Antiviral Activity of Fridericia formosa (Bureau) L. G ... · Antiviral Activity of Fridericia...

Research ArticleAntiviral Activity of Fridericia formosa (Bureau) L G Lohmann(Bignoniaceae) Extracts and Constituents

Geraldo Ceacutelio Brandatildeo1 Erna G Kroon2 Joseacute D Souza Filho3 and Alaiacutede Braga Oliveira4

1Departamento de Farmacia Escola de Farmacia Universidade Federal de Ouro Preto Campus Morro do Cruzeiro35400-000 Ouro Preto MG Brazil2Departamento de Microbiologia ICB Universidade Federal de Minas Gerais Av Antonio Carlos 662731270-901 Belo Horizonte MG Brazil3Departamento de Quımica ICEX Universidade Federal de Minas Gerais Av Antonio Carlos 662731270-901 Belo Horizonte MG Brazil4Departamento de Produtos Farmaceuticos Faculdade de Farmacia Universidade Federal de Minas GeraisAv Antonio Carlos 6627 31270-901 Belo Horizonte MG Brazil

Correspondence should be addressed to Alaıde Braga Oliveira alaidebragaterracombr

Received 16 August 2016 Revised 23 November 2016 Accepted 15 February 2017 Published 29 May 2017

Academic Editor Carlos E P Corbett

Copyright copy 2017 Geraldo Celio Brandao et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A phytochemical study of Fridericia formosa (Bignoniaceae) ethanol extracts of leaves stems and fruits was guided by in vitroassays against vaccinia virus Western Reserve (VACV-WR) human herpes virus 1 (HSV-1) murine encephalomyocarditis virus(EMCV) and dengue virus type 2 (DENV-2) by the MTT method All the ethanol extracts were active against DENV-2 HSV-1and VACV-WRwith best results for the fruits extract against DENV-2 (SI gt 382) For VACV-WR andHSV-1 EC50 values gt 200 120583gmLminus1 were determined while no inhibition of the cytopathic effect was observed with EMCV Five compounds were isolated andidentified as the C-glucosylxanthones mangiferin (1) 21015840-O-trans-caffeoylmangiferin (2) 21015840-O-trans-coumaroylmangiferin (3) 21015840-O-trans-cinnamoylmangiferin (5) and the flavonoid chrysin (4)Themost active compound was 21015840-O-trans-coumaroylmangiferin(3) with SI gt 1219 against DENV-2 and 1087 for HSV-1These results indicate that mangiferin cinnamoyl esters might be potentialantiviral drugs

1 Introduction

Viral infections represent a current problem accounting forsevere damage to human health and economic losses inlivestock [1] Some viral diseases such as dengue or denguefever (DF) herpes smallpox and encephalomyocarditis havea high impact in public health in the tropical and subtropicalregions of the world [1]

Dengue virus belonging to the Flaviviridae family Fla-vivirus genus is responsible for Dengue fever (DF) and isconsidered the most common arboviral disease of humans Itis estimated that 390 million cases occur every year aroundthe world and it is endemic in more than 100 countriesincluding the Americas Southeast Asia andWestern Pacific

regionsmost seriously affected [1 2] No effective drug as wellas no vaccine is available for human use The need for a safeand efficient approach either for treatment or prevention ofDF has been considered a global priority [1 3]

HSV belongs to the family Herpesviridae and the sub-family Alphaherpesvirinae and is characterized by neurovir-ulence latency and reactivation The prevalence of HSVinfection has increased in recent years making it a highlyrelevant public health issue Early detection and treatment areof paramount importance for disease control [4]

Encephalomyocarditis virus (EMCV) family Picornaviri-dae genus Cardiovirus is a group of closely related virusspecies with a wide host range Infections with EMCV areassociated with sporadic cases and outbreaks of myocarditis

HindawiJournal of Tropical MedicineVolume 2017 Article ID 6106959 11 pageshttpsdoiorg10115520176106959

2 Journal of Tropical Medicine

and encephalitis in domestic pigs in nonhuman primates andother mammalian species There are few reports of cases ofhuman infection by EMCV [5]

Vaccinia (VACV) is a virus of the genus Orthopoxvirusof the family Poxviridae that in humans causes nonlethalpustular and localized disease The vaccinia virus does nothave natural hosts but cases of bovine and human infectionby vaccinia virus are reported in Brazil and India causingeconomic losses and affecting health services [6 7]

As part of a bioprospecting project whose main goalis to discover potential antiviral natural products of plantsfrom Brazilian Cerrado and Atlantic Forest biomes wehave screened several species of plants collected in thestate of Minas Gerais [1 8ndash12] Among these Fridericiaformosa (Bureau) Sandwith was chosen for bioguided phy-tochemical investigation due to the good antiviral activitypresented by the ethanol extracts of leaves stems andfruits

2 Materials and Methods

21 Collection Taxonomical Determination and Processing ofPlant Materials F formosa was collected in the municipalityof Belo Horizonte Minas Gerais Brazil The plant was taxo-nomically identified by Dr J A Lombardi Departamento deBotanica Instituto de Biociencias UNESP Rio Claro BrazilA voucher specimen was deposited at the BHCBUFMGBelo Horizonte Minas Gerais Brazil under the number23885

22 Preparation of Extracts After drying in air circulatingoven at 40∘C for 72 h the plant materialmdash1363 g of leaves4615 g of stems and 138 g fruitsmdashwere ground and extractedby percolation with 96 EtOH at room temperature Thesolvent was removed in a rotary evaporator under reducedpressure at 50∘C leaving dark residuesmdashEEFFL 342 gEEFFS 600 g and EEFFF 25 gmdashfor leaves stems and fruitsrespectively which were kept in a vacuum desiccator untilconstant weight

23 HPLC Analyses In the HPLC analyses an exploratorygradient elution was used [1 9 10] Fingerprints wereregistered by RP-HPLC-DAD on a Waters 2695 apparatusequipped with a UV-DADdetector (Waters 2996) A LiChro-spher 100 RP-18 column (5 120583m 250 times 4mm id MerckDarmstadt Germany) was employed at 40∘C flow rate of10mLmin and detection at wavelengths of 220 280 and350 nm To an aliquot 100mg of dried extractfractionsand 10mg of each of the isolated compounds HPLC grademethanol was added and the mixture was dissolved bysonication in an ultrasound bath for 15min followed bycentrifugation at 10000 rpm for 10min The supernatant wasfiltered through a Millipore membrane (02 120583m) and injected(100 120583L) onto the equipment Elution was carried out with alinear gradient of water (a) and acetonitrile (b) (from 5 to95 of B in 60min)

24 Isolation of Chemical Components from Leaves ExtractTo a portion of EEFFL (100 g) MeOH was added and

an insoluble precipitate was separated by filtration throughsintered glass funnel and washed thoroughly with MeOHyielding 28 g The precipitate was recrystallized out frommethanolwater (1 1) giving 19 g of compound 1 Thefiltrate was dried in a rotary evaporator under reducedpressure at 50∘C leaving a dark residue (FFLMW 70 g)AFLMW was subjected to fractionation over a silica gel col-umn with n-hexaneCH2Cl2 (1 1) CH2Cl2 CH2Cl2EtOAc(1 1) EtOAcMeOH (2 1) EtOAcMeOH (1 2) MeOHMeOHH2O (2 1) and MeOHH2O (1 2) as eluents Aportion of EtOAcMeOH (2 1) fraction 2 (10 g) was filteredover a Sephadex LH 20 column with MeOH as eluent giving12 fractions Fractions 11 and 12 were combined leadingto a yellow solid (5540mg) which was further purifiedby HPLC employing Shim-pack PRC-ODS column (20mmid times 25 cm flow rate 50mlmin) and a gradient elutionwith MeOHH2O 45 to 95 for 50 minutes Compounds 2(223mg) and 3 (561mg) were isolated

25 Isolation of Chemical Components from Stem Extract Toa portion of EEFFS (100 g) cold MeOH was added giving aprecipitate which was separated by filtration on sintered glassfunnel and washed thoroughly with MeOH yielding 243 gThe precipitate was recrystallized out twice from MeOH togive 4050mg of compound 1 The mother liquor from thesecond recrystallization was dried in a rotary evaporatorunder reduced pressure at 50∘C leaving a yellow residue(FFSMW2 12 g) that was further subjected to fractionationby HPLC employing Shim-pack PRC-ODS column (20mmid times 25 cm flow rate 50mlmin) and a gradient elution withMeOHH2O 45 to 95 for 50 minutes affording compound 4(5110mg)

26 Isolation of Chemical Components from Fruit ExtractTo a portion of EEFFF (10 g) MeOH was added and aninsoluble precipitate was separated by filtration on sin-tered glass funnel and washed thoroughly with methanolThe precipitate was subjected to recrystallization to givecompound 1 (807mg) The filtrate was dried in a rotaryevaporator under reduced pressure at 50∘C leaving a yellowresidue (FFFMW 8097mg) that was further purified byHPLC employing Shim-pack PRC-ODS column (20mmid times 25 cm flow rate 50mlmin) and a gradient elution withMeOHH2O45 to 95 for 50minutes leading to the isolationof compounds 1 (963mg) 2 (134mg) 3 (829mg) and 5(846mg)

27 Cell Culture and Virus Vero cells (ATCC CCL-81) andLLCMK2 cells were cultured in Dulbeccorsquos modified Eaglersquosmedium (DMEM Cultilab Campinas SP Brazil) at 37∘Cin 5 CO2 atmosphere supplemented with 5 fetal bovineserum 50 120583gmL gentamicin 100UmL penicillin and5 120583gmL amphotericin B [1]

HSV-1 was obtained from the collection of Laboratoriode Virus UFMG Belo Horizonte Brazil DENV-2 EMCVand VACV-WR were kindly donated by Dr L Figueiredo(USP Ribeirao Preto Brazil) Dr I Kerr (London ResearchInstitute London UK) and Dr C Jungwirth (University of

Journal of Tropical Medicine 3

Wurzburg Wurzburg Germany) respectively The viruseswere titrated by TCID50 in Vero cells [13] and the titers were25 times 106 10 times 106 10 times 106 and 10 times 104 TCID50mLrespectively for HSV-1 EMCV VACV-WR and DENV-2

28 CytotoxicityAssay Vero andLLCMK2 cellswere exposedto different concentrations of extractsfractionscompoundsfor 48 and 72 h [1] After incubation cell viability was assessedby the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT Merck) assay at a concentration of 2mgmLin PBS [1 14] Each sample was assayed in four replicatesfor concentrations ranging from 500 to 0125 120583gmL Thecytotoxicity of each sample was expressed as CC50 that isthe concentration of sample that inhibited cell growth by50 [1]

29 Antiviral Assays The antiviral activity (EC50) of ex-tractsfractionscompounds was evaluated by theMTT assay[15] Acyclovir (Calbiochem Merck Brasil Sao Paulo SPBrazil) and 120572-2a interferon (Bergamo Brasil Sao Paulo SPBrazil) were used as positive controls [1] The cell monolayerwas infected with viral suspensions with titers of 25 times 10610 times 106 10 times 106 and 10 times 104 TCID50mL respectivelyfor HSV-1 EMCV VACV-WR and DENV-2 [1] Dilutionsof the extracts fractions and compounds in noncytotoxicconcentrations were added to the wells after viral infectionThe plates were incubated at 37∘C in humidified 5 CO2atmosphere for a period of 48 andor 72 h [1] Experimentswere carried out with eight different concentrations withinthe inhibitory range of the samples The 50 inhibitorconcentrations of the viral effect (EC50) for each of theextracts fractions and constituents were calculated fromconcentration-effect-curves after no linear regression analy-sis [1]The selectivity index (SI) is defined as CC50 over EC50Statistical calculations were carried out with the GraphPadprism 50 software package (Statistica) Results are expressedas the mean plusmn SEM of 4 independent experiments Studentrsquost-test was used for statistical analyses 119875 values gt 005 wereconsidered to be significant

210 Structural Determination The compounds isolatedwere identified on the basis of spectral analyses and com-parison with literature data 1D and 2D 1H and 13C-NMRspectra such as COSY HSQC and HMBC were obtainedon a Bruker Avance DRX400 instrument in DMSO-d6 withTMS as internal standard Chemical shifts are given as 120575(ppm) LC-MS data were obtained by electrospray ionizationmass spectrometry (ESI-MS) in an Esquire 3000 Plus BrukerDaltonics equipment capillary 4000V nebulizer 27 psi drygas 70 Lmin dry temp 320∘C andmass flux 100 uLmin inthe Central Analıtica Instituto de Quımica Universidade deSao Paulo Sao Paulo SP Brazil [1]

211 Spectroscopic Data for Isolated Compounds

Mangiferin (1 2-120573-D-Glucopyranosyl-1367-tetrahydroxy-9H-xanthen-9-one) orange powder (MeOH) mp decom-poses at 2650ndash2750 Lit 271ndash274∘C [16] UV (MeOH) 120582max

239 258 316 365 nm IR ]max 3363 3184 2935 2891 16481619 1592 1565 1520 1490 1462 1406 1351 1294 1251 11911093 1074 1032 878 822 735 cmminus1 1H NMR (DMSO-d6400MHz) 120575 1377 (s 1H 1-OH) 1055 (s 2H 67-OH) 738(s 1H H-8) 686 (s 1H H-5) 637 (s 1H H-4) 487 (s 2H3101584041015840-OH) 461 (d 100Hz 1HH-11015840) 449 (s 1H 61015840-OH) 405(t 84Hz 1H H-21015840) 370 (d 112Hz 1H H-61015840B) 341 (m 1HH-61015840A) 318 (m 1H H-31015840) 318 (m 1H H-41015840) 318 (m 1H H-51015840) 13C NMR (DMSO-d6 100MHz) 120575 1791 (C C-9) 1638(C C-3) 1618 (C C-1) 1562 (C C-4a) 1540 (C C-6) 1508(C C-10a) 1437 (C C-7) 1117 (C C-8a) 1081 (C C-8) 1075(C C-2) 1026 (C C-5) 933 (C C-4) 815 (C C-51015840) 793 (CC-31015840) 731 (C C-11015840) 706 (C C-41015840) 703 (C C-21015840) 615 (C C-61015840) HRESI-MSmz 4231024 [M minusH]minus (calcd for C19H19O114230927)

21015840-O-trans-caffeoylmangiferin (2 2-(21015840-O-trans-caffeoyl)-C-120573-D-glucopyranosyl-1367-tetrahydroxyxanthone) orangepowder (MeOH) mp decomposes at 2690ndash2780∘C UV(MeOH) 120582max 232 258 315 365 nm IR ]max 3217 1690 16041514 1471 1259 1150 1071 812 cmminus1 1H NMR (DMSO-d6400MHz) 120575 1383 (s 1H 1-OH) 733 (s 1H H-8) 723 (d160Hz 1H H-710158401015840) 692 (m 1H H-210158401015840) 687 (d 80Hz 1HH-610158401015840) 680 (s 1H H-5) 671 (d 80Hz 1H H-510158401015840) 630 (s1H H-4) 601 (d 160Hz 1H H-810158401015840) 565 (m 1H H-21015840) 495(d 120Hz 1H H-11015840) 376 (m 1H H-61015840B) 374 (m 1H H-61015840A) 349 (m 1H H-31015840) 345 (m 1H H-41015840) 327 (m 1H H-51015840) 13C NMR (DMSO-d6 100MHz) 120575 1790 (C C-9) 1652(C C-910158401015840) 1644 (C C-3) 1615 (C C-1) 1563 (C C-4a) 1545(C C-6) 1508 (C C-10a) 1482 (C C-410158401015840) 1459 (C C-310158401015840)1449 (C C-710158401015840) 1439 (C C-7) 1254 (C C-110158401015840) 1216 (C C-610158401015840) 1157 (C C-510158401015840) 1150 (C C-210158401015840) 1142 (C C-810158401015840) 1114 (CC-8a) 1078 (C C-8) 1057 (C C-2) 1025 (C C-5) 942 (C C-4) 823 (C C-51015840) 769 (C C-31015840) 724 (C C-21015840) 711 (C C-11015840)711 (C C-41015840) 619 (C C-61015840) HRESI-MS mz 5831083 [M minusH]minus (calcd for C28H23O14 5831088)

21015840-O-trans-coumaroylmangiferin (3 2-(21015840-O-trans-couma-royl)-C-120573-D-glucopyranosyl-1367-tetrahydroxyxanthone)orange powder (MeOH) mp decomposes at 2710ndash2850UV (MeOH) 120582max 233 258 315 364 nm IR ]max 3255 16941614 1472 1417 1365 1284 1230 1150 1079 1030 996 815765 706 681 cmminus1 1H NMR (DMSO-d6 400MHz) 120575 1386(s 1H 1-OH) 739 (s 1H H-8) 737 (d 160Hz 1H H-710158401015840)735 (d 80Hz 1H H-210158401015840) 735 (d 80Hz 1H H-610158401015840) 681(s 1H H-5) 676 (d 80Hz 1H H-310158401015840) 676 (d 80Hz 1HH-510158401015840) 632 (s 1H H-4) 610 (d 160Hz 1H H-810158401015840) 556(m 1H H-21015840) 501 (d 80Hz 1H H-11015840) 378 (d 80Hz 1HH-61015840B) 366 (m 1H H-61015840A) 364 (m 1H H-31015840) 347 (t100Hz 1H H-41015840) 342 (m 1H H-51015840) 13CNMR (DMSO-d6100MHz) 120575 1658 (C C-910158401015840) 1640 (C C-3) 1600 (C C-1)1600 (C C-410158401015840) 1587 (C C-4a) 1540 (C C-6) 1513 (CC-10a) 1445 (C C-710158401015840) 1439 (C C-7) 1301 (C C-210158401015840) 1301(C C-610158401015840) 1254 (C C-110158401015840) 1159 (C C-310158401015840) 1159 (C C-510158401015840)1144 (C C-810158401015840) 1122 (C C-8a) 1083 (C C-8) 1055 (C C-2)1028 (C C-5) 940 (C C-4) 814 (C C-51015840) 768 (C C-31015840)725 (C C-21015840) 714 (C C-11015840) 707 (C C-41015840) 614 (C C-61015840)HRESI-MS mz 5671144 [M minus H]minus (calcd for C28H23O135671139)


Chrysin (4 57-Dihydroxy-2-phenyl-4H-1-benzopyran-4-one)orange powder (MeOH) mp 2856ndash2879∘C Lit 289ndash291∘C[17] UV (MeOH) 120582max 267 313 (sh) nm IR ]max 3283 29201690 1610 1512 1444 1341 1230 1171 1066 1037 1012 893823 cmminus1 1H NMR (DMSO-d6 400MHz) 120575 120 (s 1H 5-OH) 806 (dd 64 and 16Hz 2H H-21015840 and H-61015840) 755ndash764(m 3H H-31015840 H-41015840 and H-51015840) 695 (s 1H H-3) 686 (s 1H7-OH) 653 (d 2Hz 1H H-8) 623 (d 2Hz 1H H-6) 13CNMR (DMSO-d6 100MHz) 120575 1819 (C C-4) 1644 (C C-7)1632 (C C-2) 1615 (C C-5) 1575 (C C-9) 1320 (C C-41015840)1307 (C C-11015840) 1294 (C C-51015840) 1291 (C C-31015840) 1264 (C C-21015840)1264 (C C-61015840) 1052 (C C-3) 1040 (C C-10) 990 (C C-6)941 (C C-8) HRESI-MS mz 2550671 [M + H]+ (calcd forC15H11O4 2550657)

21015840-O-trans-cinnamoylmangiferin (5 2-(21015840-O-trans-cinna-moyl)-C-120573-D-glucopyranosyl-1367-tetrahydroxyxanthone)orange powder (MeOH)mp decomposes at 2690ndash2790UV(MeOH) 120582max 222 257 275 (sh) 320 (sh) 366 nm IR ]max3252 1693 1614 1471 1417 1183 1150 1079 815 765 706 cmminus11H NMR (DMSO-d6 400MHz) 120575 1387 (s 1H 1-OH) 751(m 1H H-310158401015840) 751 (m 1H H-510158401015840) 733 (s 1H H-8) 723 (d160Hz 1H H-710158401015840) 680 (s 1H H-5) 636 (m 1H H-210158401015840) 636(m 1H H-610158401015840) 632 (s 1H H-4) 631 (d 160Hz 1H H-810158401015840)555 (m 1H H-21015840) 504 (d 120Hz 1H H-11015840) 378 (dd 12025Hz 1H H-61015840B) 366 (dd 120 40Hz 1H H-61015840A) 364 (m1H H-31015840) 351 (m 1H H-41015840) 344 (m 1H H-51015840) 13C NMR(DMSO-d6 100MHz) 120575 1793 (C C-9) 1650 (C C-910158401015840)1634 (C C-3) 1610 (C C-1) 1568 (C C-4a) 1539 (C C-6)1511 (C C-10a) 1439 (C C-710158401015840) 1434 (C C-7) 1339 (CC-110158401015840) 1299 (C C-410158401015840) 1285 (C C-210158401015840) 1285 (C C-610158401015840) 1277(C C-310158401015840) 1277 (C C-510158401015840) 1176 (C C-810158401015840) 1119 (C C-8a)1078 (C C-8) 1049 (C C-2) 1023 (C C-5) 1013 (C C-9a)940 (C C-4) 809 (C C-51015840) 759 (C C-31015840) 724 (C C-21015840)711 (C C-11015840) 702 (C C-41015840) 609 (C C-61015840) HRESI-MS mz5511152 [M minusH]minus (calcd for C28H23O12 5511090)

3 Results

31 HPLC Analyses Isolation and Identification of Com-pounds from Fridericia formosa HPLC-DAD analyses al-lowed identifying xanthones as major constituents in all theextracts as inferred from their UV spectra that were regis-tered online (Figure 1) UV spectra of 1367-tetraoxygenatedxanthones are characterized by the presence of three or moreabsorption bands of decreasing intensity [18] A compoundwith retention time (RT) of 78min was detected in all theethanol extracts as the major constituent in extracts of stemsand fruits

Bioguided fractionation of F formosa ethanol extracts ledto the isolation of five compounds (Figure 2) which wereidentified by comparison with literature spectroscopic data(1H and 13C NMR DEPT-135 experiment COSY HMQCHMBC IR and MS) From the EtOH extract of leaves threecompounds (1ndash3) were obtained Compound 1 was iden-tified as mangiferin a C-glucosylxanthone (120582max 239 258316 and 365 nm HPLC-DAD online) Its identification wasconfirmed by HRMS and comparison of 1H and 13C NMRspectra with literature data [19] Additionally mangiferin (1)

was also isolated from stems (EEAFS) and fruit (EEAFF)extracts Compounds 2 and 3 were purified from leavesand fruits by Sephadex LH-20 gel filtration and preparativeHPLC The presence of the mangiferin chromophore forboth compounds was indicated by their UV spectra Theprotonatedmolecular ions [M+H]+ for2 and3 (mz 5851084andmz 5691144 resp) were determined by accurate positiveHRESI-MS A comparative analysis of fragment ions detectedin experiments by HPLCESI-MSMS showed that the onlynoticeable differences were associated with the cinnamic acidmoieties attached to the mangiferin unity caffeic acid in2 and p-coumaric acid in 3 The isolation of mangiferincinnamic esters from Fridericia samydoides and F patelliferawas previously reported [20 21] and a comparative analysisof these NMR data confirmed the identification of com-pounds 2 and 3 as 21015840-O-trans-caffeoylmangiferin and 21015840-O-trans-coumaroylmangiferin respectively The position of thecinnamic ester group in the glucose unity was proved by long-range HMBC correlation between the ester carbonyl groupand the glucosyl hydrogen 120575C 1652 and H-21015840120575H 565 incompound 2 and 120575C 1658 and H-21015840 120575H 556 in compound3

Additional quantity of mangiferin (1) was obtained as anunsoluble fraction when methanol was added to the driedstem ethanol extract Fractionation of the filtrate (AFSMW2)by preparative RP-HPLC afforded compound 4 whose spec-tral data (UV IR 1H and 13C NMR) and comparison withliterature data [22] allowed its identification as the flavonoidchrysin (4)

Finally column chromatographic fractionation of theethanol fruit extract afforded four C-glucosylxanthoneswhich were shown to be identical to those obtained fromthe leaves extract mangiferin (1) caffeoyl mangiferin (2)and coumaroyl mangiferin (3) besides compound 5 andMM 542Da which might correspond to a mangiferin cin-namic ester was confirmed by NMR data Two regioi-somers 21015840-O-trans-cinnamoylmangiferin and 31015840-O-trans-cinnamoylmangiferin have been previously isolated fromstems and leaves extracts of F samydoides and F patel-lifera respectively [20 21] A comparison of 13C and 1HNMR data including two-dimensional COSY HSQC andHMBCwith those previously reported [20] allowed the iden-tification of compound 5 as 21015840-O-trans-cinnamoylmangi-ferin

32 Bioguided Fractionation of Leaves Stems and FruitsEthanol Extracts from Fridericia formosa Confirming previ-ously published results [8] the ethanol extracts from leaves(EEFFL) stems (EEFFS) and fruits (EEFFF) of F formosashowed antiviral activity against EMCV HSV-1 and VACV-WR with EC50 values in the range of 856 plusmn 41 to 1478plusmn 24 120583g mL (Table 1 and Figure 3) Furthermore theseextracts were evaluated against DENV-2 and disclosed goodantidengue activity with EC50 values ranging from 131 plusmn 16to 426 plusmn 23 120583gmL (Table 1)

An aliquot of the leaves extract (EEFFL 100 g) wassubmitted to bioguided fractionation Initially addition ofcold methanol to EEFFL led to mangiferin (1) as an


Table1Cy

totoxicity(C

C 50V

eroandLL

CMK 2

cells)in

vitro

antiv

iralactivity

(EC 50)andselectivity

index(SI)fore

thanolextractsfro

mFridericiaform

osaleaves(EEF

FL)ste

ms(EE

FFS)

fruit(EE

FFF)fractionsand

compo

unds

1ndash5

Extracts

fractio

nscom

poun

dVe

roCC50120583gmL

LLCM

K 2CC50120583gmL

a HSV

-1EC50120583gmL

SIb VAC

V-WR

EC50120583gmL

SIc EMCV

EC50120583gmL

SId D

ENV-2

EC50120583gmL

SI

EEFF

Sgt500

1739plusmn98

932plusmn54

gt54

592plusmn24

gt84

3225plusmn144

gt15

426plusmn23

41

EEFF

Fgt500

gt500

1478plusmn24

gt34

2527plusmn39

gt20

1344plusmn59

gt37

131plusmn16

gt382

EEAFL

gt500

gt500

856plusmn41

gt58

837plusmn31

gt60

1994plusmn138

gt25

163plusmn68

gt307

FFHDF(11)

2220plusmn73

867plusmn85

NA

NA

NA

NA

FFDF

2633plusmn139

951plusmn93

NA

NA

NA

NA

FFDEF

(11)

507plusmn25

138plusmn21

NA

NA

NA

39plusmn036

35

FFEF

gt500

gt500

NA

NA

NA

NA

FFEM

F(11)

gt500

gt500

1697plusmn210

gt29

1829plusmn114

gt27

1905plusmn147

gt26

318plusmn57

gt157

FFMF

gt500

gt5 00

503plusmn28

gt99

NA

NA

418plusmn56

gt120

FFMWF(21)

gt500

gt500

357plusmn20

gt140

NA

NA

228plusmn08

gt219

FFMWF(12)

gt500

gt500

NA

NA

NA

NA

Mangiferin

(1)

gt500

gt500

2679plusmn67

(6348plusmn159)gt19

1827plusmn143

(4325plusmn339)gt27

NA

2655plusmn140

(6291plusmn332)gt19

21015840-O

-Trans-caffeoylmangiferin

(2)

gt500

gt500

46plusmn15

(79plusmn26)

gt1087

238plusmn10

(407plusmn17

)gt210

NT

41plusmn

04

(70plusmn07)

gt1219

21015840-O

-Trans-cou

maroylm

angiferin

(3)gt500

gt500

474plusmn61

(834plusmn107)gt105

NA

2410plusmn318

(4243plusmn560)gt21

404plusmn42

(711plusmn74

)gt124

Chrysin

(4)

gt500

gt500

1463plusmn159

(5759plusmn626)egt34

1235plusmn105

(4862plusmn413)gt40

NA

NA

21015840-O

-Trans-cinnamoylm

angiferin

(5)gt500

gt500

774plusmn43

(1402plusmn78

)gt65

NA

NT

35plusmn05

(63plusmn09)

gt1489

Acyclovir

gt1000

gt1000

40f

Interfe

ron120572

gtg10times105gtg10times105

fg15times102

fg25times103

fg25times103

SIsele

ctivity

index

a vira

ltiterTC

ID50m

L25times106in

48h

b vira

ltiterTC

ID50m

L10times106in

48h

c vira

ltiterTC

ID50m

L10times106in

48h

d vira

ltiterTC

ID50m

L10times104in

72h

NAn

oactiv

ityin

theassayedconcentra

tionsN

Tno

test

e con

centratio

nin120583Mf 80to

100

inhibitio

nof

cytopathiceffect

g con

centratio

nin

UIm

LEE

FFL

ethano

lextract

from

Fridericiaform

osaleavesF

FHDFFridericia

form

osan-hexanedichlorom

ethane

11fraction

FFDFFridericiaform

osadichloromethane

fractio

nFF

DEFFrid

ericiaform

osadichloromethaneethylacetate11fraction

FFEFFrid

ericiaform

osaethylacetate

fractio

nFF

EMFFridericiaform

osaethylacetatem

ethano

l11

fractio

nFF

MFFridericiaform

osaethylm

ethano

lfraction

FFMWFFridericiaform

osamethano

lwater

21fraction

FFMWFFridericiaform

osa

methano

lwater

12fractio

nFridericiaform

osafractio

nsfro

mchromatograph

yof

EEFF

Lover

silicag

elcolumn


400

200

000

(AU

) (AU

)

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

010

005

(nm)(nm)25000 30000 3500025000 30000 35000

(AU

)

002

004RT = 78min RT = 118min

(a)

(AU

) (AU

)(nm)

25000 30000 35000

0020

0010

0000

000

050

100RT = 265min

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(b)

002

018

(AU

) (AU

)

000

100

200

300

RT = 136min

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

(nm)25000 30000 35000

(c)

RT = 176min

(AU

)

000

100

200

300

(AU

)

100

200

300

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(nm)25000 30000 35000

(d)

Figure 1 RP-HPLC-DAD fingerprints for the crude ethanol extracts from (a) ethanol extract from Fridericia formosa leaves (EEFFL)with mangiferin (RT = 78min) and 21015840-O-trans-caffeoylmangiferin (RT = 118min) UV spectrum registered online detection 350 nm(b) ethanol extract from Fridericia formosa stems (EEFFS) with UV spectra registered online for peak corresponding to chrysin (RT =268min) (c) ethanol extract from Fridericia formosa fruits (EEFFF) with UV spectra registered online for peak corresponding to 21015840-O-trans-coumaroylmangiferin (RT = 136min) and (d) for a mixture of the isolated compounds with UV spectra registered online for peakcorresponding to 21015840-O-trans-cinnamoylmangiferin (RT = 176min) Detection 350 nm Chromatographic conditions see Experimental


O

O

HO

OH

O

OOH

HO OH

OHO

O

OH

HO

HO

O

OOH

HO OH

OHO

OH

OH

HO

HO

R1 = R2 = OH 2998400-O-trans-caffeoylmangiferin (2)R1 = H R2 = OH 2998400-O-trans-coumaroylmangiferin (3)R1 = R2 = H 2998400-O-trans-cinnamoylmangiferin (5)

Chrysin (4)

Mangiferin (1)

R1

R2

Figure 2 Chemical structures of mangiferin (1) 21015840-O-trans-caffeoylmangiferin (2) 21015840-O-trans-coumaroylmangiferin (3) chrysin (4) and21015840-O-trans-cinnamoylmangiferin (5)

unsoluble fraction (19 g) and to a methanol soluble frac-tion (FFLMW) Mangiferin (1) was tested against all thefour virus samples and showed a low antiviral effect(Table 1) FFLMW was subjected to a chromatographicfractionation through a silica gel column employing aseluents n-hexaneCH2Cl2 (1 1) CH2Cl2 CH2Cl2EtOAc(1 1) EtOAcMeOH (2 1) EtOAcMeOH (1 2) MeOHMeOHH2O (2 1) andMeOHH2O (1 2) fractions that wereassayed against DENV-2 EMCV HSV-1 and VACV-WRThree fractions were active against HSV-1The EtOAcMeOH(2 1) fraction was the only one active against VACV-WR andEMCV Best results were observed against DENV-2 for fouractive fractions with EC50 ranging from 39 plusmn 04 to 418plusmn 56120583gmL (Table 1) Fractionation of the EtOAcMeOH(2 1) fraction through a Sephadex LH20 column andpreparative RP-HPLC afforded two xanthones 21015840-O-trans-caffeoylmangiferin (2) and 21015840-O-trans-coumaroylmangiferin(3) Xanthone 2 showed high activity against DENV-2HSV-1 and VACV-WR (EC50 of 41 plusmn 04 120583gmL 46 plusmn15 120583gmL and 238 plusmn 10 120583gmL resp) while 21015840-O-trans-coumaroylmangiferin (3) was active against DENV-2 EMCVand HSV-1 but with lower EC50 values (Table 1 andFigure 4)

EEFFS (100 g) was also submitted to bioguided fractiona-tion Initially addition of cold methanol to the crude ethanolextract led to the separation of mangiferin (1) (4050mg)as the unsoluble fraction Fractionation of the methanolsoluble fraction (FFCMW) by preparative RP-HPLC affordedchrysin (4) that showed low activity against HSV-1 andVACV-WR (Table 1 and Figure 4)

Finally bioguided fractionation of EEFFS (100 g)by extraction with cold methanol led to mangiferin (1)(807mg) as an unsoluble part and FFFMW (methanolsoluble fraction) that on fractionation by preparative RP-HPLC afforded mangiferin (1) 21015840-O-trans-caffeoylmangi-ferinn (2) 21015840-O-trans-coumaroylmangiferin (3) and 21015840-O-trans-cinnamoylmangiferin (5) The last compound (5) washighly active against DENV-2 virus (EC50 35 plusmn 05120583gmL)and moderately active against HSV-1 (EC50 774 plusmn 43)(Table 1 and Figure 4)

4 Discussion

Phytochemical investigation of EtOH extracts of leavesstems and fruits from F formosa led to the isolationof five compounds Four of them were identified as C-glucosylxanthones namely mangiferin (1) along with threecinnamoyl esters of mangiferin (2 3 and 5) and oneflavonoid chrysin (4) (Figure 2) Chrysin (4) was isolatedfrom the stems extract and showed low antiviral activityagainst VACV-WR andHSV-1 with EC50 gt 100120583gml and didnot inhibit the replication cycle of DENV-2 and EMCVGoodactivity of this flavonoid against HSV-1 with an EC50 25120583Mwas previously reported [23] and our negative result might berelated to difference in susceptibility of strainsThis flavonoidis present in other species of the Bignoniaceae family suchas F samydoides [24] Previously isolated from Oroxylumindicum (Bignoniaceae) it is reported as disclosing in vitroanti-inflammatory and anticancer effects [25ndash27] Marketedas a bodybuilding supplement it is claimed to increase


0 100 200 3000

25

50

75

100

125

inhi

bitio

n

EEFFSEEFFFEEFFL

[] (120583gmL)

(a)

EEFFSEEFFF

EEFFLAcyclovir

0 100 200 3000

25

50

75

100

125

in

hibi

tion

[] (120583gmL)

(b)

0 100 200 300 400 5000

25

50

75

100

125

in

hibi

tion

EEFFSEEFFFEEFFL

[] (120583gmL)

(c)

0 50 100 1500

25

50

75

100

in

hibi

tion

[] (120583gmL)

EEFFSEEFFFEEFFL

(d)

Figure 3 Dose-response curves for antiviral activity of ethanol extracts from Fridericia formosa (a) leaves (EEFFL) stems (EEFFS) and fruits(EEFFF) against VAC-WR (b) EEFFL EEFFS and EEFFF against HSV-1 (c) EEFFL EEFFS and EEFFF against EMCV (d) EEFFL EEAFSand EEFFF against DENV-2

testosterone levels or stimulate testosterone production how-ever clinical tests have shown no effect on testosterone levelsin men [28]

As shown in Figure 2 the four xanthones isolated fromthe extracts of leaves stems and fruits of A formosa wereidentified as mangiferin (1) 21015840-O-trans-caffeoylmangiferin(2) 21015840-O-trans-coumaroylmangiferin (3) and 21015840-O-trans-cinnamoylmangiferin (5) The antiviral effect of these com-pounds was evaluated against DENV-2 EMCV HSV-1 andVACV-WR (Figure 3) Excepting mangiferin (1) the otherthree xanthones showed good antiviral effects and inhibitedthe replication cycle of DENV-2 HSV-1 and VACV-WR(Table 1 and Figure 4)

Mangiferin (1) is mainly obtained from mango tree(Mangifera indica) and preclinical studies showed thatit exhibits antidiabetic antioxidant antiviral cardiotonic

hypotensive and anti-inflammatory properties [29] Thebiological activities of mangiferin have been attributed tomodulating expression of a large number of genes that arecritical for the regulation of apoptosis viral replicationinflammation and various autoimmune diseases [30] Fur-thermore mangiferin disclosed low cytotoxicity and goodinhibitory activity on HIV-1 replication in a dose depen-dent manner [30 31] Mechanism studies revealed thatmangiferin might inhibit the HIV-1 protease and is there-fore a novel nonpeptide protease inhibitor of HIV protease[30 31]

Recent data on biological activity of mangiferin cin-namoyl esters showed that they are antioxidant agents similarto mangiferin and are antiplasmodial with moderate activityin vitro against Plasmodium falciparum 3D7 clone which ischloroquine-sensitive (IC50 181 to 265 120583M) [21]


MangiferinCaffeoylmangiferinCoumaroylmangiferin

ChrysinCinnamoylmangiferin

0

25

50

75

100

inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

50

75

100

125

in

hibi

tion

[] (120583gmL)


Chrysin

AcyclovirCinnamoylmangiferin

(b)

0

25

50

75

100

in

hibi

tion

0 100 200 300[] (120583gmL)

MangiferinCaffeoylmangiferinChrysin

(c)

0 25 50 75 1000

25

50

75

100

in

hibi

tion

[] (120583gmL)



(d)

Figure 4 Dose-response curves for antiviral activity of mangiferin (1) 21015840-O-trans-caffeoylmangiferin (2) 21015840-O-trans-coumaroylmangiferin(3) chrysin (4) and 21015840-O-trans-cinnamoylmangiferin (5) (a) Against VAC-WR (b) against HSV-1 (c) against EMCV (d) against DENV-2

Recently a total of twenty xanthones were isolated fromSwertia mussotii (Gentianaceae) and their antiviral activ-ity was evaluated [32] Eight of these xanthones exhib-ited significant activity against hepatitis B virus inhibit-ing DNA replication with EC50 values from 001mMto 013mM [32] Additionally the xanthones norbellid-ifolin 158-trihydroxy-3-methoxyxanthone and 2-C-120573-D-glucopyranosyl-137-trihydroxyxanthone showed remark-able activity with EC50 values of 077 gt 098 and 021mMfor hepatitis B surface antigen (HBsAg) and lt062 035 and004mM for hepatitis B antigen (HBeAg) respectively [32]Besides euxanthone from Garcinia oblongifolia (Clusiaceae)leaves extract disclosed significant activity in vitro againstEnterovirus 71 with EC50 value of 122 120583M [33] In additionthe selectivity index of this compound was 30 in relation to

the cytotoxicity to Vero cells (CC50 366 120583M) [33] Moleculardocking studies of 272 xanthones for interactions with agroup of seven fungal and two viral enzymes showed thatprenylated xanthones are important hits for inhibition of theselected enzymes [34] In general prenylated xanthones wereable to establish significantly stronger complexes with thetested enzymes [34] Some compounds were pointed out aspotential inhibitors for those enzymes including nigrolineax-anthone and latisxanthone D as probably potent inhibitors ofHIV-1 reverse transcriptase [34] Xanthones have importantadvantages as potential antiviral agents because of theiravailability as natural compounds and the possibility ofbeing easily synthesized and also for the demonstratedinteraction with some important microorganisms targets[34]


5 Conclusions

Our results reveal that F formosa is a rich source of man-giferin (1) (cong47 gsdotkgminus1 of dry leaves) a C-glucosyl xanthonewith several therapeutic and cosmetic uses [30] Its contentin F formosa is higher than in mango tree (Mangifera indica)its usual source (cong17 gsdotkgminus1 of dry peel) [35] Howevermangiferin was practically inactive against the virus assayedOn the other hand minor constituents represented bymangiferin cinnamoyl esters seem to be themain responsibleconstituents for the antiviral activity previously reported forextracts of different botanical parts of this species [8] Specialattention is called for the IS of these xanthone derivativesparticularly for the caffeoyl (2) and cinnamoyl (5) esters withIS gt 100 Our findings are the first report on the chemical andantiviral activity of F formosa constituents Our results arein line with the traditional use of Fridericia species as anti-infectious agents in different South American countries [1 9]and might be of interest for the development of standardizedantiviral phytomedicines

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

Thanks are due to Dr J A Lombardi Departamento deBotanica Instituto de Biociencias UNESP Rio ClaroBrazil for collection and taxonomical determination ofF formosa This work was supported by funds fromFAPEMIG (Fundacao de Amparo a Pesquisa do Estado deMinas Gerais Brazil) Process no CDS-APQ-00270-13 andCAPES (Coordenacao de Aperfeicoamrnto de Pessoal deEnsino Superior Brazil) and CNPq (Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico Brazil) are alsoacknowledged for fellowships toGeraldoCelio Brandao ErnaG Kroon and Alaıde Braga Oliveira

References

[1] G C Brandao E G Kroon D E R Souza J D S Filho andA B Oliveira ldquoChemistry and antiviral activity of Arrabidaeapulchra (Bignoniaceae)rdquoMolecules vol 18 no 8 pp 9919ndash99322013

[2] World Health Organization Dengue and Severe Dengue 2016httpwwwwhointmediacentrefactsheetsfs117en

[3] R V Gibbons ldquoDengue conundrumsrdquo International Journal ofAntimicrobial Agents vol 36 no 1 pp S36ndashS39 2010

[4] World Health Organization ldquoHerpes simplex virusrdquo httpwwwwhointmediacentrefactsheetsfs400en

[5] M S Oberste E Gotuzzo P Blair et al ldquoHuman febrileillness caused by encephalomyocarditis virus infection perurdquoEmerging Infectious Diseases vol 15 no 4 pp 640ndash646 2009

[6] J C Quixabeira-Santos M L G Medaglia C A Pescadorand C R Damaso ldquoAnimal movement and establishment ofvaccinia virus Cantagalo Strain in Amazon Biome BrazilrdquoEmerging Infectious Diseases vol 17 no 4 pp 726ndash729 2011

[7] G S Trindade M I C Guedes B P Drumond et al ldquoZoonoticvaccinia virus Clinical and immunological characteristics in anaturally infected patientrdquo Clinical Infectious Diseases vol 48no 3 pp e37ndashe40 2009

[8] G C Brandao E G Kroon J R Dos Santos J R StehmannJ A Lombardi and A Braga De Oliveira ldquoAntiviral activityof Bignoniaceae species occurring in the state of Minas Gerais(Brazil) Part 1rdquo Letters in Applied Microbiology vol 51 no 4pp 469ndash476 2010

[9] G C Brandao E G Kroon J R dos Santos J R Stehmann JA Lombardi andA B deOliveira ldquoAntiviral activities of plantsoccurring in the state of Minas Gerais Brazil Part 2 Screeningbignoniaceae speciesrdquo Brazilian Journal of Pharmacognosy vol20 no 5 pp 742ndash750 2010

[10] G C Brandao E G Kroon J R dos Santos J R StehmannJ A Lombardi and A B Oliveira ldquoAntiviral activity of plantsoccurring in the state of minas gerais (Brazil) Part IIIrdquo Journalof Chemical and Pharmaceutical Research vol 3 no 4 pp 223ndash236 2011

[11] L R Simoes G M Maciel G C Brandao E G Kroon RO Castilho and A B Oliveira ldquoAntiviral activity of Distictellaelongata (Vahl) Urb (Bignoniaceae) a potentially useful sourceof anti-dengue drugs from the state of Minas Gerais BrazilrdquoLetters in Applied Microbiology vol 53 no 6 pp 602ndash607 2011

[12] G C Brandao E G Kroon A W Matosa J D SouzaFilho and A B Oliveira ldquoBioguided isolation of an antiviralcompound from Xylophragma myrianthum (Cham) Sprague(Bignoniaceae Juss)rdquo Revista Fitos vol 8 no 2 pp 125ndash1362013

[13] D J De Rodriguez J Chulia C M O Simoes M Amoros AM Mariotte and L Girre ldquoSearch for rsquoin vitrorsquo antiviral activityof a new isoflavonec glycoside from ulex europaeusrdquo PlantaMedica vol 56 no 1 pp 59ndash62 1990

[14] P R Twentyman and M Luscombe ldquoA study of some variablesin a tetrazolium dye (MTT) based assay for cell growth andchemosensitivityrdquo British Journal of Cancer vol 56 no 3 pp279ndash285 1987

[15] L A Betancur-Galvis S J H Granados A Salazar and J EOssa ldquoAntitumor and antiviral activity of colombian medicinalplant extractsrdquoMemorias do Instituto Oswaldo Cruz vol 94 no4 pp 531ndash535 1999

[16] R A Finnegan R A Stephani G Ganguli S N Gangulyand A K Bhattacharya ldquoOccurrence of mangiferin in Hiptagemadablota geartnrdquo Journal of Pharmaceutical Sciences vol 57no 6 pp 1039-1040 1968

[17] M P Yuldashev and A Karimov ldquoFlavonoids of Scutellariaimmaculata rootsrdquoChemistry ofNatural Compounds vol 41 no1 pp 32ndash34 2005

[18] R K Chaudhuri and S Ghosal ldquoXanthones of Canscoradecussata schultrdquo Phytochemistry vol 10 no 10 pp 2425ndash24321971

[19] S Catalano S Luschi G Flamini P L Cioni E M Nieriand I Morelli ldquoA xanthone from Senecio mikanioides leavesrdquoPhytochemistry vol 42 no 6 pp 1605ndash1607 1996

[20] P Mendonca Pauletti I Castro-Gamboa D H Siqueira Silvaet al ldquoNew Antioxidant C-Glucosylxanthones from the Stemsof Arrabidaea samydoidesrdquo Journal of Natural Products vol 66no 10 pp 1384ndash1387 2003

[21] F Martin A-E Hay D Cressend et al ldquoAntioxidant C-glucosylxanthones from the leaves of Arrabidaea patelliferardquoJournal of Natural Products vol 71 no 11 pp 1887ndash1890 2008


[22] P K Agrawal Carbon-13 NMR of Flavonoids vol 28 ElsevierAmsterdam Nederland IST edition 1989

[23] S-Y Lyu J-Y Rhim andW-B Park ldquoAntiherpetic activities offlavonoids against herpes simplex virus type 1 (HSV-1) and type2 (HSV-2) in vitrordquo Archives of Pharmacal Research vol 28 no11 pp 1293ndash1301 2005

[24] P M Pauletti V s Bolzani and M C M Young ldquoChemicalconstituents of Arrabidaea samydoides (Bignoniaceae)rdquo Quim-ica Nova vol 26 no 5 pp 641ndash643 2003

[25] L-J Chen D E Games and J Jones ldquoIsolation and identifica-tion of four flavonoid constituents from the seeds of Oroxylumindicum by high-speed counter-current chromatographyrdquo Jour-nal of Chromatography A vol 988 no 1 pp 95ndash105 2003

[26] K J Woo Y-J Jeong H Inoue J-W Park and T K KwonldquoChrysin suppresses lipopolysaccharide-induced cyclooxygen-ase-2 expression through the inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activityrdquo FEBS Letters vol 579 no 3pp 705ndash711 2005

[27] K J Woo Y-J Jeong J-W Park and T K Kwon ldquoChrysin-induced apoptosis is mediated through caspase activation andAkt inactivation in U937 leukemia cellsrdquo Biochemical andBiophysical Research Communications vol 325 no 4 pp 1215ndash1222 2004

[28] C Gambelunghe R Rossi M Sommavilla et al ldquoEffects ofchrysin on urinary testosterone levels in humanmalesrdquo Journalof Medicinal Food vol 6 no 4 pp 387ndash390 2003

[29] K Shah M Patel R Patel and P Parmar ldquoMangifera indica(mango)rdquo Pharmacognosy Reviews vol 4 no 7 pp 42ndash48 2010

[30] M Telang S Dhulap A Mandhare and R Hirwani ldquoTher-apeutic and cosmetic applications of mangiferin A patentreviewrdquo Expert Opinion on Therapeutic Patents vol 23 no 12pp 1561ndash1580 2013

[31] R-R Wang Y-D Gao C-H Ma et al ldquoMangiferin an anti-HIV-1 agent targeting protease and effective against resistantstrainsrdquoMolecules vol 16 no 5 pp 4264ndash4277 2011

[32] T-W Cao C-A Geng Y-B Ma et al ldquoXanthones with anti-hepatitis B virus activity from Swertia mussotiirdquo PlantaMedicavol 79 no 8 pp 697ndash700 2013

[33] H Zhang L Tao andW-W Fu ldquoPrenylated benzoylphloroglu-cinols and xanthones from the leaves of Garcinia oblongifoliawith antienteroviral activityrdquo Journal of Natural Products vol77 no 4 pp 1037ndash1046 2014

[34] F A Bernal and E Coy-Barrera ldquoMolecular docking andmultivariate analysis of xanthones as antimicrobial and antiviralagentsrdquoMolecules vol 20 no 7 pp 13165ndash13204 2015

[35] M Masibo and H Qian ldquoMajor mango polyphenols and theirpotential significance to human healthrdquoComprehensive Reviewsin Food Science and Food Safety vol 7 no 4 pp 309ndash319 2008


and encephalitis in domestic pigs in nonhuman primates andother mammalian species There are few reports of cases ofhuman infection by EMCV [5]

Vaccinia (VACV) is a virus of the genus Orthopoxvirusof the family Poxviridae that in humans causes nonlethalpustular and localized disease The vaccinia virus does nothave natural hosts but cases of bovine and human infectionby vaccinia virus are reported in Brazil and India causingeconomic losses and affecting health services [6 7]

As part of a bioprospecting project whose main goalis to discover potential antiviral natural products of plantsfrom Brazilian Cerrado and Atlantic Forest biomes wehave screened several species of plants collected in thestate of Minas Gerais [1 8ndash12] Among these Fridericiaformosa (Bureau) Sandwith was chosen for bioguided phy-tochemical investigation due to the good antiviral activitypresented by the ethanol extracts of leaves stems andfruits

2 Materials and Methods

21 Collection Taxonomical Determination and Processing ofPlant Materials F formosa was collected in the municipalityof Belo Horizonte Minas Gerais Brazil The plant was taxo-nomically identified by Dr J A Lombardi Departamento deBotanica Instituto de Biociencias UNESP Rio Claro BrazilA voucher specimen was deposited at the BHCBUFMGBelo Horizonte Minas Gerais Brazil under the number23885

22 Preparation of Extracts After drying in air circulatingoven at 40∘C for 72 h the plant materialmdash1363 g of leaves4615 g of stems and 138 g fruitsmdashwere ground and extractedby percolation with 96 EtOH at room temperature Thesolvent was removed in a rotary evaporator under reducedpressure at 50∘C leaving dark residuesmdashEEFFL 342 gEEFFS 600 g and EEFFF 25 gmdashfor leaves stems and fruitsrespectively which were kept in a vacuum desiccator untilconstant weight

23 HPLC Analyses In the HPLC analyses an exploratorygradient elution was used [1 9 10] Fingerprints wereregistered by RP-HPLC-DAD on a Waters 2695 apparatusequipped with a UV-DADdetector (Waters 2996) A LiChro-spher 100 RP-18 column (5 120583m 250 times 4mm id MerckDarmstadt Germany) was employed at 40∘C flow rate of10mLmin and detection at wavelengths of 220 280 and350 nm To an aliquot 100mg of dried extractfractionsand 10mg of each of the isolated compounds HPLC grademethanol was added and the mixture was dissolved bysonication in an ultrasound bath for 15min followed bycentrifugation at 10000 rpm for 10min The supernatant wasfiltered through a Millipore membrane (02 120583m) and injected(100 120583L) onto the equipment Elution was carried out with alinear gradient of water (a) and acetonitrile (b) (from 5 to95 of B in 60min)

24 Isolation of Chemical Components from Leaves ExtractTo a portion of EEFFL (100 g) MeOH was added and

an insoluble precipitate was separated by filtration throughsintered glass funnel and washed thoroughly with MeOHyielding 28 g The precipitate was recrystallized out frommethanolwater (1 1) giving 19 g of compound 1 Thefiltrate was dried in a rotary evaporator under reducedpressure at 50∘C leaving a dark residue (FFLMW 70 g)AFLMW was subjected to fractionation over a silica gel col-umn with n-hexaneCH2Cl2 (1 1) CH2Cl2 CH2Cl2EtOAc(1 1) EtOAcMeOH (2 1) EtOAcMeOH (1 2) MeOHMeOHH2O (2 1) and MeOHH2O (1 2) as eluents Aportion of EtOAcMeOH (2 1) fraction 2 (10 g) was filteredover a Sephadex LH 20 column with MeOH as eluent giving12 fractions Fractions 11 and 12 were combined leadingto a yellow solid (5540mg) which was further purifiedby HPLC employing Shim-pack PRC-ODS column (20mmid times 25 cm flow rate 50mlmin) and a gradient elutionwith MeOHH2O 45 to 95 for 50 minutes Compounds 2(223mg) and 3 (561mg) were isolated

25 Isolation of Chemical Components from Stem Extract Toa portion of EEFFS (100 g) cold MeOH was added giving aprecipitate which was separated by filtration on sintered glassfunnel and washed thoroughly with MeOH yielding 243 gThe precipitate was recrystallized out twice from MeOH togive 4050mg of compound 1 The mother liquor from thesecond recrystallization was dried in a rotary evaporatorunder reduced pressure at 50∘C leaving a yellow residue(FFSMW2 12 g) that was further subjected to fractionationby HPLC employing Shim-pack PRC-ODS column (20mmid times 25 cm flow rate 50mlmin) and a gradient elution withMeOHH2O 45 to 95 for 50 minutes affording compound 4(5110mg)

26 Isolation of Chemical Components from Fruit ExtractTo a portion of EEFFF (10 g) MeOH was added and aninsoluble precipitate was separated by filtration on sin-tered glass funnel and washed thoroughly with methanolThe precipitate was subjected to recrystallization to givecompound 1 (807mg) The filtrate was dried in a rotaryevaporator under reduced pressure at 50∘C leaving a yellowresidue (FFFMW 8097mg) that was further purified byHPLC employing Shim-pack PRC-ODS column (20mmid times 25 cm flow rate 50mlmin) and a gradient elution withMeOHH2O45 to 95 for 50minutes leading to the isolationof compounds 1 (963mg) 2 (134mg) 3 (829mg) and 5(846mg)

27 Cell Culture and Virus Vero cells (ATCC CCL-81) andLLCMK2 cells were cultured in Dulbeccorsquos modified Eaglersquosmedium (DMEM Cultilab Campinas SP Brazil) at 37∘Cin 5 CO2 atmosphere supplemented with 5 fetal bovineserum 50 120583gmL gentamicin 100UmL penicillin and5 120583gmL amphotericin B [1]

HSV-1 was obtained from the collection of Laboratoriode Virus UFMG Belo Horizonte Brazil DENV-2 EMCVand VACV-WR were kindly donated by Dr L Figueiredo(USP Ribeirao Preto Brazil) Dr I Kerr (London ResearchInstitute London UK) and Dr C Jungwirth (University of














3 Results









Table1Cy

totoxicity(C

C 50V

eroandLL

CMK 2

cells)in

vitro

antiv

iralactivity


index(SI)fore

thanolextractsfro

mFridericiaform

osaleaves(EEF

FL)ste

ms(EE

FFS)

fruit(EE

FFF)fractionsand

compo

unds

1ndash5

Extracts

fractio

nscom

poun

dVe

roCC50120583gmL

LLCM

K 2CC50120583gmL

a HSV

-1EC50120583gmL

SIb VAC

V-WR

EC50120583gmL

SIc EMCV

EC50120583gmL

SId D

ENV-2

EC50120583gmL

SI

EEFF

Sgt500

1739plusmn98

932plusmn54

gt54

592plusmn24

gt84

3225plusmn144

gt15

426plusmn23

41

EEFF

Fgt500

gt500

1478plusmn24

gt34

2527plusmn39

gt20

1344plusmn59

gt37

131plusmn16

gt382

EEAFL

gt500

gt500

856plusmn41

gt58

837plusmn31

gt60

1994plusmn138

gt25

163plusmn68

gt307

FFHDF(11)

2220plusmn73

867plusmn85

NA

NA

NA

NA

FFDF

2633plusmn139

951plusmn93

NA

NA

NA

NA

FFDEF

(11)

507plusmn25

138plusmn21

NA

NA

NA

39plusmn036

35

FFEF

gt500

gt500

NA

NA

NA

NA

FFEM

F(11)

gt500

gt500

1697plusmn210

gt29

1829plusmn114

gt27

1905plusmn147

gt26

318plusmn57

gt157

FFMF

gt500

gt5 00

503plusmn28

gt99

NA

NA

418plusmn56

gt120

FFMWF(21)

gt500

gt500

357plusmn20

gt140

NA

NA

228plusmn08

gt219

FFMWF(12)

gt500

gt500

NA

NA

NA

NA

Mangiferin

(1)

gt500

gt500

2679plusmn67

(6348plusmn159)gt19

1827plusmn143

(4325plusmn339)gt27

NA

2655plusmn140

(6291plusmn332)gt19

21015840-O


(2)

gt500

gt500

46plusmn15

(79plusmn26)

gt1087

238plusmn10

(407plusmn17

)gt210

NT

41plusmn

04

(70plusmn07)

gt1219

21015840-O

-Trans-cou

maroylm

angiferin

(3)gt500

gt500

474plusmn61

(834plusmn107)gt105

NA

2410plusmn318

(4243plusmn560)gt21

404plusmn42

(711plusmn74

)gt124

Chrysin

(4)

gt500

gt500

1463plusmn159


1235plusmn105

(4862plusmn413)gt40

NA

NA

21015840-O

-Trans-cinnamoylm

angiferin

(5)gt500

gt500

774plusmn43

(1402plusmn78

)gt65

NA

NT

35plusmn05

(63plusmn09)

gt1489

Acyclovir

gt1000

gt1000

40f

Interfe

ron120572


fg15times102

fg25times103

fg25times103

SIsele

ctivity

index

a vira

ltiterTC

ID50m

L25times106in

48h

b vira

ltiterTC

ID50m

L10times106in

48h

c vira

ltiterTC

ID50m

L10times106in

48h

d vira

ltiterTC

ID50m

L10times104in

72h

NAn

oactiv

ityin

theassayedconcentra

tionsN

Tno

test

e con

centratio

nin120583Mf 80to

100

inhibitio

nof

cytopathiceffect

g con

centratio

nin

UIm

LEE

FFL

ethano

lextract

from

Fridericiaform

osaleavesF

FHDFFridericia

form


ethane

11fraction

FFDFFridericiaform

osadichloromethane

fractio

nFF

DEFFrid

ericiaform


FFEFFrid

ericiaform

osaethylacetate

fractio

nFF

EMFFridericiaform

osaethylacetatem

ethano

l11

fractio

nFF

MFFridericiaform

osaethylm

ethano

lfraction

FFMWFFridericiaform

osamethano

lwater

21fraction

FFMWFFridericiaform

osa

methano

lwater

12fractio

nFridericiaform

osafractio

nsfro

mchromatograph

yof

EEFF

Lover

silicag

elcolumn


400

200

000

(AU

) (AU

)

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

010

005

(nm)(nm)25000 30000 3500025000 30000 35000

(AU

)

002


(a)

(AU

) (AU

)(nm)

25000 30000 35000

0020

0010

0000

000

050

100RT = 265min

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(b)

002

018

(AU

) (AU

)

000

100

200

300

RT = 136min

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

(nm)25000 30000 35000

(c)

RT = 176min

(AU

)

000

100

200

300

(AU

)

100

200

300

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(nm)25000 30000 35000

(d)



O

O

HO

OH

O

OOH

HO OH

OHO

O

OH

HO

HO

O

OOH

HO OH

OHO

OH

OH

HO

HO


Chrysin (4)

Mangiferin (1)

R1

R2





4 Discussion



0 100 200 3000

25

50

75

100

125

inhi

bitio

n

EEFFSEEFFFEEFFL

[] (120583gmL)

(a)

EEFFSEEFFF

EEFFLAcyclovir

0 100 200 3000

25

50

75

100

125

in

hibi

tion

[] (120583gmL)

(b)

0 100 200 300 400 5000

25

50

75

100

125

in

hibi

tion

EEFFSEEFFFEEFFL

[] (120583gmL)

(c)

0 50 100 1500

25

50

75

100

in

hibi

tion

[] (120583gmL)

EEFFSEEFFFEEFFL

(d)










0

25

50

75

100

inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

50

75

100

125

in

hibi

tion

[] (120583gmL)


Chrysin


(b)

0

25

50

75

100

in

hibi

tion

0 100 200 300[] (120583gmL)


(c)

0 25 50 75 1000

25

50

75

100

in

hibi

tion

[] (120583gmL)



(d)





5 Conclusions




Acknowledgments


References


















































3 Results









Table1Cy

totoxicity(C

C 50V

eroandLL

CMK 2

cells)in

vitro

antiv

iralactivity


index(SI)fore

thanolextractsfro

mFridericiaform

osaleaves(EEF

FL)ste

ms(EE

FFS)

fruit(EE

FFF)fractionsand

compo

unds

1ndash5

Extracts

fractio

nscom

poun

dVe

roCC50120583gmL

LLCM

K 2CC50120583gmL

a HSV

-1EC50120583gmL

SIb VAC

V-WR

EC50120583gmL

SIc EMCV

EC50120583gmL

SId D

ENV-2

EC50120583gmL

SI

EEFF

Sgt500

1739plusmn98

932plusmn54

gt54

592plusmn24

gt84

3225plusmn144

gt15

426plusmn23

41

EEFF

Fgt500

gt500

1478plusmn24

gt34

2527plusmn39

gt20

1344plusmn59

gt37

131plusmn16

gt382

EEAFL

gt500

gt500

856plusmn41

gt58

837plusmn31

gt60

1994plusmn138

gt25

163plusmn68

gt307

FFHDF(11)

2220plusmn73

867plusmn85

NA

NA

NA

NA

FFDF

2633plusmn139

951plusmn93

NA

NA

NA

NA

FFDEF

(11)

507plusmn25

138plusmn21

NA

NA

NA

39plusmn036

35

FFEF

gt500

gt500

NA

NA

NA

NA

FFEM

F(11)

gt500

gt500

1697plusmn210

gt29

1829plusmn114

gt27

1905plusmn147

gt26

318plusmn57

gt157

FFMF

gt500

gt5 00

503plusmn28

gt99

NA

NA

418plusmn56

gt120

FFMWF(21)

gt500

gt500

357plusmn20

gt140

NA

NA

228plusmn08

gt219

FFMWF(12)

gt500

gt500

NA

NA

NA

NA

Mangiferin

(1)

gt500

gt500

2679plusmn67

(6348plusmn159)gt19

1827plusmn143

(4325plusmn339)gt27

NA

2655plusmn140

(6291plusmn332)gt19

21015840-O


(2)

gt500

gt500

46plusmn15

(79plusmn26)

gt1087

238plusmn10

(407plusmn17

)gt210

NT

41plusmn

04

(70plusmn07)

gt1219

21015840-O

-Trans-cou

maroylm

angiferin

(3)gt500

gt500

474plusmn61

(834plusmn107)gt105

NA

2410plusmn318

(4243plusmn560)gt21

404plusmn42

(711plusmn74

)gt124

Chrysin

(4)

gt500

gt500

1463plusmn159


1235plusmn105

(4862plusmn413)gt40

NA

NA

21015840-O

-Trans-cinnamoylm

angiferin

(5)gt500

gt500

774plusmn43

(1402plusmn78

)gt65

NA

NT

35plusmn05

(63plusmn09)

gt1489

Acyclovir

gt1000

gt1000

40f

Interfe

ron120572


fg15times102

fg25times103

fg25times103

SIsele

ctivity

index

a vira

ltiterTC

ID50m

L25times106in

48h

b vira

ltiterTC

ID50m

L10times106in

48h

c vira

ltiterTC

ID50m

L10times106in

48h

d vira

ltiterTC

ID50m

L10times104in

72h

NAn

oactiv

ityin

theassayedconcentra

tionsN

Tno

test

e con

centratio

nin120583Mf 80to

100

inhibitio

nof

cytopathiceffect

g con

centratio

nin

UIm

LEE

FFL

ethano

lextract

from

Fridericiaform

osaleavesF

FHDFFridericia

form


ethane

11fraction

FFDFFridericiaform

osadichloromethane

fractio

nFF

DEFFrid

ericiaform


FFEFFrid

ericiaform

osaethylacetate

fractio

nFF

EMFFridericiaform

osaethylacetatem

ethano

l11

fractio

nFF

MFFridericiaform

osaethylm

ethano

lfraction

FFMWFFridericiaform

osamethano

lwater

21fraction

FFMWFFridericiaform

osa

methano

lwater

12fractio

nFridericiaform

osafractio

nsfro

mchromatograph

yof

EEFF

Lover

silicag

elcolumn


400

200

000

(AU

) (AU

)

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

010

005

(nm)(nm)25000 30000 3500025000 30000 35000

(AU

)

002


(a)

(AU

) (AU

)(nm)

25000 30000 35000

0020

0010

0000

000

050

100RT = 265min

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(b)

002

018

(AU

) (AU

)

000

100

200

300

RT = 136min

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

(nm)25000 30000 35000

(c)

RT = 176min

(AU

)

000

100

200

300

(AU

)

100

200

300

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(nm)25000 30000 35000

(d)



O

O

HO

OH

O

OOH

HO OH

OHO

O

OH

HO

HO

O

OOH

HO OH

OHO

OH

OH

HO

HO


Chrysin (4)

Mangiferin (1)

R1

R2





4 Discussion



0 100 200 3000

25

50

75

100

125

inhi

bitio

n

EEFFSEEFFFEEFFL

[] (120583gmL)

(a)

EEFFSEEFFF

EEFFLAcyclovir

0 100 200 3000

25

50

75

100

125

in

hibi

tion

[] (120583gmL)

(b)

0 100 200 300 400 5000

25

50

75

100

125

in

hibi

tion

EEFFSEEFFFEEFFL

[] (120583gmL)

(c)

0 50 100 1500

25

50

75

100

in

hibi

tion

[] (120583gmL)

EEFFSEEFFFEEFFL

(d)










0

25

50

75

100

inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

50

75

100

125

in

hibi

tion

[] (120583gmL)


Chrysin


(b)

0

25

50

75

100

in

hibi

tion

0 100 200 300[] (120583gmL)


(c)

0 25 50 75 1000

25

50

75

100

in

hibi

tion

[] (120583gmL)



(d)





5 Conclusions




Acknowledgments


References






































Table1Cy

totoxicity(C

C 50V

eroandLL

CMK 2

cells)in

vitro

antiv

iralactivity


index(SI)fore

thanolextractsfro

mFridericiaform

osaleaves(EEF

FL)ste

ms(EE

FFS)

fruit(EE

FFF)fractionsand

compo

unds

1ndash5

Extracts

fractio

nscom

poun

dVe

roCC50120583gmL

LLCM

K 2CC50120583gmL

a HSV

-1EC50120583gmL

SIb VAC

V-WR

EC50120583gmL

SIc EMCV

EC50120583gmL

SId D

ENV-2

EC50120583gmL

SI

EEFF

Sgt500

1739plusmn98

932plusmn54

gt54

592plusmn24

gt84

3225plusmn144

gt15

426plusmn23

41

EEFF

Fgt500

gt500

1478plusmn24

gt34

2527plusmn39

gt20

1344plusmn59

gt37

131plusmn16

gt382

EEAFL

gt500

gt500

856plusmn41

gt58

837plusmn31

gt60

1994plusmn138

gt25

163plusmn68

gt307

FFHDF(11)

2220plusmn73

867plusmn85

NA

NA

NA

NA

FFDF

2633plusmn139

951plusmn93

NA

NA

NA

NA

FFDEF

(11)

507plusmn25

138plusmn21

NA

NA

NA

39plusmn036

35

FFEF

gt500

gt500

NA

NA

NA

NA

FFEM

F(11)

gt500

gt500

1697plusmn210

gt29

1829plusmn114

gt27

1905plusmn147

gt26

318plusmn57

gt157

FFMF

gt500

gt5 00

503plusmn28

gt99

NA

NA

418plusmn56

gt120

FFMWF(21)

gt500

gt500

357plusmn20

gt140

NA

NA

228plusmn08

gt219

FFMWF(12)

gt500

gt500

NA

NA

NA

NA

Mangiferin

(1)

gt500

gt500

2679plusmn67

(6348plusmn159)gt19

1827plusmn143

(4325plusmn339)gt27

NA

2655plusmn140

(6291plusmn332)gt19

21015840-O


(2)

gt500

gt500

46plusmn15

(79plusmn26)

gt1087

238plusmn10

(407plusmn17

)gt210

NT

41plusmn

04

(70plusmn07)

gt1219

21015840-O

-Trans-cou

maroylm

angiferin

(3)gt500

gt500

474plusmn61

(834plusmn107)gt105

NA

2410plusmn318

(4243plusmn560)gt21

404plusmn42

(711plusmn74

)gt124

Chrysin

(4)

gt500

gt500

1463plusmn159


1235plusmn105

(4862plusmn413)gt40

NA

NA

21015840-O

-Trans-cinnamoylm

angiferin

(5)gt500

gt500

774plusmn43

(1402plusmn78

)gt65

NA

NT

35plusmn05

(63plusmn09)

gt1489

Acyclovir

gt1000

gt1000

40f

Interfe

ron120572


fg15times102

fg25times103

fg25times103

SIsele

ctivity

index

a vira

ltiterTC

ID50m

L25times106in

48h

b vira

ltiterTC

ID50m

L10times106in

48h

c vira

ltiterTC

ID50m

L10times106in

48h

d vira

ltiterTC

ID50m

L10times104in

72h

NAn

oactiv

ityin

theassayedconcentra

tionsN

Tno

test

e con

centratio

nin120583Mf 80to

100

inhibitio

nof

cytopathiceffect

g con

centratio

nin

UIm

LEE

FFL

ethano

lextract

from

Fridericiaform

osaleavesF

FHDFFridericia

form


ethane

11fraction

FFDFFridericiaform

osadichloromethane

fractio

nFF

DEFFrid

ericiaform


FFEFFrid

ericiaform

osaethylacetate

fractio

nFF

EMFFridericiaform

osaethylacetatem

ethano

l11

fractio

nFF

MFFridericiaform

osaethylm

ethano

lfraction

FFMWFFridericiaform

osamethano

lwater

21fraction

FFMWFFridericiaform

osa

methano

lwater

12fractio

nFridericiaform

osafractio

nsfro

mchromatograph

yof

EEFF

Lover

silicag

elcolumn


400

200

000

(AU

) (AU

)

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

010

005

(nm)(nm)25000 30000 3500025000 30000 35000

(AU

)

002


(a)

(AU

) (AU

)(nm)

25000 30000 35000

0020

0010

0000

000

050

100RT = 265min

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(b)

002

018

(AU

) (AU

)

000

100

200

300

RT = 136min

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

(nm)25000 30000 35000

(c)

RT = 176min

(AU

)

000

100

200

300

(AU

)

100

200

300

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(nm)25000 30000 35000

(d)



O

O

HO

OH

O

OOH

HO OH

OHO

O

OH

HO

HO

O

OOH

HO OH

OHO

OH

OH

HO

HO


Chrysin (4)

Mangiferin (1)

R1

R2





4 Discussion



0 100 200 3000

25

50

75

100

125

inhi

bitio

n

EEFFSEEFFFEEFFL

[] (120583gmL)

(a)

EEFFSEEFFF

EEFFLAcyclovir

0 100 200 3000

25

50

75

100

125

in

hibi

tion

[] (120583gmL)

(b)

0 100 200 300 400 5000

25

50

75

100

125

in

hibi

tion

EEFFSEEFFFEEFFL

[] (120583gmL)

(c)

0 50 100 1500

25

50

75

100

in

hibi

tion

[] (120583gmL)

EEFFSEEFFFEEFFL

(d)










0

25

50

75

100

inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

50

75

100

125

in

hibi

tion

[] (120583gmL)


Chrysin


(b)

0

25

50

75

100

in

hibi

tion

0 100 200 300[] (120583gmL)


(c)

0 25 50 75 1000

25

50

75

100

in

hibi

tion

[] (120583gmL)



(d)





5 Conclusions




Acknowledgments


References






































400

200

000

(AU

) (AU

)

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

010

005

(nm)(nm)25000 30000 3500025000 30000 35000

(AU

)

002


(a)

(AU

) (AU

)(nm)

25000 30000 35000

0020

0010

0000

000

050

100RT = 265min

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(b)

002

018

(AU

) (AU

)

000

100

200

300

RT = 136min

000 1000 2000 3000 4000 5000 6000 7000

(Minutes)

(nm)25000 30000 35000

(c)

RT = 176min

(AU

)

000

100

200

300

(AU

)

100

200

300

000 1000 2000 3000 4000 5000 6000 7000(Minutes)

(nm)25000 30000 35000

(d)



O

O

HO

OH

O

OOH

HO OH

OHO

O

OH

HO

HO

O

OOH

HO OH

OHO

OH

OH

HO

HO


Chrysin (4)

Mangiferin (1)

R1

R2





4 Discussion



0 100 200 3000

25

50

75

100

125

inhi

bitio

n

EEFFSEEFFFEEFFL

[] (120583gmL)

(a)

EEFFSEEFFF

EEFFLAcyclovir

0 100 200 3000

25

50

75

100

125

in

hibi

tion

[] (120583gmL)

(b)

0 100 200 300 400 5000

25

50

75

100

125

in

hibi

tion

EEFFSEEFFFEEFFL

[] (120583gmL)

(c)

0 50 100 1500

25

50

75

100

in

hibi

tion

[] (120583gmL)

EEFFSEEFFFEEFFL

(d)










0

25

50

75

100

inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

50

75

100

125

in

hibi

tion

[] (120583gmL)


Chrysin


(b)

0

25

50

75

100

in

hibi

tion

0 100 200 300[] (120583gmL)


(c)

0 25 50 75 1000

25

50

75

100

in

hibi

tion

[] (120583gmL)



(d)





5 Conclusions




Acknowledgments


References






































O

O

HO

OH

O

OOH

HO OH

OHO

O

OH

HO

HO

O

OOH

HO OH

OHO

OH

OH

HO

HO


Chrysin (4)

Mangiferin (1)

R1

R2





4 Discussion



0 100 200 3000

25

50

75

100

125

inhi

bitio

n

EEFFSEEFFFEEFFL

[] (120583gmL)

(a)

EEFFSEEFFF

EEFFLAcyclovir

0 100 200 3000

25

50

75

100

125

in

hibi

tion

[] (120583gmL)

(b)

0 100 200 300 400 5000

25

50

75

100

125

in

hibi

tion

EEFFSEEFFFEEFFL

[] (120583gmL)

(c)

0 50 100 1500

25

50

75

100

in

hibi

tion

[] (120583gmL)

EEFFSEEFFFEEFFL

(d)










0

25

50

75

100

inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

50

75

100

125

in

hibi

tion

[] (120583gmL)


Chrysin


(b)

0

25

50

75

100

in

hibi

tion

0 100 200 300[] (120583gmL)


(c)

0 25 50 75 1000

25

50

75

100

in

hibi

tion

[] (120583gmL)



(d)





5 Conclusions




Acknowledgments


References






































0 100 200 3000

25

50

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125

inhi

bitio

n

EEFFSEEFFFEEFFL

[] (120583gmL)

(a)

EEFFSEEFFF

EEFFLAcyclovir

0 100 200 3000

25

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125

in

hibi

tion

[] (120583gmL)

(b)

0 100 200 300 400 5000

25

50

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125

in

hibi

tion

EEFFSEEFFFEEFFL

[] (120583gmL)

(c)

0 50 100 1500

25

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in

hibi

tion

[] (120583gmL)

EEFFSEEFFFEEFFL

(d)










0

25

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inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

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125

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hibi

tion

[] (120583gmL)


Chrysin


(b)

0

25

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100

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hibi

tion

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(c)

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tion

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(d)





5 Conclusions




Acknowledgments


References








































0

25

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inhi

bitio

n

0 100 200 300[] (120583gmL)

(a)

0 25 50 75 100 125 1500

25

50

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100

125

in

hibi

tion

[] (120583gmL)


Chrysin


(b)

0

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100

in

hibi

tion

0 100 200 300[] (120583gmL)


(c)

0 25 50 75 1000

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in

hibi

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[] (120583gmL)



(d)





5 Conclusions




Acknowledgments


References






































5 Conclusions




Acknowledgments


References





































Antiviral Activity of Fridericia formosa (Bureau) L. G ... · Antiviral Activity of Fridericia...

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