimicrobial activity of henrin A (1) against biofilm of two dental bacteria and the athelete’s Growth Inhibition Rate (%) foot fungus. Compounds Bacterial Biofilm Formation Fungus S. mutans S. sobrinus T. rubrum Growth Inhibition Rate (%) Henrin A (1) a 15.55 ± 5.66 11.20 ± 2.23 9.73 ± 8.75 Compounds Penicillin G b 80.44Bacterial ± 3.14 Biofilm Formation 76.82 ± 3.93 Fungus - Chlorhexidine c 77.26S. mutans ± 6.30 S. 71.49sobrinus ± 5.21 T. rubrum 83.12 ± 0.47 Miconazole a - - 80.69 ± 0.10 Henrin A (1) a 15.55 ˘ 5.66 11.20 ˘ 2.23 9.73 ˘ 8.75 a Compound assay concentration at 20 µg/mL; b Compound assay concentration at 10 µg/mL; Penicillin G b 80.44 ˘ 3.14 76.82 ˘ 3.93 - c Compound assayChlorhexidine concentrationc at 77.2612 µg/mL.˘ 6.30 71.49 ˘ 5.21 83.12 ˘ 0.47 Miconazole a - - 80.69 ˘ 0.10 4 a Compound assay concentration at 20 µg/mL; b Compound assay concentration at 10 µg/mL; c Compound assay concentration at 12 µg/mL.

27981 Int. J. Mol. Sci. 2015, 16, 27978–27987 Int. J. Mol. Sci. 2015, 16, page–page

HenrinHenrin A A (1 )(1 was) was then then evaluated evaluated for for its its anti-HIV anti-HIV activity activity using using our our previously previously established established “One-Stone-Two-Birds”“One-Stone-Two-Birds” assay assay evaluation evaluation system system [25 ].[25]. This This protocol protocol allows allows us us to to identify identify potential potential inhibitorsinhibitors for for HIV HIV replication replication (post-entry (post-entry steps). steps). This This protocol protocol is is an an easy, easy, safe, safe, and and efficient efficient HIV HIV vector-basedvector-based assay assay system system to to evaluate evaluate and and identify identify potential potential inhibitors inhibitors against against HIV HIV replication replication (Figure(Figure5 and 5 and Table Table3). Henrin3). Henrin A ( A1 )(1 was) was found found to to exhibit exhibit anti-HIV anti-HIV activity activity with with an an IC 50ICvalue50 value of of 9.19.1µM µM with with selective selective index index of 12.2.of 12.2.

A 80 B 100 60 80

40 60 40 20 Cell viability (%)

Inhibition rate (%)Inhibition 20

0 0 110 110 Log (μM) Log (μM)

FigureFigure 5. 5. InhibitionInhibition of of HIV/VSV-G HIV/VSV-G by bycompound compound 1. (A1.() TheA )inhibitory The inhibitory effect of effect 1 on HIV/VSV-G of 1 on HIV/VSV-Ginfectivity infectivitywas investigated, was investigated, and it displayed and it displayed dose-dependent dose-dependent inhibitory inhibitory activities; activities; (B) The (B)cytotoxicity The cytotoxicity of 1 was of 1testedwas on tested A549 on target A549 cells. target Three cells. independent Three independent experiments experiments were performed were to performeddetermine to determinethe effect of the the effect compound. of the compound.

TableTable 3. Anti-HIV 3. Anti-HIV activity activity of compoundof compound1. 1.

Compound IC50 (µM) CC50 (µM) SI Compound IC50 (µM) CC50 (µM) SI Henrin A 9.1 110.5 12.2 Henrin A 9.1 110.5 12.2 AZTAZT (zidovudine) (zidovudine) 0.03 0.03 >100 >100 >3333 >3333

Many kaurane compounds have been found to possess cytotoxic activity due to the presence of Many kaurane compounds have been found to possess cytotoxic activity due to the presence of an α, β-unsaturated ketone group in their structures [26]. However, for those without this structural an α, β-unsaturated ketone group in their structures [26]. However, for those without this structural feature, low or no cytotoxicity activity is expected [27–30], and these compounds thus may be feature, low or no cytotoxicity activity is expected [27–30], and these compounds thus may be further further explored for their antimicrobial potential. Henrin A (1) is an ent-kaurane diterpene belonging explored for their antimicrobial potential. Henrin A (1) is an ent-kaurane diterpene belonging to this to this category, and our assay determined that this compound was not toxic against a panel of category, and our assay determined that this compound was not toxic against a panel of human human cell lines but indeed displayed anti-HIV activity. More than 1000 different types of cell lines but indeed displayed anti-HIV activity. More than 1000 different types of ent-kaurane ent-kaurane compounds have been discovered from plants [30]. These diterpenes can be a valuable compounds have been discovered from plants [30]. These diterpenes can be a valuable source source for the discovery of antiviral agents by testing the natural or modified compounds without an for the discovery of antiviral agents by testing the natural or modified compounds without an α, α, β-unsaturated ketone group. β-unsaturated ketone group. 3. Experimental Section 3. Experimental Section

3.1.3.1. General General Experimental Experimental Procedures Procedures OpticalOptical rotation rotation was was measured measured with with a Rudolph a Rudolph digital digital polarimeter. polarimeter. IR IR spectrum spectrum was was recorded recorded onon a VECTOR22a VECTOR22 spectrophotometer spectrophotometer (Bruker, (Bruker, Rheinstetten, Rheinstetten, Germany) Germany) with with KBr KBr pellets. pellets. 1D 1D (one (one dimentiona)dimentiona) and and 2D (two2D dimentional)(two dimentional) NMR spectra NMR werespectra recorded were on recorded a JEOL 500MHz on a spectrometerJEOL 500MHz (JEOLspectrometer Ltd., Tokyo, (JEOL Japan). Ltd., Unless Tokyo, otherwise Japan). specified,Unless otherwise chemical specified, shifts (δ) werechemical expressed shifts in(δ ppm) were withexpressed reference in toppm the with solvent reference signals. to the High-resolution solvent signals. mass High-resolution spectrum (HR-EIMS) mass spectrum was performed (HR-EIMS) onwas a VG performed Autospec-3000 on a VG spectrometer Autospec-3000 (VG, Manchester,spectrometer UK) (VG, under Manchester, 70 eV. Column UK) under chromatography 70 eV. Column waschromatography performed with was silica performed gel (200–300 with mesh; silica Qingdaogel (200–300 Marine mesh; Chemical, Qingdao Inc., Marine Qingdao, Chemical, China). Inc., FractionsQingdao, were China). monitored Fractions by TLC were and monitored spots were by visualized TLC and spots by heating were silicavisualized gel plates by heating sprayed silica with gel plates sprayed with 10% H2SO4 in EtOH. All solvents including˝ petroleum ether (60–90 °C) were 10% H2SO4 in EtOH. All solvents including petroleum ether (60–90 C) were distilled prior to use. distilled prior to use. 27982 5 Int. J. Mol. Sci. 2015, 16, 27978–27987

3.2. Plant Material The plant materials of the leaves of P. henryi Chirst were collected in Anshun, Guizhou Province, China, in September 2010. The voucher specimen was identified by Professor Junhua Zhao of the Guiyang College of Traditional Chinese Medicine, and deposited at Guiyang College of Traditional Chinese Medicine, with the number of the voucher specimen as No. 20101003.

3.3. Extraction and Isolation The air-dried powder of the leaves of P. henryi Chirst (5 kg) was percolated with 95% MeOH at room temperature (3 ˆ 5 L), and the MeOH crude extract (266 g) was subjected to silica gel chromatography separation (10 cm ˆ 100 cm), eluting with a gradient solvent system of CHCl3/MeOH (from 10/1 to 0/1, v/v) to afford six fractions (A–F). Fraction C was separated over an additional chromatographic column of silica gel (30 mm ˆ 300 mm), eluting with a gradient solvent system of CHCl3/MeOH (from 5/1 to 1/1, v/v) to afford fraction G, which was further separated by a Sephadex LH-20 column (300 mm ˆ 1000 mm), eluding with the solvent CHCl3/MeOH (1/1, v/v) to afford herin A (30 mg). ˝ 11 ˝ Henrin A (1): Colorless crystals (MeOH); mp 246–248 C; rαsD ´ 5.66 (c 2.12, MeOH); UV (MeOH) λmax (log ε) 204 (1.60) nm; IR (KBr) νmax 3386, 2940, 2865, 1447, 1468, 1334, 1146, 1099, 1049, 601 cm´1; 1H and 13C NMR, see Table1; HR-EIMS ([M] + m/z 345.2405 [M + Na]+ (calcd. 345.2400 for C20H34O3Na)).

3.4. X-ray Data of Henrin A (1) CCDC 1039988 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre (Available online: www.ccdc.cam.ac.uk/data_request/cif). Crystal data of 1 (from MeOH): space group ˝ P212121,C20H34O3, M = 322.25, a = 6.462(2) Å, b = 12.320(4) Å, c = 22.311(7) Å, α = β = γ = 90.00 , V = 1776.3(9) Å3, T = 293(2) K, Z = 4. µ (Mo Kα) = 0.71073 mm´1. A crystal of dimensions of 0.26 mm ˆ 0.25 mm ˆ 0.24 mm was used for measurements on an APEX DUO diffractometer ˝ (Bruker, Rheinstetten, Germany) with a graphite monochromator (ω-κ scans, 2θmax = 50.00 ), Mo Kα radiation. The total number of independent reflections measured was 3113, of which 2250 were observed (|F|2 ě 2σ|F|2). The crystal structure was solved and refined by the direct method SHELXS-97 [31], expanded using difference Fourier techniques and full-matrix least-squares calculations. Final indices: R1 = 0.0601, wR2 = 0.1458 (w = 1/σ|F|2), s = 1.062. Flack parameter = ´0.3(2).

3.5. Biological Activity of Henrin A (1)

3.5.1. Cytotoxicity Assay Cytotoxicity assays involving oral epidermoid (KB: a Hela derivative previously referred as oral epidermis), colon (HCT116), breast (MCF-7), and lung (A549) carcinoma cell lines (ATCC, Manassas, VA, USA) were performed using sulforhodamine B based on the slightly modified protocols used for individual cell lines [32]. KB and A549 cells were maintained in Dulbecco's modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA). HCT116 cells were maintained in McCoy’s 5A medium (Life Technologies). MCF-7 cells were maintained in DMEM medium containing 10 mg/L of insulin. Briefly, medium was supplemented with 10% fetal bovine serum (FBS) (Life Technologies). Serial dilutions of test samples were prepared using 10% aqueous DMSO as solvent. The cell suspension was added into 96-well microliter plates in 190 µL at plating densities of 5000 cells/well. One plate was fixed in situ with TCA to represent a no growth control at the time of drug addition (day 0). Then 10 µL 10% aqueous DMSO was used as control group. After 72 h incubation, the cells were fixed to plastic substratum by the addition of 50 µL cold 50% aqueous trichloroacetic acid and washed with water after incubation at 4 ˝C for 30 min. After staining cells with 100 µL of 0.4%

27983 Int. J. Mol. Sci. 2015, 16, 27978–27987 sulforhodamine B in 1% aqueous AcOH for 30 min, unbound dye was removed by washing four times with 1% aqueous AcOH. Allowed the plates to dry at room temperature, then the bound dye was solubilized with 200 µL 10 mM unbuffered Tris base, pH 10. Shaken for 5 min or until the dye was completely solubilized and the optical density was measured at 515 nm using an ELISA plate reader (Bio-Rad, Hercules, CA, USA). The average data were expressed as a percentage, relative to the control. Percentage growth inhibition was calculated as: (OD (cells + samples) ´ OD (day 0 only cells))/(OD (cells + 10% DMSO) ´ OD (day 0 only cells)) = % survival, Cytotoxicity = 1 ´ % survival.

3.5.2. Anti-Biofilm Activity Test The anti-dental bacterial activity was evaluated against Streptococcus mutans (ATCC35668) and S. sobrinus (ATCC33478) (both strains were obtained from Rory Munro Watt’s laborary of Faculty of Dentistry, Hong Kong University, Hong Kong), and the antifungal activity was evaluated against Trichophyton rubrum (ATCC MYA-4438) (obtained from Institute of Dermatology, Chinese Academy of Medical Science, Nanjing, China). Biofilm formation was quantified according to a method previously described [33], with minor modifications. Briefly, the bacteria were suspended in BHI broth until turbidity was equal to a 0.5 McFarland Standard [34], and then bacterial suspension was diluted 1:100 into fresh BHI broth in microtiter wells (SPL Lifesciences Co., Gyeonggi-do, Korea) supplemented with the compound at a final concentration of 20 µg/mL or with penicillin G, chlorhexidine, and DMSO as the two positive and one negative controls, respectively. After 24 h of incubation at 37 ˝C without agitation, the content of each well was removed, and each well was washed three times with 250 µL of sterile physiological saline. The plates were vigorously shaken in order to remove all non-adherent bacteria. The remaining attached bacteria were fixed with 200 µL of methanol per well, and after 15 min plates were emptied and left to dry. Then, 50 µL of the crystal violet solution (1%, wt/vol) was added to each sample well, and the mixture was incubated at room temperature for 15 min. Wells were washed four times with distilled water and were filled with 200 µL of 95% ethanol to solubilize crystal violet in the solvent. The eluent (150 µL) was transferred to a new microtiter well, and the absorbance was determined with a multimode microplate reader (Bio-Rad) at 570 nm.

3.5.3. Antifungal Microtiter Assay A 96-flat-bottom-well microtiter plate (SPL Lifesciences Co., Gyeonggi-do, Korea) was used in experiments to evaluate the effectiveness of the compound in inhibiting T. rubrum. The antifungal activity tests were performed using the broth micro dilution method as described in M38-A2, with modifications [35,36]. The medium used was Roswell Park Memorial Institute medium (RPMI) 1640 (Life Technologies Inc., Grand Island, NE, USA) with L-glutamine buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS), supplemented with 2% glucose. The cell suspension was prepared in growth medium. Each test well received 190 µL of conidia at 2.0 ˆ 104/mL, and 10 µL of the compound solution at 4 mg/mL, where the final concentrations in the well were 20 µg/mL. Positive (10 µL of miconazole with 190 µL of inoculum) and negative (200 µL of medium) controls were included in all experiments. The plates were incubated at 30 ˝C for 72 h. Then, 20 µL 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was added into the 96-well plate, then cultured 10 more hours. The pate was centrifuged at 2500 rpm for 10 min. Remove the supernatant and add 150 µL DMSO to dissolve the formazan by shaking for 30 min. The 96-well plate was centrifuged and the supernatant was transferred to a new 96-well plate, and OD reading was measured at 510 nm by using the microplate reader (Bio-Rad).

3.5.4. Inhibitory HIV Activity Assay HIV/VSV-G were produced by co-transfecting 3 g of VSV-G envelope expression plasmid with 21 g of a replication-defective HIV vector (pNL4-3.Luc.R‚E) [37,38] into human embryonic kidney 293T cells (90% confluent) in 10 cm plates with PEI (polyethylenimine) (Invitrogen, Carlsbad, CA, USA), as previously described [26]. Eight hours post-transfection, all media was replaced with

27984 Int. J. Mol. Sci. 2015, 16, 27978–27987 fresh, complete DMEM. Forty-eight hours post-transfection, the supernatants were collected and filtered through a 0.45-µm-pore-size filter (Millipore, Billerica, MA, USA) and the pseudovirions were directly used for infection. Target A549 cells were seeded at 104 cells per well (96-well plate) in complete DMEM. Ten microliter compound for serial concentrations (20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 µg/mL) and 190 µL of the pseudovirus were incubated with target cells. Forty-eight hours post-infection, cells were lysed and prepared for luciferase assay (Promega, Madison, WI, USA).

4. Conclusions A new ent-kaurane diterpene (henrin A, 1) was isolated from the leaves of P. henryi. The chemical structure was elucidated by analysis of the spectroscopic data and was further confirmed by the X-ray crystallographic analysis. The compound was evaluated for its biological activities against a panel of cancer cell lines, dental bacterial biofilm formation, and HIV. Our assay results showed that henrin A (1) had low cytotoxicity due to its lack of an α,β-unsaturated ketone group in the structure, but the compound has been determined as a potential anti-HIV agent in our antiviral assay study.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/ 16/11/26071/s1. Acknowledgments: The work described in this paper was collaborative efforts within multi-disciplinary cooperative programs supported by grants from by the Natural Science Foundation Committee of China (81160496), the Science and Technology Fund of Guizhou Province (No. [2009]2145), the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKBU 262912 and HKBU12103014), HKBU Interdisciplinary Research Matching Scheme (RC-IRMS/12-13/03), and Faculty Research Grants, Hong Kong Baptist University (FRG2/14-15/047 and FRG1/13-14/029). Author Contributions: Wan-Fei Li and Juan Zou performed most of the chemistry-related experiments including separation, structure determination of the reported compound with support of Lu-Tai Pan and Jing-Jie Zhang; Juan Wang, Xun Song and Chuen-Fai Ku performed most of the biology-related experiments including cytotoxic activity and antimicrobial evaluation with support of Hong-Jie Zhang and Li-Jun Rong; Lu-Tai Pan and Ji-Xin Li collected the plant materials; Lu-Tai Pan and Jing-Jie Zhang designed the separation study; Hong-Jie Zhang and Li-Jun Rong designed the bioassay study; Wan-Fei Li, Lu-Tai Pan and Hong-Jie Zhang co-wrote the manuscript with the assistance of Jing-Jie Zhang and Li-Jun Rong. All authors discussed the results and commented on the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Huang, S.X.; Xiao, W.L.; Li, L.M.; Li, S.H.; Zhou, Y.; Ding, L.S.; Lou, L.G.; Sun, H.D. Bisrubescensins A–C: Three new dimeric ent-kauranoids isolated from Isodon rubescens. Org. Lett. 2006, 8, 1157–1160. [CrossRef] [PubMed] 2. Sun, H.D.; Xu, Y.L.; Jiang, B. Diterpenoids from Isodon Species; SciPress, Ltd.: Beijing, China, 2001. 3. Sun, H.D.; Huang, S.X.; Han, Q.B. Diterpenoids from Isodon species and their biological activities. Nat. Prod. Rep. 2006, 23, 673–698. [CrossRef][PubMed] 4. Chen, S.; Gao, J.; Halicka, H.D.; Huang, X.; Traganos, F.; Darzynkiewicz, Z. The cytostatic and cytotoxic effects of oridonin (Rubescenin), a diterpenoid from Rabdosia rubescens, on tumor cells of different lineage. Int. J. Oncol. 2005, 26, 579–588. [CrossRef][PubMed] 5. Wang, S.P.; Zhong, Z.F.; Wan, J.B.; Tan, W.; Wu, G.S.; Chen, M.W.; Wang, Y.T. Oridonin induces apoptosis, inhibits migration and invasion on highly-metastatic human breast cancer cells. Am. J. Chin. Med. 2013, 41, 177–196. [CrossRef][PubMed] 6. Ikezoe, T.; Yang, Y.; Bandobashi, K.; Saito, T.; Takemoto, S.; Machida, H.; Togitani, K.; Koeffler, H.P.; Taguchi, H. Oridonin, a diterpenoid purified from Rabdosia rubescens, inhibits the proliferation of cells from lymphoid malignancies in association with blockade of the NF-κB signal pathways. Mol. Cancer Ther. 2005, 4, 578–586. [CrossRef][PubMed] 7. Fuji, K.; Xu, H.J.; Tatsumi, H.; Imahori, H.; Ito, N.; Node, M.; Inaba, M. Design and synthesis of antitumor compounds based on the cytotoxic diterpenoids from the genus Rabdosia. Chem. Pharm. Bull. 1991, 39, 685–689. [CrossRef][PubMed]

27985 Int. J. Mol. Sci. 2015, 16, 27978–27987

8. Murakami, T.; Maehashi, H.; Tanaka, N.; Satake, T.; Kuraishi, T.; Komazawa, Y.; Saiki, Y.; Chen, C.M. Chemical and chemotaxonomical studies on filices. LV. Studies on the constituents of several species of Pteris. Yakugaku Zasshi 1985, 105, 640–648. 9. Murakami, T.; Iida, H.; Tanaka, N.; Saiki, Y.; Chen, C.M.; Iitaka, Y. Chemical and chemotaxonomic study of filicales. 33. Chemical studies of the components of Pteris longipes Don. Chem. Pharm. Bull. 1981, 29, 657–662. [CrossRef] 10. Liu, Q.F.; Qin, M.Z. Chemical studies of the components of Pteris multifida Poir. Chin. Tradit. Herb. Drugs (Zhong Cao Yao) 2002, 33, 113–114. 11. Tanaka, N.; Hata, M.; Murakami, T.; Saiki, Y.; Chen, C.M. Chemical and chemotaxonomic studies of Pteris and related genera (Pteridaceae). XIII. Additional components of Pteris dispar Kunze. Chem. Pharm. Bull. 1976, 24, 1965–1966. [CrossRef] 12. Murakami, T.; Tanaka, N.; Komazawa, Y.; Saiki, Y.; Chen, C.M. Chemical and chemotaxonomic studies of ferns. XLI. Additional contents of Pteris purpureorachis Copel. Chem. Pharm. Bull. 1983, 31, 1502–1504. [CrossRef] 13. Tanaka, N.; Murakami, T.; Saiki, Y.; Chen, C.M.; Iitaka, Y. Chemical and chemo taxonomic study of filices. XXXIV. chemical studies of the components of Pteris purpureorachis Copel. Chem. Pharm. Bull. 1981, 29, 663–666. [CrossRef] 14. Zhang, X.; Li, J.H.; He, C.W.; Tanaka, N. Study on the diterpenoid constituents and anticancer action of Pteris semipinnata. Chin. Pharm. J. 1999, 34, 512–514. 15. Cheng, H.E.; Liang, N.; Li, M.O. Inhibitory effect of compound 6F isolated from Pteris semipinnata L. on biosynthesis of DNA, RNA and protein in HL-60 cells. J. Guangdong Med. Coll. 2002, 20, 247–250. 16. Zhu, Y.L.; Song, L.D. The research of Pteris genus in China. China Prac. Med. 2014, 9, 256. 17. Murakami, T.; Tanaka, N. Occurrence, structure and taxonomic implications of fern constituents. In Fortschritte der Chemie organischer Naturstoffe/Progress in the Chemistry of Organic Natural Products; Herz, W., Grisebach, H., Kirby, G.W., Tamm, C., Eds.; Springer Vienna: Tokyo, Japan, 1988; pp. 1–310. 18. The Editorial Committee of Flora of Guizhou. Flora of Guizhou (Guizhou Zhiwu Zhi Volume 8); Guizhou People Press: Guiyang, China, 1986; pp. 491–492. 19. Pan, L.T.; Zhao, J.H.; Sun, Q.W. Medicinal Pteridophytes of Guizhou (Guizhou Juelei Zhiwu zhi); Guizhou Science and Technology Press: Guiyang, China, 2012; p. 113. 20. Flack, H.D. On enantiomorph-polarity estimation. Act. Crystallogr. 1983, A39, 876–881. [CrossRef] 21. Flack, H.D.; Bernardinelli, G. The use of X-ray crystallography to determine absolute configuration. Chirality 2008, 20, 681–690. [CrossRef][PubMed] 22. Hanson, J.R. The tetracyclic diterpenes. In International Series of Monographs in Organic Chemistry; Pergamon Press: London, UK, 1968; Volume 9, p. 8. 23. Huang, S.X. Tetracyclic diterpenes. In Chemistry of Diterpenes; Sun, H.D., Ed.; Chemical Industry Press: Beijing, China, 2011; pp. 203–227. 24. Dewick, P.M. Medicinal Natural Products, A Biosynthetic Approach; John Wiley & Sons Ltd.: West Sussex, UK, 2009; pp. 228–229. 25. Rumschlag-Booms, E.; Zhang, H.J.; Soejarto, D.D.; Fong, H.H.; Rong, L.J. Development of an antiviral screening protocol: One-Stone-Two-Birds. J. Antivir. Antiretrovir. 2011, 3, 8–10. [CrossRef][PubMed] 26. Rosselli, S.; Bruno, M.; Maggio, A.; Bellone, G.; Chen, T.H.; Bastow, K.F.; Lee, K.H. Cytotoxic activity of some natural and synthetic ent-kauranes. J. Nat. Prod. 2007, 70, 347–352. [CrossRef][PubMed] 27. Chen, K.; Shi, Q.; Fujioka, T.; Zhang, D.C.; Hu, C.Q.; Jin, J.Q.; Kilkuskie, R.E.; Lee, K.H. Anti-AIDS agents, 4. Tripterifordin, a novel anti-HIV principle from Tripterygium wilfordii: Isolation and structural elucidation. J. Nat. Prod. 1992, 55, 88–92. [CrossRef][PubMed] 28. Chang, F.R.; Yang, P.Y.; Lin, J.Y.; Lee, K.H.; Wu, Y.C. Bioactive kaurane diterpenoids from Annona glabra. J. Nat. Prod. 1998, 61, 437–439. [CrossRef][PubMed] 29. Wu, Y.C.; Hung, Y.C.; Chang, F.R.; Cosentino, M.; Wang, H.K.; Lee, K.H. Identification of ent-16β,17-dihydroxykauran-19-oic acid as an anti-HIV principle and isolation of the new diterpenoids annosquamosins A and B from Annona squamosal. J. Nat. Prod. 1996, 59, 635–637. [CrossRef][PubMed] 30. Sun, H.D. Diterpenoid Chemistry (Ertie Huaxue); Chemical Industry Press: Beijing, China, 2011; p. 206. 31. Sheldrick, G.M. SHELXL-97, Program for X-ray Crystal Structure Refinement; University of Gottingen: Gottingen, Germany, 1997.

27986 Int. J. Mol. Sci. 2015, 16, 27978–27987

32. Zhang, H.J.; Ma, C.Y.; Hung, N.V.; Cuong, N.M.; Tan, G.T.; Santarsiero, B.D.; Mesecar, A.D.; Soejarto, D.D.; Pezzuto, J.M.; Fong, H.H.S. Miliusanes, a class of cytotoxic agents from Miliusa sinensis. J. Med. Chem. 2006, 49, 693–708. [CrossRef][PubMed] 33. O’Toole, G.A.; Kolter, R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: A genetic analysis. Mol. Microbiol. 1998, 28, 449–461. [CrossRef] [PubMed] 34. Koneman, E.W.; Allen, S.D.; Janda, W.M.; Schreckenberger, P.C.; Winn, W.C. Color Atlas And Textbook of Diagnostic Microbiology, 5th ed.; Lippincott: Philadelphia, PA, USA, 1997; pp. 803–841. 35. Yang, H.C.; Mikami, Y.; Yazawa, K.; Taguchi, H.; Nishimura, K.; Miyaji, M.; Branchini, M.L.; Aoki, F.H.; Yamamoto, K. Colorimetric MTT assessment of antifungal activity of D0870 against fluconazole-resistant Candida albicans. Mycoses 1998, 41, 477–480. [CrossRef][PubMed] 36. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi, Approved Standard, CLSI document M38-A2, 2nd ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2008. 37. He, J.; Choe, S.; Walker, R.; di Marzio, P.; Morgan, D.O.; Landau, N.R. Human immunodeficiency virus type I viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J. Virol. 1995, 69, 6705–6711. [PubMed] 38. Connor, R.I.; Chen, B.K.; Choe, S.; Landau, N.R. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 1995, 206, 935–944. [CrossRef] [PubMed]

© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

27987