Research ArticleAntiviral Action of Hydromethanolic Extract ofGeopropolis from Scaptotrigona postica againstAntiherpes Simplex Virus (HSV-1)

Guilherme Rabelo Coelho1 Ronaldo Zucatelli Mendonccedila1

Karina de Senna Vilar1 Cristina Adelaide Figueiredo2 Juliana Cuoco Badari1

Noemi Taniwaki2 Gisleine Namiyama2 Maria Isabel de Oliveira2

Suely Pires Curti2 Patricia Evelyn Silva2 and Giuseppina Negri3

1 Instituto Butantan Avenida Vital Brasil No 1500 Butanta 05503-900 Sao Paulo SP Brazil2Instituto Adolfo Lutz Avenida Dr Arnaldo 355 01246-900 Sao Paulo SP Brazil3CEBRID Departamento de Medicina Preventiva Universidade Federal de Sao Paulo Rua Botucatu 740Vila Clementino 04023-900 Sao Paulo SP Brazil

Correspondence should be addressed to Giuseppina Negri gnegriterracombr

Received 26 November 2014 Revised 13 February 2015 Accepted 15 February 2015

Academic Editor Andreas Sandner-Kiesling

Copyright copy 2015 Guilherme Rabelo Coelho et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The studies on chemical composition and biological activity of propolis had focused mainly on species Apis mellifera L(Hymenoptera Apidae) There are few studies about the uncommon propolis collected by stingless bees of the Meliponini tribeknown as geopropolis The geopropolis from Scaptotrigona postica was collected in the region of Barra do Corda Maranhao stateBrazil The chemical analysis of hydromethanolic extract of this geopropolis (HMG) was carried out through HPLC-DAD-ESI-MSMS and the main constituents found were pyrrolizidine alkaloids and C-glycosyl flavonesThe presence of alkaloids in extractsof propolis is detected for the first time in this sampleThe antiviral activity ofHMGwas evaluated through viral DNAquantificationexperiments and electron microscopy experiments Quantification of viral DNA from herpes virus showed reduction of about 98in all conditions and concentration tested of the HMG extract The results obtained were corroborated by transmission electronmicroscopy in which the images did not show particle or viral replication complexThe antiviral activity of C-glycosyl flavones wasreported for a variety of viruses being observed at different points in the viral replication This work is the first report about theantiviral activity of geopropolis from Scaptotrigona postica in vitro against antiherpes simplex virus (HSV)

1 Introduction

Propolis is a resinous material comprising plant exudatesand wax used by bees for sealing the hive and as protectionagainst microorganisms [1 2] The studies about chemicalcomposition and biological activity of propolis had focusedmainly on species Apis mellifera L (Hymenoptera Apidae)The uncommon propolis collected by stingless bees of theMeliponini tribe is a mixture of resin wax and soil known asgeopropolis Stingless bees are widely found in tropical andsubtropical areas worldwide [3 4]

Thegeopropolis from Scaptotrigona posticahad beenusedpopularly in the region of Barra do Corda Maranhao stateBrazil in the form of ointment in the treatment of tumorsand wound healing [5 6] but there is no information on itschemical composition and biological activity The chemicalcomposition of the geopropolis of some countries includingBrazil was analyzed recently Eleven compounds belongingto the classes of phenolic acids and hydrolyzable tannins (gal-lotannins and ellagitannins) [4] and benzophenones [7] werefound from geopropolis of Melipona scutellaris Flavonoidsglycosides were found in geopropolis from two species of

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 296086 10 pageshttpdxdoiorg1011552015296086

2 Evidence-Based Complementary and Alternative Medicine

stingless Amazonian beesMelipona interrupta andMeliponaseminigra [8] Phenylpropanoids and flavonoids were foundin geopropolis from Melipona subnitida (jandaira) stinglessbee [9] and aromatic acids phenolic compounds and ter-penes are detected from geopropolis of the stingless beeMelipona orbignyi (Hymenoptera Apidae) found in MatoGrosso do Sul Brazil [10]

Flavones-di-C-glycosides caffeoylquinic acid derivativesand polyprenylated benzophenones had been reported inpropolis [11ndash13] The similarity in chemical composition ofpropolis and geopropolis was attributed to the fact that thetwo bees (Africanized and stingless bees) produce this beeproduct using resin collected from plants The C-methylatedflavanones that were detected in geopropolis from Australianstingless bees (Tetragonula carbonaria Meliponini) were alsofound in extracts from Corymbia torelliana (Myrtaceae) fruitresins of which probably T carbonaria collected the resin forthe production of its geopropolis [14]

Pyrrolizidine alkaloids a diverse class of monoestersare generally found in plants from the families AsteraceaeBoraginaceae and Fabaceae and are present approximately in6000 flowering plants species worldwide [15]The presence of12-dihydropyrrolizidine alkaloids had been observed in beeproducts such as honeys and pollen Echimidine is one ofthe main alkaloids reported in honey [15ndash18] There are noreports about the presence of alkaloids in propolis [2]

Herpes simplex viruses (HSV) are part of the alphaher-pesvirus subfamily of herpes viruses The incidence of dis-eases caused by herpes simplex virus (HSV) types 1 and 2 hasincreased in recent years [19] HSV-1 and HSV-2 are closelyrelated to ancient human pathogens responsible for a numberof diseases including oral and genital ulcerations virallyinduced blindness viral encephalitis and disseminated infec-tions of neonates [19 20] HSV-1 suppresses the interferon(IFN) signaling pathway of infection at multiple sites inorder to evade host defense mechanisms [19] There arethree ways to control HSV infections using anti-HSV drugsmicrobicides and vaccine Nowadays the standard therapyfor themanagement of HSV infections includes acyclovir andpenciclovir with their respective prodrugs valacyclovir andfamciclovir [19 20] The development of the novel strategiesto control HSV is a global public health priority

The aim of this work was to evaluate the chemicalcomposition and antiviral activity of the hydromethanolicextract of geopropolis (HMG) from Scaptotrigona posticaagainst antiherpes simplex virus (HSV)

2 Material and Methods

21 Cells The Vero cells (African green monkey kidneymdashATCC CCL-81) were grown in 75 cm2 plastic cell cultureflasks inDMEMmedium (DulbeccorsquosMinimumEagle Essen-tial Medium) supplemented with 10 inactive fetal bovineserum (FBS) and 20mM L-glutamine (Invitrogen USA)

22 Determination of the Virus Infectious Dose The con-fluent monolayers were dispersed with 02 trypsin and002 versene and added in DMEM growth medium with100 IUmL penicillin G and 100mgmL streptomycin For

the preparation of 96-well plates the cell suspension wasdiluted to 20 times 104 cellsmL Plates were seeded with 200 120583Lof suspension and incubated at 37∘C in a humidified 5 CO

2

atmosphere Herpes simplex virus strain (McIntyre) stockvirus was quantified by medium tissuemdashculture infectionswith 001 moi (multiplicity of infection) on cell cultures Theconfluent cell cultures were inoculated with 100120583L dilutedvirus in quadruplicates After 1 hour adsorption at roomtemperature each well received 200120583L of DMEM mediumwith 2 FBS Uninfected cultures were also prepared andtreated identically as controls Plate cultures were observedfor CPE daily during seven days when the test was concludedFifty percent infectivity end points were calculated by themethod of Reed andMuench [21] All titers were given as log10 TCID

50per 01mL of virus

23 MTT Assay The cell viability assay was based on MTTreduction in the mitochondria by enzymatic action whichgenerated a colored product Vero cells were cultured in96-well plates in DMEM supplemented with 5 of fetalbovine serum After 48 h HMG with concentrations rangingfrom 10mgmL to 01mgmL was added to the wells culturemedium After 24 hours of exposure the supernatant wasdiscarded and MTT at concentration of 500 microgrammLwas added to the growth medium for 4 hours After thisthe culture medium was removed and 100mL of DMSO wasadded After addition of dimethyl sulfoxide (DMSO) theplates were shaken for 30 minutes and then were read on aspectrophotometer at 570 nm

24 Antiviral Effect of Geopropolis on Infected Cells Verocells were grown to approximately 90 confluence in 96-wellplates in DMEM supplemented with 2mM L-glutamine and10 of FBS Plates were incubated at 37∘C in a humidified5 CO

2atmosphere The confluent cells were infected with

HSV-1 at a concentration of 10minus8 and monitored for cyto-pathic effects during 3 days HMGwas added to the cells at 3 hprior to virus infections 1 h after virus infection and virucidaThese antiviral screenings were repeated independently threetimes with three concentrations of HMG (100 10 and1 120583gmL) After this the determination of the geopropoliseffect on the infected cells was carried out using Real-TimePCR

25 Quantitative Real-Time PCR Assay Genomic DNAsincluding viral DNAs were isolated from the harvested cellsusing the MagNA Pure extractor (Roche Basel Switzerland)according to the manufacturerrsquos instructions The forwardprimer sequence for HSV-1 is 51015840-TGGGACACATGCCTT-CTTGG while the reverse sequence is 51015840-ACCCTTAGT-CAGACTCTGTTACTTACCC with amplicon size of 147 bp[22] For the Real-Time PCR the 20 120583L reaction mixturecontained 12 120583L of the SYBRGreen PCRMasterMix (AppliedBiosystems Foster City CA USA) 10120583M of each primer65 120583L H

2O and 5 120583L of cDNA The assay was performed

using the ABI 7300 Real-Time PCR Systems (Applied Biosys-tems Foster City USA) under the following conditions94∘C for 5min followed by 35 cycles of 94∘C for 30 s 57∘Cfor 20 s and 72∘C for 40 s Standard curves were prepared

Evidence-Based Complementary and Alternative Medicine 3

by qPCR using serial dilution of known copies number ofpurified amplification product for HSV-1 The copy numberof the samples was calculated from the standard curves[23] Percentage of reduction was defined as [copy numberof infected minus copy number of treated]copy number ofinfected times 100 All experiments were made in triplicate

26 Transmission Electron Microscopy (TEM) The Vero cellswere cultivated on Aclar film [24] and after two days cellswere infected with the sample from virucida (virus incubatedwith propolis during 1 h) and with HSV at concentration of10minus8 After 48 h the cultures were fixed in situ using 25glutaraldehyde (Sigma USA) in 01M sodium cacodylatebuffer and pH 72 for 1 h at 4∘C After they were rinsedtwice with cacodylate buffer the cultures were post-fixed ina solution containing 1 osmium tetroxide 08 potassiumferrocyanide and 5mM calcium chloride washed in 01Msodium cacodylate buffer dehydrated in graded acetone andembedded in epoxy resin Ultrathin sections were stainedusing uranyl acetate and lead citrate and examined througha transmission electron microscope JEM-1011 (JEOL Japan)

27 Direct Electron Microscopy (DEM) The supernatant cellsinfectedwith herpes virus at concentration of 10minus8 and treatedwith virucida were resuspended in 50mL of phosphate-buffered saline (PBS) at pH 72 One drop of the suspensionwas put on EM grid and submitted to negative staining tech-nique with 2 potassium phosphotungstate (PTK) at pH 64The grids were examined and the viruses were documentedin a JEM-1011 (JEOL Japan) electron microscope [22]

28 Separation and Identification ofthe Constituents of the Hydromethanolic Extract ofGeopropolis (HMG) from Scaptotrigona postica

281 Geopropolis Sample from Scaptotrigona postica Thegeopropolis sample from Scaptotrigona postica was collectedin the region of Barra do Corda Maranhao state BrazilThe ethanolic extract of geopropolis was provided by bee-keeper The geopropolis (1 Kg) was extracted by macerationin 1 L of cereal alcohol during 3 months and after this thecrude preparation was filtered first through cotton and thenthrough filter The alcoholic extract was concentrated andstored at freezer until sample workup For biological teststhe ethanolic extract prepared by beekeeper was dissolved inhexane ethyl acetate and a mixture of watermethanol (1 2)that obtained a fraction rich of hydrossoluble compoundswhich was dried and after this dissolved in water Milli-Q

282 Analysis of theHydromethanolic Extract fromGeopropo-lis (HMG) Using Fourier Transform Infrared Spectrometry(FTIR) FTIR spectrum was recorded at room temperature(ca 25∘C) using a Bomem spectrometer by scanning overthe frequency range 4000ndash400 cmminus1 at a resolution of 5 cmminus1using pastilles of KBr

283 Reversed Phase HPLC-DAD-ESI-MSMS AnalysisThe HPLC-DAD-ESI-MSMS analysis was conducted on

DADSPD-M10AVP Shimadzu system equipped with a pho-todiode array detector coupled to Esquire 3000 Plus (BrukerDaltonics) which consisted of two LC-20AD pumps SPD-20A diode array detector CTO-20A column oven and SIL20AC autoinjector (Shimadzu Corporation Kyoto Japan)The mass detector was a quadrupole ion trap equipped withatmospheric pressure ionization source through electrosprayionization interface which was operated in the full scanMSMS mode All the operations acquisition and dataanalysis were controlled by CBM-20A software The HMGwas dissolved in methanol water (80 20) vv and filteredwith 045120583mfilter before 312 120583L was injected into the HPLCsystem The peaks were monitored with diode array detec-tion at wavelengths of 254 and 300 nm The mobile phasesconsisted of two solvents eluent A (01 aq formic acid inMilli-Q water) and eluent B (acetonitrile) Constituents wereseparated using a reverse phase Phenomenex Gemini C-18(250 times 46mm 5 um) connected to a guard column Theelution started with 10 B in A at 10min it reached 20B in A at 20min 40 B in A at 30min 60 B in A at40min 80 B in A at 50min 100 B in A and finally itreturned to the initial conditions (10 B) to reequilibrate thecolumn prior to another runThe flow rate was kept constantat 10mLminminus1 and the temperature of the column wasmaintained at 40∘C The ionization conditions were adjustedas follows electrospray ionization was performed using anion source voltage of 40V and a capillary offset voltage of4000V The full scan mass acquisition was performed usingelectrospray ionization in the positive ionization mode byscanning from 119898119911 100ndash1200 u Helium was used as thecollision gas and nitrogen as the nebulizing gas Nebulizationwas aided with a coaxial nitrogen sheath gas provided at apressure of 27 psi Desolvation was assisted using a countercurrent nitrogen flow set at a flux of 70 Lmin and a capillarytemperature of 320∘C The data dependent MSMS eventswere performed on the most intense ions detected in fullscan MS Maximum accumulation time of ion trap and thenumbers ofMS to obtain theMS average spectrawere set at 30and 3MS respectively All the compoundswere characterizedby the interpretation of their UV spectra and mass spectradata obtained through ESI-MS and ESI-MSMS and alsotaking into account the MS data provided by the literature

3 Results

31 Determination of the Virus Infectious Dose The exposureof cell cultures to concentrations up to 25mgmL of theHMG did not show significant reduction in cell viability

32 Quantitative Real-Time PCR Assay The antiviral activ-ity of geopropolis against HSV-1 was confirmed by qPCRReal-Time The HMG was added into each designed wellat 3 h before infection (pretreatment) 1 h after infection(posttreatment) and virucida at the concentrations (100 10and 1 120583gmL) The results showed that HMG significantlyreduced the number of copies of HSV-1 genomic DNA inthe supernatant and in the lysate cell (Figures 1(a) and 1(b))All concentrations tested against HSV-1 through pre- post-and virucida treatment were found to be most effective in

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Infection Virucida Pretreatment Posttreatment

Cells

100120583gmL10120583gmL

1120583gmL

Inhi

bitio

n (

)

(a)

Infection Virucida Pretreatment Posttreatment

Supernatant

10120583gmL1120583gmL100120583gmL

0

20

40

60

80

100

120

Inhi

bitio

n (

)(b)

Figure 1 Herpes transcription level cells treated with propolis andmeasured by Real-Time PCR Total DNAwas performed at 3 hours beforeinfection (pretreatment) 1 hour postinfection (posttreatment) and virucida (a) Cells lysate (b) Supernatant Antiviral activity percentageand relative DNA viral quantification

inhibiting HSV-1 viral replication This suggested that thegeopropolis inhibited the events in the early infection suchas viral binding and viral entry into cells as well as the viralreplication Beside this in the cells inoculated with virucidasample and processed by DEM and TEM was not detectedthe HSV like particles into the cytoplasm from Vero cellsanalyzed as can be seen in Figure 2

33 Chemical Characterization ofConstituents from Geopropolis

331 Analysis of Hydromethanolic Extract from Geopropo-lis (HMG) Using Fourier Transform Infrared Spectrometry(FTIR) Fourier Transform Infrared Spectroscopy (FTIR) isa fast accurate and nondestructive technique used as apowerful analytical tool in many fieldsThe absorption bandsobserved in the FTIR spectrum [120592Max cmminus1 (KBr)] for HMGwere 3365 2919 2362 2343 1609 1414 1077 1048 864 and675 cmminus1 The OndashH stretching vibrations are sensitive tohydrogen bonding and the existence of an intermolecularhydrogen bond can lower theOndashH stretchingwavenumber to3500ndash3200 cmminus1The broad peak at 3365 cmminus1 was attributedto hydroxyl stretching vibration of flavones [25 26]

The peak at 1609 cmminus1 was attributed to a carbonyl stretchof flavones that occur at lower vibrational energies as aresult of decreased bond strengths of the carbonyl bonddue to resonance structures with a C=C bond adjacent toa carbonyl group resulting in the electron delocalization inthe carbonyl and the double bond [25] and at 1048 cmminus1 wasattributed to the vibration of CndashOH The band at 1077 cmminus1is a vibration attributed to the CndashOndashC asymmetric stretchingand at 1414 cmminus1 it was assigned toC=C stretch of the flavones

phenyl rings [25 26] In apigenin derivatives the CndashC ringstretching vibrations give rise to characteristic bands in IRspectrum covering the spectral range from 1610 to 1300 cmminus1[26] The presence of nitrogen compounds was attributed toabsorption bands at 2362 and 2343 cmminus1

332 Analyses of Chemical Constituents through HPLC-DAD-ESI-MSMS Mass spectrometry data were acquiredthrough electrospray ionization positive mode Table 1 sum-marizes the following information on peaks observed duringRPHPLC-DAD-ESI-MSMS analyses retention times (Rt)MS data for protonated molecules and proposed structuresConstituents mainly pyrrolizidine alkaloids were tentativelyidentified by the similarity of their fragmentation patternwith known compounds reported in the literature data

The sugar residues of flavonoids glycosides are mainlyhexoses (glucose and galactose) 6-deoxyhexoses (rhamnoseand furanose) pentoses (arabinose and xylose) and uronicacids (glucuronic acid and galacturonic acid) Glucose rham-nose xylose and arabinose are the more common sugar[27 28] Flavonoid O-glycosides are bounded to sugar withformation of an acid labile glycosidic OndashC bond and theirfragmentations patterns involve the heterocyclic cleavageat the glycosidic O-linkage (hemiacetal OndashC bond) with aconcomitant H-rearrangement leading to the elimination ofthe saccharide residue [27 28]

Glycosylation also occurs by direct linkage of the sugarto the basic nucleus of the flavonoid which is stable towardsacid hydrolysis The main fragmentations take place in thesugar which possess the weakest bonds that observed thecross-ring cleavages of the saccharide residue through thesuccessive loss of molecules of water [29ndash32] Two sugarscan be attached to the flavonoid aglycone either at two

Evidence-Based Complementary and Alternative Medicine 5

(a) (b)

GC

V

(c)

Figure 2 Vero cells were cultivated on Aclar film and were inoculated with HSV-1 or virucida sample after 48 hrs (a) Vero cells wereinoculated with HSV-1 and processed by DEM Note particle viral (b) Vero cells were inoculated with HSV-1 and processed by TEM Notethe presence of a typical particle viral (c) Vero cells were inoculated with virucida sample and processed by TEM Note Complex the Golgivesicles Not found the HSV like particles

different positions (di-O-glycosides di-C-glycosides and di-CO-glycosides) or at the same position (O-diglycosidesCO-diglycosides) [27ndash32] Sodium adduct ions [M + Na]+at 22 u are often detected in first-order mass spectra obtainedwith ESI in the positive ion mode [27]

The main flavonoids found in HMG were flavones di-C-glycosides (Table 1) The ESI-MSMS spectra of protonatedmolecules of flavones di-C-glycosides 6ndash11 13 14 and 17displayed neutral losses of water (18 u) as fragments [27ndash32]The main flavone di-C-glycoside compound 14 exhibitedprotonated molecule at119898119911 595 and sodiated molecule [M +Na]+ at 119898119911 617 respectively Its ESI-MSMS spectrum inpositive ion mode produced fragment ions at 577 559 541529 511 499 475 and 457 obtained through the loss of water(Table 1) Compound 17 exhibited protonated molecule at119898119911 565 which afterMSMS experiments exhibited fragmentions at 119898119911 547 529 511 and 427 According to literature

data [29ndash32] compound 14 was characterized as apigenin-68-di-C-glucoside also known as vicenin-2 and compound17was characterized as apigenin-68-di-C-arabinoside gluco-side respectively

Apigenin-di-C-malonyl trihexoside isomers (compounds7ndash9 11 and 13) showed different retention times but the samesodiated molecule at 119898119911 865 and protonated molecule at119898119911 843 (Table 1) respectively The malonyl moiety corre-sponds to 86 u Besides this the ESI-MSMS spectra of pro-tonated molecule showed the same fragmentation pattern at119898119911 825 807 789 771 723 705 and 687 attributed to the lossof water from glucose andor galactose as saccharides [29ndash32] which possess different intensities for each compoundas can be seen in Table 1 what could be used to discriminatebetween these apigenin-di-C-malonyl trihexoside isomers

Other flavones-di-C-glycosides are acacetin and luteolinderivatives Compound 6 showed protonated molecule at

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Results obtained by RPHPLC-DAD-ESI-MSMS analyses of geopropolis from Scaptotrigona postica retention times (Rt) MS datafor positive ion mode and structure proposed

Rt [M + Na]+ [M + H]+ Structure proposed

1 39 mdash [M + H]+ 443MSMSmdash291 Catechin-3-O-gallate

2 56 [M + Na]+mdash365 [M + H]+mdash343 Caffeoyl glucoside

3 65 [M + H]+mdash415MSMSmdash397 (100) 379 (40) 361 (30) 331 (50)

7-Methoxy-5-hydroxy-8-C-flavonerhamnoside

4 155 [M + H]+ 430MSMSmdash412 (100) 385 (70) 367 (10) 315 (20)

7(3-Methoxy-2-methylbutyryl)-9-echimidinylretronecine or methoxyechimidine derivative

5 160 [M + H]+ 444MSMSmdash426 (100) 399 (20)

7(3-Ethoxy-2-methylbutyryl)-9-echimidinylretronecine or ethoxyechimidine derivative

6 165 [M + H]+ 619MSMSmdash601 (80) 583 (20) 565 (20) 535 (100) Acacetin-di-C-acetyl dirhamnoside

7 179 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (40) 789 (20) 771 (30)723 (60) 705 (50)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

8 192 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (80) 807 (40) 789 (30) 771 (20)723 (70) 705 (100) 687 (40)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

9 202 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (20) 771 (30)723 (60) 705 (70) 687 (40)

Apigenin-di-C-malonyl trihexosideisomer

10 204 [M + H]+ 857MSMSmdash839 (100) 803 (30) 737 (40) 719 (30) Acacetin-di-C-malonyl trihexoside

11 211 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (90) 807 (50) 789 (30) 771 (25)723 (100) 705 (60) 687 (10)

Apigenin-di-C-malonyl trihexosideisomer

12 226 [M + H]+ 563MSMSmdash417

Acacetin-8-C-arabinoside-7-O-rhamnoside

13 245 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (10) 771 (20)723 (70) 705 (60)

apigenin-di-C-malonyl trihexosideisomer

14 270[M + Na]+ 617MSMSmdash599(100) 581 (20)

[M + H]+ 595MSMSmdash577 (100) 559 (30) 541 (20) 529 (40)511 (50) 499 (30) 475 (30) 457 (60)

Vicenin-2

15 275[M + Na]+mdash445MSMSmdash427(100) 409 (18)

[M + H]+mdash423MSMSmdash405 (100) 387 (30) 369 (30) 357 (60)327 (30)

Catechin arabinoside

16 280 [M + Na]+ 459MSMSmdash441

[M + H]+mdash437MSMSmdash419 (100) 401 (20) 383 (10) 371 (40)341 (20)

Catechin rhamnoside

17 285 [M + H]+ 565MSMSmdash547 (100) 529 (70) 511 (50) 427 (60)

Apigenin-68-di-C-arabinosideglucoside

18 294 [M + H]+ 547MSMSmdash401

Chrysin-8-C-rhamnoside-7-O-rhamnoside

19 298 [M + H]+ 517MSMSmdash355 (80) 337 (100) 35-Dicaffeoyl quinic acid

20 310 [M + H]+ 595MSMSmdash577 (100) 567 (70) 551 (90) Luteolin-8-C-caffeoyl rhamnoside

21 334 [M + H]+ 487 Caffeoylquinic acid-O-arabinoside

Evidence-Based Complementary and Alternative Medicine 7

119898119911 619 which afterMSMS experiments produced fragmentions at 119898119911 601 583 565 and 535 Compound 10 exhibitedprotonated molecule at 119898119911 857 which after MSMS exper-iments produced fragment ions at 119898119911 839 803 737 and719 Compound 20 showed protonated molecule at 119898119911 595which after MSMS experiments produced fragment ions at119898119911 577 567 and 551 Compounds 6 10 and 20 were ten-tatively characterized as acacetin-di-C-acetyl dirhamnosideacacetin-di-C-malonyl trihexoside and luteolin-8-C-caffeoylrhamnoside respectively

Flavones di-CO-glycosides were also found in low con-tent The ESI-MS spectrum of compound 12 exhibited proto-nated molecule at 119898119911 563 which after MSMS experimentsproduced high fragment ion at119898119911 417 [MndashHminus 146]minus whichwas attributed to the loss of the O-deoxyhexosyl radical(rhamnose moiety) probably linked to phenolic hydroxylgroup in 7-position of aglycone [27ndash32] In the same waycompound 18 exhibited protonated molecule at 119898119911 547which afterMSMS experiments produced high fragment ionat119898119911 401 [M ndash H minus 146]minus Compounds 12 and 18 presentedO-glycosylation on the phenolic hydroxyl group of aglyconewhich was corroborated by literature data [29ndash32] and weresuggested as acacetin-8-C-arabinoside-7-O-rhamnoside andchrysin-8-C-rhamnoside-7-O-rhamnoside respectively

The trans-cinnamic acids are esterified at one or moreof the hydroxyls at positions 1 3 4 and 5 of quinic acidoriginating a series of positional isomers [33ndash35] For com-pound 19 the ESI-MS spectrum showed protonatedmoleculeat 119898119911 517 which after MSMS experiments gave fragmentions at 119898119911 355 (caffeoylquinic acid moiety) and 119898119911 337suggesting di-O-caffeoylquinic acid positional isomers TheESI-MS spectrum of compound 21 exhibited protonatedmolecule at119898119911 487 According to literature data [28 33ndash35]compound 19 was characterized as 35-di-O-caffeoylquinicacid and compound 21 as caffeoylquinic acid-O-arabinosiderespectively

Compound 1 was characterized as catechin-3-O-gallatebased on ESI-MS data that showed protonated molecule at119898119911 443 which after MSMS experiments produced frag-ment ion at 119898119911 291 (protonated catechin) attributed to theloss of galloyl moiety (152 u) [36 37] Other catechin deriva-tives compounds 15 and 16 exhibited sodiated moleculesat 119898119911 445 and 119898119911 459 and protonated molecules at 119898119911423 and 119898119911 437 respectively After MSMS experimentsboth compounds exhibited fragment ions generated by theloss of water molecules indicating the presence of the sugarmolecules linked to the catechin The main fragmentationstake place in the sugar which possess the weakest bondsand the cross-ring cleavages of the saccharide residue areobserved through the successive loss of molecules of wateras shown in Table 1 According to literature data [36 37]compounds 15 and 16 are tentatively characterized as catechinarabinoside and catechin rhamnoside

The presence of 12-dihydropyrrolizidine alkaloids hadbeen observed in bee products and echimidine is the mainalkaloid reported in honey [15ndash18 38] According to Boppreet al [15ndash17] these compounds are transferred to the honeyby bees that visit the flowers to forage nectar and pollenHowever there are no reports about human poisoning

from honey by 12-dihydropyrrolizidine alkaloids [15ndash18 38]Pyrrolizidine alkaloids similar to echimidine are detected forthe first time in this geopropolis from Scaptotrigona postica

TheESI-MS spectrumof compound4 showed protonatedmolecule at 119898119911 430 which after fragmentation producedfragment ions at119898119911 412 attributed to the loss of water (18 u)and at 119898119911 385 attributed to the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid The fragment ion [M +H minus 115]+ at 119898119911 315 was attributed to the loss of methoxydihydro angelic acid moiety that is present on the C-7 ofopen chain diester type of pyrrolizidine alkaloid The lossof angelic acid moiety from echimidine was observed byBoppre et al [15ndash17] For compound 5 the ESI-MS spectrumexhibited protonated molecule at 119898119911 444 while its ESI-MSMS spectrum exhibited the same fragmentation patternas 4 with fragment ions at 119898119911 426 and 119898119911 399 attributedto the loss of water (18 u) and the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid According to literatureMS data [15ndash18 38] the echimidine derivatives 4 and 5were tentatively identified as 7(3-methoxy-2-methylbutyryl)-9-echimidinylretronecine and 7(3-ethoxy-2-methylbutyryl)-9-echimidinylretronecine respectively

4 Discussion

Herpes simplex virus type 1 (HSV-1) is an important pathogenfor humans and the discovery of novel effective antiherpeticdrugs without adverse effects is of great interest [39] Acy-clovir is the first-line treatment for themanagement of herpessimplex virus 1 (HSV-1) and virus 2 (HSV-2) diseases How-ever long-term administration of this drug for the treatmentof chronic infections in the immunocompromised host canlead to the development of acyclovir resistance [19 20 39 40]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about biological activities In order to examinethe antiviral activity of HMG against antiherpes simplexvirus the cells were treated with HMG 1 hour before infec-tion 3 hours after infection and virucidal condition andit was observed that in all conditions and concentrationstested the HMG showed great potential in inhibition ofreplication of HSV-1 Virucida test results suggest that HMGinterfered with virion envelope structures or are maskingviral compounds which are necessary for adsorption or entryinto host cells Apparently free herpes virus is very sensitiveto HMG and the inhibition of HSV-1 appears to occurbefore entering into the cell In our study in all conditionsand concentrations tested EMG showed great potential inreducing the number of copies of viral mRNA Recently ithas been reported that essential oils demonstrated a similarantiviral effect on herpes viruses [41 42]

Besides this HMG showed the highest antiviral activitywhen added during the intracellular replication period Ourresults are in accordance with that observed by Amoros et al[43] during intracellular replication Huleihel and Isanu(2002) [44] observed strong interaction between the propolisextracts and the surface of Vero cells but no direct interactionwith herpes virus particles The studies about the antiviral

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

stingless Amazonian beesMelipona interrupta andMeliponaseminigra [8] Phenylpropanoids and flavonoids were foundin geopropolis from Melipona subnitida (jandaira) stinglessbee [9] and aromatic acids phenolic compounds and ter-penes are detected from geopropolis of the stingless beeMelipona orbignyi (Hymenoptera Apidae) found in MatoGrosso do Sul Brazil [10]

Flavones-di-C-glycosides caffeoylquinic acid derivativesand polyprenylated benzophenones had been reported inpropolis [11ndash13] The similarity in chemical composition ofpropolis and geopropolis was attributed to the fact that thetwo bees (Africanized and stingless bees) produce this beeproduct using resin collected from plants The C-methylatedflavanones that were detected in geopropolis from Australianstingless bees (Tetragonula carbonaria Meliponini) were alsofound in extracts from Corymbia torelliana (Myrtaceae) fruitresins of which probably T carbonaria collected the resin forthe production of its geopropolis [14]

Pyrrolizidine alkaloids a diverse class of monoestersare generally found in plants from the families AsteraceaeBoraginaceae and Fabaceae and are present approximately in6000 flowering plants species worldwide [15]The presence of12-dihydropyrrolizidine alkaloids had been observed in beeproducts such as honeys and pollen Echimidine is one ofthe main alkaloids reported in honey [15ndash18] There are noreports about the presence of alkaloids in propolis [2]

Herpes simplex viruses (HSV) are part of the alphaher-pesvirus subfamily of herpes viruses The incidence of dis-eases caused by herpes simplex virus (HSV) types 1 and 2 hasincreased in recent years [19] HSV-1 and HSV-2 are closelyrelated to ancient human pathogens responsible for a numberof diseases including oral and genital ulcerations virallyinduced blindness viral encephalitis and disseminated infec-tions of neonates [19 20] HSV-1 suppresses the interferon(IFN) signaling pathway of infection at multiple sites inorder to evade host defense mechanisms [19] There arethree ways to control HSV infections using anti-HSV drugsmicrobicides and vaccine Nowadays the standard therapyfor themanagement of HSV infections includes acyclovir andpenciclovir with their respective prodrugs valacyclovir andfamciclovir [19 20] The development of the novel strategiesto control HSV is a global public health priority

The aim of this work was to evaluate the chemicalcomposition and antiviral activity of the hydromethanolicextract of geopropolis (HMG) from Scaptotrigona posticaagainst antiherpes simplex virus (HSV)

2 Material and Methods

21 Cells The Vero cells (African green monkey kidneymdashATCC CCL-81) were grown in 75 cm2 plastic cell cultureflasks inDMEMmedium (DulbeccorsquosMinimumEagle Essen-tial Medium) supplemented with 10 inactive fetal bovineserum (FBS) and 20mM L-glutamine (Invitrogen USA)

22 Determination of the Virus Infectious Dose The con-fluent monolayers were dispersed with 02 trypsin and002 versene and added in DMEM growth medium with100 IUmL penicillin G and 100mgmL streptomycin For

the preparation of 96-well plates the cell suspension wasdiluted to 20 times 104 cellsmL Plates were seeded with 200 120583Lof suspension and incubated at 37∘C in a humidified 5 CO

2

atmosphere Herpes simplex virus strain (McIntyre) stockvirus was quantified by medium tissuemdashculture infectionswith 001 moi (multiplicity of infection) on cell cultures Theconfluent cell cultures were inoculated with 100120583L dilutedvirus in quadruplicates After 1 hour adsorption at roomtemperature each well received 200120583L of DMEM mediumwith 2 FBS Uninfected cultures were also prepared andtreated identically as controls Plate cultures were observedfor CPE daily during seven days when the test was concludedFifty percent infectivity end points were calculated by themethod of Reed andMuench [21] All titers were given as log10 TCID

50per 01mL of virus

23 MTT Assay The cell viability assay was based on MTTreduction in the mitochondria by enzymatic action whichgenerated a colored product Vero cells were cultured in96-well plates in DMEM supplemented with 5 of fetalbovine serum After 48 h HMG with concentrations rangingfrom 10mgmL to 01mgmL was added to the wells culturemedium After 24 hours of exposure the supernatant wasdiscarded and MTT at concentration of 500 microgrammLwas added to the growth medium for 4 hours After thisthe culture medium was removed and 100mL of DMSO wasadded After addition of dimethyl sulfoxide (DMSO) theplates were shaken for 30 minutes and then were read on aspectrophotometer at 570 nm

24 Antiviral Effect of Geopropolis on Infected Cells Verocells were grown to approximately 90 confluence in 96-wellplates in DMEM supplemented with 2mM L-glutamine and10 of FBS Plates were incubated at 37∘C in a humidified5 CO

2atmosphere The confluent cells were infected with

HSV-1 at a concentration of 10minus8 and monitored for cyto-pathic effects during 3 days HMGwas added to the cells at 3 hprior to virus infections 1 h after virus infection and virucidaThese antiviral screenings were repeated independently threetimes with three concentrations of HMG (100 10 and1 120583gmL) After this the determination of the geopropoliseffect on the infected cells was carried out using Real-TimePCR

25 Quantitative Real-Time PCR Assay Genomic DNAsincluding viral DNAs were isolated from the harvested cellsusing the MagNA Pure extractor (Roche Basel Switzerland)according to the manufacturerrsquos instructions The forwardprimer sequence for HSV-1 is 51015840-TGGGACACATGCCTT-CTTGG while the reverse sequence is 51015840-ACCCTTAGT-CAGACTCTGTTACTTACCC with amplicon size of 147 bp[22] For the Real-Time PCR the 20 120583L reaction mixturecontained 12 120583L of the SYBRGreen PCRMasterMix (AppliedBiosystems Foster City CA USA) 10120583M of each primer65 120583L H

2O and 5 120583L of cDNA The assay was performed

using the ABI 7300 Real-Time PCR Systems (Applied Biosys-tems Foster City USA) under the following conditions94∘C for 5min followed by 35 cycles of 94∘C for 30 s 57∘Cfor 20 s and 72∘C for 40 s Standard curves were prepared

Evidence-Based Complementary and Alternative Medicine 3

by qPCR using serial dilution of known copies number ofpurified amplification product for HSV-1 The copy numberof the samples was calculated from the standard curves[23] Percentage of reduction was defined as [copy numberof infected minus copy number of treated]copy number ofinfected times 100 All experiments were made in triplicate

26 Transmission Electron Microscopy (TEM) The Vero cellswere cultivated on Aclar film [24] and after two days cellswere infected with the sample from virucida (virus incubatedwith propolis during 1 h) and with HSV at concentration of10minus8 After 48 h the cultures were fixed in situ using 25glutaraldehyde (Sigma USA) in 01M sodium cacodylatebuffer and pH 72 for 1 h at 4∘C After they were rinsedtwice with cacodylate buffer the cultures were post-fixed ina solution containing 1 osmium tetroxide 08 potassiumferrocyanide and 5mM calcium chloride washed in 01Msodium cacodylate buffer dehydrated in graded acetone andembedded in epoxy resin Ultrathin sections were stainedusing uranyl acetate and lead citrate and examined througha transmission electron microscope JEM-1011 (JEOL Japan)

27 Direct Electron Microscopy (DEM) The supernatant cellsinfectedwith herpes virus at concentration of 10minus8 and treatedwith virucida were resuspended in 50mL of phosphate-buffered saline (PBS) at pH 72 One drop of the suspensionwas put on EM grid and submitted to negative staining tech-nique with 2 potassium phosphotungstate (PTK) at pH 64The grids were examined and the viruses were documentedin a JEM-1011 (JEOL Japan) electron microscope [22]

28 Separation and Identification ofthe Constituents of the Hydromethanolic Extract ofGeopropolis (HMG) from Scaptotrigona postica

281 Geopropolis Sample from Scaptotrigona postica Thegeopropolis sample from Scaptotrigona postica was collectedin the region of Barra do Corda Maranhao state BrazilThe ethanolic extract of geopropolis was provided by bee-keeper The geopropolis (1 Kg) was extracted by macerationin 1 L of cereal alcohol during 3 months and after this thecrude preparation was filtered first through cotton and thenthrough filter The alcoholic extract was concentrated andstored at freezer until sample workup For biological teststhe ethanolic extract prepared by beekeeper was dissolved inhexane ethyl acetate and a mixture of watermethanol (1 2)that obtained a fraction rich of hydrossoluble compoundswhich was dried and after this dissolved in water Milli-Q

282 Analysis of theHydromethanolic Extract fromGeopropo-lis (HMG) Using Fourier Transform Infrared Spectrometry(FTIR) FTIR spectrum was recorded at room temperature(ca 25∘C) using a Bomem spectrometer by scanning overthe frequency range 4000ndash400 cmminus1 at a resolution of 5 cmminus1using pastilles of KBr

283 Reversed Phase HPLC-DAD-ESI-MSMS AnalysisThe HPLC-DAD-ESI-MSMS analysis was conducted on

DADSPD-M10AVP Shimadzu system equipped with a pho-todiode array detector coupled to Esquire 3000 Plus (BrukerDaltonics) which consisted of two LC-20AD pumps SPD-20A diode array detector CTO-20A column oven and SIL20AC autoinjector (Shimadzu Corporation Kyoto Japan)The mass detector was a quadrupole ion trap equipped withatmospheric pressure ionization source through electrosprayionization interface which was operated in the full scanMSMS mode All the operations acquisition and dataanalysis were controlled by CBM-20A software The HMGwas dissolved in methanol water (80 20) vv and filteredwith 045120583mfilter before 312 120583L was injected into the HPLCsystem The peaks were monitored with diode array detec-tion at wavelengths of 254 and 300 nm The mobile phasesconsisted of two solvents eluent A (01 aq formic acid inMilli-Q water) and eluent B (acetonitrile) Constituents wereseparated using a reverse phase Phenomenex Gemini C-18(250 times 46mm 5 um) connected to a guard column Theelution started with 10 B in A at 10min it reached 20B in A at 20min 40 B in A at 30min 60 B in A at40min 80 B in A at 50min 100 B in A and finally itreturned to the initial conditions (10 B) to reequilibrate thecolumn prior to another runThe flow rate was kept constantat 10mLminminus1 and the temperature of the column wasmaintained at 40∘C The ionization conditions were adjustedas follows electrospray ionization was performed using anion source voltage of 40V and a capillary offset voltage of4000V The full scan mass acquisition was performed usingelectrospray ionization in the positive ionization mode byscanning from 119898119911 100ndash1200 u Helium was used as thecollision gas and nitrogen as the nebulizing gas Nebulizationwas aided with a coaxial nitrogen sheath gas provided at apressure of 27 psi Desolvation was assisted using a countercurrent nitrogen flow set at a flux of 70 Lmin and a capillarytemperature of 320∘C The data dependent MSMS eventswere performed on the most intense ions detected in fullscan MS Maximum accumulation time of ion trap and thenumbers ofMS to obtain theMS average spectrawere set at 30and 3MS respectively All the compoundswere characterizedby the interpretation of their UV spectra and mass spectradata obtained through ESI-MS and ESI-MSMS and alsotaking into account the MS data provided by the literature

3 Results

31 Determination of the Virus Infectious Dose The exposureof cell cultures to concentrations up to 25mgmL of theHMG did not show significant reduction in cell viability

32 Quantitative Real-Time PCR Assay The antiviral activ-ity of geopropolis against HSV-1 was confirmed by qPCRReal-Time The HMG was added into each designed wellat 3 h before infection (pretreatment) 1 h after infection(posttreatment) and virucida at the concentrations (100 10and 1 120583gmL) The results showed that HMG significantlyreduced the number of copies of HSV-1 genomic DNA inthe supernatant and in the lysate cell (Figures 1(a) and 1(b))All concentrations tested against HSV-1 through pre- post-and virucida treatment were found to be most effective in

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Infection Virucida Pretreatment Posttreatment

Cells

100120583gmL10120583gmL

1120583gmL

Inhi

bitio

n (

)

(a)

Infection Virucida Pretreatment Posttreatment

Supernatant

10120583gmL1120583gmL100120583gmL

0

20

40

60

80

100

120

Inhi

bitio

n (

)(b)

Figure 1 Herpes transcription level cells treated with propolis andmeasured by Real-Time PCR Total DNAwas performed at 3 hours beforeinfection (pretreatment) 1 hour postinfection (posttreatment) and virucida (a) Cells lysate (b) Supernatant Antiviral activity percentageand relative DNA viral quantification

inhibiting HSV-1 viral replication This suggested that thegeopropolis inhibited the events in the early infection suchas viral binding and viral entry into cells as well as the viralreplication Beside this in the cells inoculated with virucidasample and processed by DEM and TEM was not detectedthe HSV like particles into the cytoplasm from Vero cellsanalyzed as can be seen in Figure 2

33 Chemical Characterization ofConstituents from Geopropolis

331 Analysis of Hydromethanolic Extract from Geopropo-lis (HMG) Using Fourier Transform Infrared Spectrometry(FTIR) Fourier Transform Infrared Spectroscopy (FTIR) isa fast accurate and nondestructive technique used as apowerful analytical tool in many fieldsThe absorption bandsobserved in the FTIR spectrum [120592Max cmminus1 (KBr)] for HMGwere 3365 2919 2362 2343 1609 1414 1077 1048 864 and675 cmminus1 The OndashH stretching vibrations are sensitive tohydrogen bonding and the existence of an intermolecularhydrogen bond can lower theOndashH stretchingwavenumber to3500ndash3200 cmminus1The broad peak at 3365 cmminus1 was attributedto hydroxyl stretching vibration of flavones [25 26]

The peak at 1609 cmminus1 was attributed to a carbonyl stretchof flavones that occur at lower vibrational energies as aresult of decreased bond strengths of the carbonyl bonddue to resonance structures with a C=C bond adjacent toa carbonyl group resulting in the electron delocalization inthe carbonyl and the double bond [25] and at 1048 cmminus1 wasattributed to the vibration of CndashOH The band at 1077 cmminus1is a vibration attributed to the CndashOndashC asymmetric stretchingand at 1414 cmminus1 it was assigned toC=C stretch of the flavones

phenyl rings [25 26] In apigenin derivatives the CndashC ringstretching vibrations give rise to characteristic bands in IRspectrum covering the spectral range from 1610 to 1300 cmminus1[26] The presence of nitrogen compounds was attributed toabsorption bands at 2362 and 2343 cmminus1

332 Analyses of Chemical Constituents through HPLC-DAD-ESI-MSMS Mass spectrometry data were acquiredthrough electrospray ionization positive mode Table 1 sum-marizes the following information on peaks observed duringRPHPLC-DAD-ESI-MSMS analyses retention times (Rt)MS data for protonated molecules and proposed structuresConstituents mainly pyrrolizidine alkaloids were tentativelyidentified by the similarity of their fragmentation patternwith known compounds reported in the literature data

The sugar residues of flavonoids glycosides are mainlyhexoses (glucose and galactose) 6-deoxyhexoses (rhamnoseand furanose) pentoses (arabinose and xylose) and uronicacids (glucuronic acid and galacturonic acid) Glucose rham-nose xylose and arabinose are the more common sugar[27 28] Flavonoid O-glycosides are bounded to sugar withformation of an acid labile glycosidic OndashC bond and theirfragmentations patterns involve the heterocyclic cleavageat the glycosidic O-linkage (hemiacetal OndashC bond) with aconcomitant H-rearrangement leading to the elimination ofthe saccharide residue [27 28]

Glycosylation also occurs by direct linkage of the sugarto the basic nucleus of the flavonoid which is stable towardsacid hydrolysis The main fragmentations take place in thesugar which possess the weakest bonds that observed thecross-ring cleavages of the saccharide residue through thesuccessive loss of molecules of water [29ndash32] Two sugarscan be attached to the flavonoid aglycone either at two

Evidence-Based Complementary and Alternative Medicine 5

(a) (b)

GC

V

(c)

Figure 2 Vero cells were cultivated on Aclar film and were inoculated with HSV-1 or virucida sample after 48 hrs (a) Vero cells wereinoculated with HSV-1 and processed by DEM Note particle viral (b) Vero cells were inoculated with HSV-1 and processed by TEM Notethe presence of a typical particle viral (c) Vero cells were inoculated with virucida sample and processed by TEM Note Complex the Golgivesicles Not found the HSV like particles

different positions (di-O-glycosides di-C-glycosides and di-CO-glycosides) or at the same position (O-diglycosidesCO-diglycosides) [27ndash32] Sodium adduct ions [M + Na]+at 22 u are often detected in first-order mass spectra obtainedwith ESI in the positive ion mode [27]

The main flavonoids found in HMG were flavones di-C-glycosides (Table 1) The ESI-MSMS spectra of protonatedmolecules of flavones di-C-glycosides 6ndash11 13 14 and 17displayed neutral losses of water (18 u) as fragments [27ndash32]The main flavone di-C-glycoside compound 14 exhibitedprotonated molecule at119898119911 595 and sodiated molecule [M +Na]+ at 119898119911 617 respectively Its ESI-MSMS spectrum inpositive ion mode produced fragment ions at 577 559 541529 511 499 475 and 457 obtained through the loss of water(Table 1) Compound 17 exhibited protonated molecule at119898119911 565 which afterMSMS experiments exhibited fragmentions at 119898119911 547 529 511 and 427 According to literature

data [29ndash32] compound 14 was characterized as apigenin-68-di-C-glucoside also known as vicenin-2 and compound17was characterized as apigenin-68-di-C-arabinoside gluco-side respectively

Apigenin-di-C-malonyl trihexoside isomers (compounds7ndash9 11 and 13) showed different retention times but the samesodiated molecule at 119898119911 865 and protonated molecule at119898119911 843 (Table 1) respectively The malonyl moiety corre-sponds to 86 u Besides this the ESI-MSMS spectra of pro-tonated molecule showed the same fragmentation pattern at119898119911 825 807 789 771 723 705 and 687 attributed to the lossof water from glucose andor galactose as saccharides [29ndash32] which possess different intensities for each compoundas can be seen in Table 1 what could be used to discriminatebetween these apigenin-di-C-malonyl trihexoside isomers

Other flavones-di-C-glycosides are acacetin and luteolinderivatives Compound 6 showed protonated molecule at

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Results obtained by RPHPLC-DAD-ESI-MSMS analyses of geopropolis from Scaptotrigona postica retention times (Rt) MS datafor positive ion mode and structure proposed

Rt [M + Na]+ [M + H]+ Structure proposed

1 39 mdash [M + H]+ 443MSMSmdash291 Catechin-3-O-gallate

2 56 [M + Na]+mdash365 [M + H]+mdash343 Caffeoyl glucoside

3 65 [M + H]+mdash415MSMSmdash397 (100) 379 (40) 361 (30) 331 (50)

7-Methoxy-5-hydroxy-8-C-flavonerhamnoside

4 155 [M + H]+ 430MSMSmdash412 (100) 385 (70) 367 (10) 315 (20)

7(3-Methoxy-2-methylbutyryl)-9-echimidinylretronecine or methoxyechimidine derivative

5 160 [M + H]+ 444MSMSmdash426 (100) 399 (20)

7(3-Ethoxy-2-methylbutyryl)-9-echimidinylretronecine or ethoxyechimidine derivative

6 165 [M + H]+ 619MSMSmdash601 (80) 583 (20) 565 (20) 535 (100) Acacetin-di-C-acetyl dirhamnoside

7 179 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (40) 789 (20) 771 (30)723 (60) 705 (50)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

8 192 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (80) 807 (40) 789 (30) 771 (20)723 (70) 705 (100) 687 (40)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

9 202 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (20) 771 (30)723 (60) 705 (70) 687 (40)

Apigenin-di-C-malonyl trihexosideisomer

10 204 [M + H]+ 857MSMSmdash839 (100) 803 (30) 737 (40) 719 (30) Acacetin-di-C-malonyl trihexoside

11 211 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (90) 807 (50) 789 (30) 771 (25)723 (100) 705 (60) 687 (10)

Apigenin-di-C-malonyl trihexosideisomer

12 226 [M + H]+ 563MSMSmdash417

Acacetin-8-C-arabinoside-7-O-rhamnoside

13 245 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (10) 771 (20)723 (70) 705 (60)

apigenin-di-C-malonyl trihexosideisomer

14 270[M + Na]+ 617MSMSmdash599(100) 581 (20)

[M + H]+ 595MSMSmdash577 (100) 559 (30) 541 (20) 529 (40)511 (50) 499 (30) 475 (30) 457 (60)

Vicenin-2

15 275[M + Na]+mdash445MSMSmdash427(100) 409 (18)

[M + H]+mdash423MSMSmdash405 (100) 387 (30) 369 (30) 357 (60)327 (30)

Catechin arabinoside

16 280 [M + Na]+ 459MSMSmdash441

[M + H]+mdash437MSMSmdash419 (100) 401 (20) 383 (10) 371 (40)341 (20)

Catechin rhamnoside

17 285 [M + H]+ 565MSMSmdash547 (100) 529 (70) 511 (50) 427 (60)

Apigenin-68-di-C-arabinosideglucoside

18 294 [M + H]+ 547MSMSmdash401

Chrysin-8-C-rhamnoside-7-O-rhamnoside

19 298 [M + H]+ 517MSMSmdash355 (80) 337 (100) 35-Dicaffeoyl quinic acid

20 310 [M + H]+ 595MSMSmdash577 (100) 567 (70) 551 (90) Luteolin-8-C-caffeoyl rhamnoside

21 334 [M + H]+ 487 Caffeoylquinic acid-O-arabinoside

Evidence-Based Complementary and Alternative Medicine 7

119898119911 619 which afterMSMS experiments produced fragmentions at 119898119911 601 583 565 and 535 Compound 10 exhibitedprotonated molecule at 119898119911 857 which after MSMS exper-iments produced fragment ions at 119898119911 839 803 737 and719 Compound 20 showed protonated molecule at 119898119911 595which after MSMS experiments produced fragment ions at119898119911 577 567 and 551 Compounds 6 10 and 20 were ten-tatively characterized as acacetin-di-C-acetyl dirhamnosideacacetin-di-C-malonyl trihexoside and luteolin-8-C-caffeoylrhamnoside respectively

Flavones di-CO-glycosides were also found in low con-tent The ESI-MS spectrum of compound 12 exhibited proto-nated molecule at 119898119911 563 which after MSMS experimentsproduced high fragment ion at119898119911 417 [MndashHminus 146]minus whichwas attributed to the loss of the O-deoxyhexosyl radical(rhamnose moiety) probably linked to phenolic hydroxylgroup in 7-position of aglycone [27ndash32] In the same waycompound 18 exhibited protonated molecule at 119898119911 547which afterMSMS experiments produced high fragment ionat119898119911 401 [M ndash H minus 146]minus Compounds 12 and 18 presentedO-glycosylation on the phenolic hydroxyl group of aglyconewhich was corroborated by literature data [29ndash32] and weresuggested as acacetin-8-C-arabinoside-7-O-rhamnoside andchrysin-8-C-rhamnoside-7-O-rhamnoside respectively

The trans-cinnamic acids are esterified at one or moreof the hydroxyls at positions 1 3 4 and 5 of quinic acidoriginating a series of positional isomers [33ndash35] For com-pound 19 the ESI-MS spectrum showed protonatedmoleculeat 119898119911 517 which after MSMS experiments gave fragmentions at 119898119911 355 (caffeoylquinic acid moiety) and 119898119911 337suggesting di-O-caffeoylquinic acid positional isomers TheESI-MS spectrum of compound 21 exhibited protonatedmolecule at119898119911 487 According to literature data [28 33ndash35]compound 19 was characterized as 35-di-O-caffeoylquinicacid and compound 21 as caffeoylquinic acid-O-arabinosiderespectively

Compound 1 was characterized as catechin-3-O-gallatebased on ESI-MS data that showed protonated molecule at119898119911 443 which after MSMS experiments produced frag-ment ion at 119898119911 291 (protonated catechin) attributed to theloss of galloyl moiety (152 u) [36 37] Other catechin deriva-tives compounds 15 and 16 exhibited sodiated moleculesat 119898119911 445 and 119898119911 459 and protonated molecules at 119898119911423 and 119898119911 437 respectively After MSMS experimentsboth compounds exhibited fragment ions generated by theloss of water molecules indicating the presence of the sugarmolecules linked to the catechin The main fragmentationstake place in the sugar which possess the weakest bondsand the cross-ring cleavages of the saccharide residue areobserved through the successive loss of molecules of wateras shown in Table 1 According to literature data [36 37]compounds 15 and 16 are tentatively characterized as catechinarabinoside and catechin rhamnoside

The presence of 12-dihydropyrrolizidine alkaloids hadbeen observed in bee products and echimidine is the mainalkaloid reported in honey [15ndash18 38] According to Boppreet al [15ndash17] these compounds are transferred to the honeyby bees that visit the flowers to forage nectar and pollenHowever there are no reports about human poisoning

from honey by 12-dihydropyrrolizidine alkaloids [15ndash18 38]Pyrrolizidine alkaloids similar to echimidine are detected forthe first time in this geopropolis from Scaptotrigona postica

TheESI-MS spectrumof compound4 showed protonatedmolecule at 119898119911 430 which after fragmentation producedfragment ions at119898119911 412 attributed to the loss of water (18 u)and at 119898119911 385 attributed to the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid The fragment ion [M +H minus 115]+ at 119898119911 315 was attributed to the loss of methoxydihydro angelic acid moiety that is present on the C-7 ofopen chain diester type of pyrrolizidine alkaloid The lossof angelic acid moiety from echimidine was observed byBoppre et al [15ndash17] For compound 5 the ESI-MS spectrumexhibited protonated molecule at 119898119911 444 while its ESI-MSMS spectrum exhibited the same fragmentation patternas 4 with fragment ions at 119898119911 426 and 119898119911 399 attributedto the loss of water (18 u) and the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid According to literatureMS data [15ndash18 38] the echimidine derivatives 4 and 5were tentatively identified as 7(3-methoxy-2-methylbutyryl)-9-echimidinylretronecine and 7(3-ethoxy-2-methylbutyryl)-9-echimidinylretronecine respectively

4 Discussion

Herpes simplex virus type 1 (HSV-1) is an important pathogenfor humans and the discovery of novel effective antiherpeticdrugs without adverse effects is of great interest [39] Acy-clovir is the first-line treatment for themanagement of herpessimplex virus 1 (HSV-1) and virus 2 (HSV-2) diseases How-ever long-term administration of this drug for the treatmentof chronic infections in the immunocompromised host canlead to the development of acyclovir resistance [19 20 39 40]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about biological activities In order to examinethe antiviral activity of HMG against antiherpes simplexvirus the cells were treated with HMG 1 hour before infec-tion 3 hours after infection and virucidal condition andit was observed that in all conditions and concentrationstested the HMG showed great potential in inhibition ofreplication of HSV-1 Virucida test results suggest that HMGinterfered with virion envelope structures or are maskingviral compounds which are necessary for adsorption or entryinto host cells Apparently free herpes virus is very sensitiveto HMG and the inhibition of HSV-1 appears to occurbefore entering into the cell In our study in all conditionsand concentrations tested EMG showed great potential inreducing the number of copies of viral mRNA Recently ithas been reported that essential oils demonstrated a similarantiviral effect on herpes viruses [41 42]

Besides this HMG showed the highest antiviral activitywhen added during the intracellular replication period Ourresults are in accordance with that observed by Amoros et al[43] during intracellular replication Huleihel and Isanu(2002) [44] observed strong interaction between the propolisextracts and the surface of Vero cells but no direct interactionwith herpes virus particles The studies about the antiviral

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

by qPCR using serial dilution of known copies number ofpurified amplification product for HSV-1 The copy numberof the samples was calculated from the standard curves[23] Percentage of reduction was defined as [copy numberof infected minus copy number of treated]copy number ofinfected times 100 All experiments were made in triplicate

26 Transmission Electron Microscopy (TEM) The Vero cellswere cultivated on Aclar film [24] and after two days cellswere infected with the sample from virucida (virus incubatedwith propolis during 1 h) and with HSV at concentration of10minus8 After 48 h the cultures were fixed in situ using 25glutaraldehyde (Sigma USA) in 01M sodium cacodylatebuffer and pH 72 for 1 h at 4∘C After they were rinsedtwice with cacodylate buffer the cultures were post-fixed ina solution containing 1 osmium tetroxide 08 potassiumferrocyanide and 5mM calcium chloride washed in 01Msodium cacodylate buffer dehydrated in graded acetone andembedded in epoxy resin Ultrathin sections were stainedusing uranyl acetate and lead citrate and examined througha transmission electron microscope JEM-1011 (JEOL Japan)

27 Direct Electron Microscopy (DEM) The supernatant cellsinfectedwith herpes virus at concentration of 10minus8 and treatedwith virucida were resuspended in 50mL of phosphate-buffered saline (PBS) at pH 72 One drop of the suspensionwas put on EM grid and submitted to negative staining tech-nique with 2 potassium phosphotungstate (PTK) at pH 64The grids were examined and the viruses were documentedin a JEM-1011 (JEOL Japan) electron microscope [22]

28 Separation and Identification ofthe Constituents of the Hydromethanolic Extract ofGeopropolis (HMG) from Scaptotrigona postica

281 Geopropolis Sample from Scaptotrigona postica Thegeopropolis sample from Scaptotrigona postica was collectedin the region of Barra do Corda Maranhao state BrazilThe ethanolic extract of geopropolis was provided by bee-keeper The geopropolis (1 Kg) was extracted by macerationin 1 L of cereal alcohol during 3 months and after this thecrude preparation was filtered first through cotton and thenthrough filter The alcoholic extract was concentrated andstored at freezer until sample workup For biological teststhe ethanolic extract prepared by beekeeper was dissolved inhexane ethyl acetate and a mixture of watermethanol (1 2)that obtained a fraction rich of hydrossoluble compoundswhich was dried and after this dissolved in water Milli-Q

282 Analysis of theHydromethanolic Extract fromGeopropo-lis (HMG) Using Fourier Transform Infrared Spectrometry(FTIR) FTIR spectrum was recorded at room temperature(ca 25∘C) using a Bomem spectrometer by scanning overthe frequency range 4000ndash400 cmminus1 at a resolution of 5 cmminus1using pastilles of KBr

283 Reversed Phase HPLC-DAD-ESI-MSMS AnalysisThe HPLC-DAD-ESI-MSMS analysis was conducted on

DADSPD-M10AVP Shimadzu system equipped with a pho-todiode array detector coupled to Esquire 3000 Plus (BrukerDaltonics) which consisted of two LC-20AD pumps SPD-20A diode array detector CTO-20A column oven and SIL20AC autoinjector (Shimadzu Corporation Kyoto Japan)The mass detector was a quadrupole ion trap equipped withatmospheric pressure ionization source through electrosprayionization interface which was operated in the full scanMSMS mode All the operations acquisition and dataanalysis were controlled by CBM-20A software The HMGwas dissolved in methanol water (80 20) vv and filteredwith 045120583mfilter before 312 120583L was injected into the HPLCsystem The peaks were monitored with diode array detec-tion at wavelengths of 254 and 300 nm The mobile phasesconsisted of two solvents eluent A (01 aq formic acid inMilli-Q water) and eluent B (acetonitrile) Constituents wereseparated using a reverse phase Phenomenex Gemini C-18(250 times 46mm 5 um) connected to a guard column Theelution started with 10 B in A at 10min it reached 20B in A at 20min 40 B in A at 30min 60 B in A at40min 80 B in A at 50min 100 B in A and finally itreturned to the initial conditions (10 B) to reequilibrate thecolumn prior to another runThe flow rate was kept constantat 10mLminminus1 and the temperature of the column wasmaintained at 40∘C The ionization conditions were adjustedas follows electrospray ionization was performed using anion source voltage of 40V and a capillary offset voltage of4000V The full scan mass acquisition was performed usingelectrospray ionization in the positive ionization mode byscanning from 119898119911 100ndash1200 u Helium was used as thecollision gas and nitrogen as the nebulizing gas Nebulizationwas aided with a coaxial nitrogen sheath gas provided at apressure of 27 psi Desolvation was assisted using a countercurrent nitrogen flow set at a flux of 70 Lmin and a capillarytemperature of 320∘C The data dependent MSMS eventswere performed on the most intense ions detected in fullscan MS Maximum accumulation time of ion trap and thenumbers ofMS to obtain theMS average spectrawere set at 30and 3MS respectively All the compoundswere characterizedby the interpretation of their UV spectra and mass spectradata obtained through ESI-MS and ESI-MSMS and alsotaking into account the MS data provided by the literature

3 Results

31 Determination of the Virus Infectious Dose The exposureof cell cultures to concentrations up to 25mgmL of theHMG did not show significant reduction in cell viability

32 Quantitative Real-Time PCR Assay The antiviral activ-ity of geopropolis against HSV-1 was confirmed by qPCRReal-Time The HMG was added into each designed wellat 3 h before infection (pretreatment) 1 h after infection(posttreatment) and virucida at the concentrations (100 10and 1 120583gmL) The results showed that HMG significantlyreduced the number of copies of HSV-1 genomic DNA inthe supernatant and in the lysate cell (Figures 1(a) and 1(b))All concentrations tested against HSV-1 through pre- post-and virucida treatment were found to be most effective in

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Infection Virucida Pretreatment Posttreatment

Cells

100120583gmL10120583gmL

1120583gmL

Inhi

bitio

n (

)

(a)

Infection Virucida Pretreatment Posttreatment

Supernatant

10120583gmL1120583gmL100120583gmL

0

20

40

60

80

100

120

Inhi

bitio

n (

)(b)

Figure 1 Herpes transcription level cells treated with propolis andmeasured by Real-Time PCR Total DNAwas performed at 3 hours beforeinfection (pretreatment) 1 hour postinfection (posttreatment) and virucida (a) Cells lysate (b) Supernatant Antiviral activity percentageand relative DNA viral quantification

inhibiting HSV-1 viral replication This suggested that thegeopropolis inhibited the events in the early infection suchas viral binding and viral entry into cells as well as the viralreplication Beside this in the cells inoculated with virucidasample and processed by DEM and TEM was not detectedthe HSV like particles into the cytoplasm from Vero cellsanalyzed as can be seen in Figure 2

33 Chemical Characterization ofConstituents from Geopropolis

331 Analysis of Hydromethanolic Extract from Geopropo-lis (HMG) Using Fourier Transform Infrared Spectrometry(FTIR) Fourier Transform Infrared Spectroscopy (FTIR) isa fast accurate and nondestructive technique used as apowerful analytical tool in many fieldsThe absorption bandsobserved in the FTIR spectrum [120592Max cmminus1 (KBr)] for HMGwere 3365 2919 2362 2343 1609 1414 1077 1048 864 and675 cmminus1 The OndashH stretching vibrations are sensitive tohydrogen bonding and the existence of an intermolecularhydrogen bond can lower theOndashH stretchingwavenumber to3500ndash3200 cmminus1The broad peak at 3365 cmminus1 was attributedto hydroxyl stretching vibration of flavones [25 26]

The peak at 1609 cmminus1 was attributed to a carbonyl stretchof flavones that occur at lower vibrational energies as aresult of decreased bond strengths of the carbonyl bonddue to resonance structures with a C=C bond adjacent toa carbonyl group resulting in the electron delocalization inthe carbonyl and the double bond [25] and at 1048 cmminus1 wasattributed to the vibration of CndashOH The band at 1077 cmminus1is a vibration attributed to the CndashOndashC asymmetric stretchingand at 1414 cmminus1 it was assigned toC=C stretch of the flavones

phenyl rings [25 26] In apigenin derivatives the CndashC ringstretching vibrations give rise to characteristic bands in IRspectrum covering the spectral range from 1610 to 1300 cmminus1[26] The presence of nitrogen compounds was attributed toabsorption bands at 2362 and 2343 cmminus1

332 Analyses of Chemical Constituents through HPLC-DAD-ESI-MSMS Mass spectrometry data were acquiredthrough electrospray ionization positive mode Table 1 sum-marizes the following information on peaks observed duringRPHPLC-DAD-ESI-MSMS analyses retention times (Rt)MS data for protonated molecules and proposed structuresConstituents mainly pyrrolizidine alkaloids were tentativelyidentified by the similarity of their fragmentation patternwith known compounds reported in the literature data

The sugar residues of flavonoids glycosides are mainlyhexoses (glucose and galactose) 6-deoxyhexoses (rhamnoseand furanose) pentoses (arabinose and xylose) and uronicacids (glucuronic acid and galacturonic acid) Glucose rham-nose xylose and arabinose are the more common sugar[27 28] Flavonoid O-glycosides are bounded to sugar withformation of an acid labile glycosidic OndashC bond and theirfragmentations patterns involve the heterocyclic cleavageat the glycosidic O-linkage (hemiacetal OndashC bond) with aconcomitant H-rearrangement leading to the elimination ofthe saccharide residue [27 28]

Glycosylation also occurs by direct linkage of the sugarto the basic nucleus of the flavonoid which is stable towardsacid hydrolysis The main fragmentations take place in thesugar which possess the weakest bonds that observed thecross-ring cleavages of the saccharide residue through thesuccessive loss of molecules of water [29ndash32] Two sugarscan be attached to the flavonoid aglycone either at two

Evidence-Based Complementary and Alternative Medicine 5

(a) (b)

GC

V

(c)

Figure 2 Vero cells were cultivated on Aclar film and were inoculated with HSV-1 or virucida sample after 48 hrs (a) Vero cells wereinoculated with HSV-1 and processed by DEM Note particle viral (b) Vero cells were inoculated with HSV-1 and processed by TEM Notethe presence of a typical particle viral (c) Vero cells were inoculated with virucida sample and processed by TEM Note Complex the Golgivesicles Not found the HSV like particles

different positions (di-O-glycosides di-C-glycosides and di-CO-glycosides) or at the same position (O-diglycosidesCO-diglycosides) [27ndash32] Sodium adduct ions [M + Na]+at 22 u are often detected in first-order mass spectra obtainedwith ESI in the positive ion mode [27]

The main flavonoids found in HMG were flavones di-C-glycosides (Table 1) The ESI-MSMS spectra of protonatedmolecules of flavones di-C-glycosides 6ndash11 13 14 and 17displayed neutral losses of water (18 u) as fragments [27ndash32]The main flavone di-C-glycoside compound 14 exhibitedprotonated molecule at119898119911 595 and sodiated molecule [M +Na]+ at 119898119911 617 respectively Its ESI-MSMS spectrum inpositive ion mode produced fragment ions at 577 559 541529 511 499 475 and 457 obtained through the loss of water(Table 1) Compound 17 exhibited protonated molecule at119898119911 565 which afterMSMS experiments exhibited fragmentions at 119898119911 547 529 511 and 427 According to literature

data [29ndash32] compound 14 was characterized as apigenin-68-di-C-glucoside also known as vicenin-2 and compound17was characterized as apigenin-68-di-C-arabinoside gluco-side respectively

Apigenin-di-C-malonyl trihexoside isomers (compounds7ndash9 11 and 13) showed different retention times but the samesodiated molecule at 119898119911 865 and protonated molecule at119898119911 843 (Table 1) respectively The malonyl moiety corre-sponds to 86 u Besides this the ESI-MSMS spectra of pro-tonated molecule showed the same fragmentation pattern at119898119911 825 807 789 771 723 705 and 687 attributed to the lossof water from glucose andor galactose as saccharides [29ndash32] which possess different intensities for each compoundas can be seen in Table 1 what could be used to discriminatebetween these apigenin-di-C-malonyl trihexoside isomers

Other flavones-di-C-glycosides are acacetin and luteolinderivatives Compound 6 showed protonated molecule at

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Results obtained by RPHPLC-DAD-ESI-MSMS analyses of geopropolis from Scaptotrigona postica retention times (Rt) MS datafor positive ion mode and structure proposed

Rt [M + Na]+ [M + H]+ Structure proposed

1 39 mdash [M + H]+ 443MSMSmdash291 Catechin-3-O-gallate

2 56 [M + Na]+mdash365 [M + H]+mdash343 Caffeoyl glucoside

3 65 [M + H]+mdash415MSMSmdash397 (100) 379 (40) 361 (30) 331 (50)

7-Methoxy-5-hydroxy-8-C-flavonerhamnoside

4 155 [M + H]+ 430MSMSmdash412 (100) 385 (70) 367 (10) 315 (20)

7(3-Methoxy-2-methylbutyryl)-9-echimidinylretronecine or methoxyechimidine derivative

5 160 [M + H]+ 444MSMSmdash426 (100) 399 (20)

7(3-Ethoxy-2-methylbutyryl)-9-echimidinylretronecine or ethoxyechimidine derivative

6 165 [M + H]+ 619MSMSmdash601 (80) 583 (20) 565 (20) 535 (100) Acacetin-di-C-acetyl dirhamnoside

7 179 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (40) 789 (20) 771 (30)723 (60) 705 (50)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

8 192 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (80) 807 (40) 789 (30) 771 (20)723 (70) 705 (100) 687 (40)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

9 202 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (20) 771 (30)723 (60) 705 (70) 687 (40)

Apigenin-di-C-malonyl trihexosideisomer

10 204 [M + H]+ 857MSMSmdash839 (100) 803 (30) 737 (40) 719 (30) Acacetin-di-C-malonyl trihexoside

11 211 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (90) 807 (50) 789 (30) 771 (25)723 (100) 705 (60) 687 (10)

Apigenin-di-C-malonyl trihexosideisomer

12 226 [M + H]+ 563MSMSmdash417

Acacetin-8-C-arabinoside-7-O-rhamnoside

13 245 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (10) 771 (20)723 (70) 705 (60)

apigenin-di-C-malonyl trihexosideisomer

14 270[M + Na]+ 617MSMSmdash599(100) 581 (20)

[M + H]+ 595MSMSmdash577 (100) 559 (30) 541 (20) 529 (40)511 (50) 499 (30) 475 (30) 457 (60)

Vicenin-2

15 275[M + Na]+mdash445MSMSmdash427(100) 409 (18)

[M + H]+mdash423MSMSmdash405 (100) 387 (30) 369 (30) 357 (60)327 (30)

Catechin arabinoside

16 280 [M + Na]+ 459MSMSmdash441

[M + H]+mdash437MSMSmdash419 (100) 401 (20) 383 (10) 371 (40)341 (20)

Catechin rhamnoside

17 285 [M + H]+ 565MSMSmdash547 (100) 529 (70) 511 (50) 427 (60)

Apigenin-68-di-C-arabinosideglucoside

18 294 [M + H]+ 547MSMSmdash401

Chrysin-8-C-rhamnoside-7-O-rhamnoside

19 298 [M + H]+ 517MSMSmdash355 (80) 337 (100) 35-Dicaffeoyl quinic acid

20 310 [M + H]+ 595MSMSmdash577 (100) 567 (70) 551 (90) Luteolin-8-C-caffeoyl rhamnoside

21 334 [M + H]+ 487 Caffeoylquinic acid-O-arabinoside

Evidence-Based Complementary and Alternative Medicine 7

119898119911 619 which afterMSMS experiments produced fragmentions at 119898119911 601 583 565 and 535 Compound 10 exhibitedprotonated molecule at 119898119911 857 which after MSMS exper-iments produced fragment ions at 119898119911 839 803 737 and719 Compound 20 showed protonated molecule at 119898119911 595which after MSMS experiments produced fragment ions at119898119911 577 567 and 551 Compounds 6 10 and 20 were ten-tatively characterized as acacetin-di-C-acetyl dirhamnosideacacetin-di-C-malonyl trihexoside and luteolin-8-C-caffeoylrhamnoside respectively

Flavones di-CO-glycosides were also found in low con-tent The ESI-MS spectrum of compound 12 exhibited proto-nated molecule at 119898119911 563 which after MSMS experimentsproduced high fragment ion at119898119911 417 [MndashHminus 146]minus whichwas attributed to the loss of the O-deoxyhexosyl radical(rhamnose moiety) probably linked to phenolic hydroxylgroup in 7-position of aglycone [27ndash32] In the same waycompound 18 exhibited protonated molecule at 119898119911 547which afterMSMS experiments produced high fragment ionat119898119911 401 [M ndash H minus 146]minus Compounds 12 and 18 presentedO-glycosylation on the phenolic hydroxyl group of aglyconewhich was corroborated by literature data [29ndash32] and weresuggested as acacetin-8-C-arabinoside-7-O-rhamnoside andchrysin-8-C-rhamnoside-7-O-rhamnoside respectively

The trans-cinnamic acids are esterified at one or moreof the hydroxyls at positions 1 3 4 and 5 of quinic acidoriginating a series of positional isomers [33ndash35] For com-pound 19 the ESI-MS spectrum showed protonatedmoleculeat 119898119911 517 which after MSMS experiments gave fragmentions at 119898119911 355 (caffeoylquinic acid moiety) and 119898119911 337suggesting di-O-caffeoylquinic acid positional isomers TheESI-MS spectrum of compound 21 exhibited protonatedmolecule at119898119911 487 According to literature data [28 33ndash35]compound 19 was characterized as 35-di-O-caffeoylquinicacid and compound 21 as caffeoylquinic acid-O-arabinosiderespectively

Compound 1 was characterized as catechin-3-O-gallatebased on ESI-MS data that showed protonated molecule at119898119911 443 which after MSMS experiments produced frag-ment ion at 119898119911 291 (protonated catechin) attributed to theloss of galloyl moiety (152 u) [36 37] Other catechin deriva-tives compounds 15 and 16 exhibited sodiated moleculesat 119898119911 445 and 119898119911 459 and protonated molecules at 119898119911423 and 119898119911 437 respectively After MSMS experimentsboth compounds exhibited fragment ions generated by theloss of water molecules indicating the presence of the sugarmolecules linked to the catechin The main fragmentationstake place in the sugar which possess the weakest bondsand the cross-ring cleavages of the saccharide residue areobserved through the successive loss of molecules of wateras shown in Table 1 According to literature data [36 37]compounds 15 and 16 are tentatively characterized as catechinarabinoside and catechin rhamnoside

The presence of 12-dihydropyrrolizidine alkaloids hadbeen observed in bee products and echimidine is the mainalkaloid reported in honey [15ndash18 38] According to Boppreet al [15ndash17] these compounds are transferred to the honeyby bees that visit the flowers to forage nectar and pollenHowever there are no reports about human poisoning

from honey by 12-dihydropyrrolizidine alkaloids [15ndash18 38]Pyrrolizidine alkaloids similar to echimidine are detected forthe first time in this geopropolis from Scaptotrigona postica

TheESI-MS spectrumof compound4 showed protonatedmolecule at 119898119911 430 which after fragmentation producedfragment ions at119898119911 412 attributed to the loss of water (18 u)and at 119898119911 385 attributed to the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid The fragment ion [M +H minus 115]+ at 119898119911 315 was attributed to the loss of methoxydihydro angelic acid moiety that is present on the C-7 ofopen chain diester type of pyrrolizidine alkaloid The lossof angelic acid moiety from echimidine was observed byBoppre et al [15ndash17] For compound 5 the ESI-MS spectrumexhibited protonated molecule at 119898119911 444 while its ESI-MSMS spectrum exhibited the same fragmentation patternas 4 with fragment ions at 119898119911 426 and 119898119911 399 attributedto the loss of water (18 u) and the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid According to literatureMS data [15ndash18 38] the echimidine derivatives 4 and 5were tentatively identified as 7(3-methoxy-2-methylbutyryl)-9-echimidinylretronecine and 7(3-ethoxy-2-methylbutyryl)-9-echimidinylretronecine respectively

4 Discussion

Herpes simplex virus type 1 (HSV-1) is an important pathogenfor humans and the discovery of novel effective antiherpeticdrugs without adverse effects is of great interest [39] Acy-clovir is the first-line treatment for themanagement of herpessimplex virus 1 (HSV-1) and virus 2 (HSV-2) diseases How-ever long-term administration of this drug for the treatmentof chronic infections in the immunocompromised host canlead to the development of acyclovir resistance [19 20 39 40]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about biological activities In order to examinethe antiviral activity of HMG against antiherpes simplexvirus the cells were treated with HMG 1 hour before infec-tion 3 hours after infection and virucidal condition andit was observed that in all conditions and concentrationstested the HMG showed great potential in inhibition ofreplication of HSV-1 Virucida test results suggest that HMGinterfered with virion envelope structures or are maskingviral compounds which are necessary for adsorption or entryinto host cells Apparently free herpes virus is very sensitiveto HMG and the inhibition of HSV-1 appears to occurbefore entering into the cell In our study in all conditionsand concentrations tested EMG showed great potential inreducing the number of copies of viral mRNA Recently ithas been reported that essential oils demonstrated a similarantiviral effect on herpes viruses [41 42]

Besides this HMG showed the highest antiviral activitywhen added during the intracellular replication period Ourresults are in accordance with that observed by Amoros et al[43] during intracellular replication Huleihel and Isanu(2002) [44] observed strong interaction between the propolisextracts and the surface of Vero cells but no direct interactionwith herpes virus particles The studies about the antiviral

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Infection Virucida Pretreatment Posttreatment

Cells

100120583gmL10120583gmL

1120583gmL

Inhi

bitio

n (

)

(a)

Infection Virucida Pretreatment Posttreatment

Supernatant

10120583gmL1120583gmL100120583gmL

0

20

40

60

80

100

120

Inhi

bitio

n (

)(b)

Figure 1 Herpes transcription level cells treated with propolis andmeasured by Real-Time PCR Total DNAwas performed at 3 hours beforeinfection (pretreatment) 1 hour postinfection (posttreatment) and virucida (a) Cells lysate (b) Supernatant Antiviral activity percentageand relative DNA viral quantification

inhibiting HSV-1 viral replication This suggested that thegeopropolis inhibited the events in the early infection suchas viral binding and viral entry into cells as well as the viralreplication Beside this in the cells inoculated with virucidasample and processed by DEM and TEM was not detectedthe HSV like particles into the cytoplasm from Vero cellsanalyzed as can be seen in Figure 2

33 Chemical Characterization ofConstituents from Geopropolis

331 Analysis of Hydromethanolic Extract from Geopropo-lis (HMG) Using Fourier Transform Infrared Spectrometry(FTIR) Fourier Transform Infrared Spectroscopy (FTIR) isa fast accurate and nondestructive technique used as apowerful analytical tool in many fieldsThe absorption bandsobserved in the FTIR spectrum [120592Max cmminus1 (KBr)] for HMGwere 3365 2919 2362 2343 1609 1414 1077 1048 864 and675 cmminus1 The OndashH stretching vibrations are sensitive tohydrogen bonding and the existence of an intermolecularhydrogen bond can lower theOndashH stretchingwavenumber to3500ndash3200 cmminus1The broad peak at 3365 cmminus1 was attributedto hydroxyl stretching vibration of flavones [25 26]

The peak at 1609 cmminus1 was attributed to a carbonyl stretchof flavones that occur at lower vibrational energies as aresult of decreased bond strengths of the carbonyl bonddue to resonance structures with a C=C bond adjacent toa carbonyl group resulting in the electron delocalization inthe carbonyl and the double bond [25] and at 1048 cmminus1 wasattributed to the vibration of CndashOH The band at 1077 cmminus1is a vibration attributed to the CndashOndashC asymmetric stretchingand at 1414 cmminus1 it was assigned toC=C stretch of the flavones

phenyl rings [25 26] In apigenin derivatives the CndashC ringstretching vibrations give rise to characteristic bands in IRspectrum covering the spectral range from 1610 to 1300 cmminus1[26] The presence of nitrogen compounds was attributed toabsorption bands at 2362 and 2343 cmminus1

332 Analyses of Chemical Constituents through HPLC-DAD-ESI-MSMS Mass spectrometry data were acquiredthrough electrospray ionization positive mode Table 1 sum-marizes the following information on peaks observed duringRPHPLC-DAD-ESI-MSMS analyses retention times (Rt)MS data for protonated molecules and proposed structuresConstituents mainly pyrrolizidine alkaloids were tentativelyidentified by the similarity of their fragmentation patternwith known compounds reported in the literature data

The sugar residues of flavonoids glycosides are mainlyhexoses (glucose and galactose) 6-deoxyhexoses (rhamnoseand furanose) pentoses (arabinose and xylose) and uronicacids (glucuronic acid and galacturonic acid) Glucose rham-nose xylose and arabinose are the more common sugar[27 28] Flavonoid O-glycosides are bounded to sugar withformation of an acid labile glycosidic OndashC bond and theirfragmentations patterns involve the heterocyclic cleavageat the glycosidic O-linkage (hemiacetal OndashC bond) with aconcomitant H-rearrangement leading to the elimination ofthe saccharide residue [27 28]

Glycosylation also occurs by direct linkage of the sugarto the basic nucleus of the flavonoid which is stable towardsacid hydrolysis The main fragmentations take place in thesugar which possess the weakest bonds that observed thecross-ring cleavages of the saccharide residue through thesuccessive loss of molecules of water [29ndash32] Two sugarscan be attached to the flavonoid aglycone either at two

Evidence-Based Complementary and Alternative Medicine 5

(a) (b)

GC

V

(c)

Figure 2 Vero cells were cultivated on Aclar film and were inoculated with HSV-1 or virucida sample after 48 hrs (a) Vero cells wereinoculated with HSV-1 and processed by DEM Note particle viral (b) Vero cells were inoculated with HSV-1 and processed by TEM Notethe presence of a typical particle viral (c) Vero cells were inoculated with virucida sample and processed by TEM Note Complex the Golgivesicles Not found the HSV like particles

different positions (di-O-glycosides di-C-glycosides and di-CO-glycosides) or at the same position (O-diglycosidesCO-diglycosides) [27ndash32] Sodium adduct ions [M + Na]+at 22 u are often detected in first-order mass spectra obtainedwith ESI in the positive ion mode [27]

The main flavonoids found in HMG were flavones di-C-glycosides (Table 1) The ESI-MSMS spectra of protonatedmolecules of flavones di-C-glycosides 6ndash11 13 14 and 17displayed neutral losses of water (18 u) as fragments [27ndash32]The main flavone di-C-glycoside compound 14 exhibitedprotonated molecule at119898119911 595 and sodiated molecule [M +Na]+ at 119898119911 617 respectively Its ESI-MSMS spectrum inpositive ion mode produced fragment ions at 577 559 541529 511 499 475 and 457 obtained through the loss of water(Table 1) Compound 17 exhibited protonated molecule at119898119911 565 which afterMSMS experiments exhibited fragmentions at 119898119911 547 529 511 and 427 According to literature

data [29ndash32] compound 14 was characterized as apigenin-68-di-C-glucoside also known as vicenin-2 and compound17was characterized as apigenin-68-di-C-arabinoside gluco-side respectively

Apigenin-di-C-malonyl trihexoside isomers (compounds7ndash9 11 and 13) showed different retention times but the samesodiated molecule at 119898119911 865 and protonated molecule at119898119911 843 (Table 1) respectively The malonyl moiety corre-sponds to 86 u Besides this the ESI-MSMS spectra of pro-tonated molecule showed the same fragmentation pattern at119898119911 825 807 789 771 723 705 and 687 attributed to the lossof water from glucose andor galactose as saccharides [29ndash32] which possess different intensities for each compoundas can be seen in Table 1 what could be used to discriminatebetween these apigenin-di-C-malonyl trihexoside isomers

Other flavones-di-C-glycosides are acacetin and luteolinderivatives Compound 6 showed protonated molecule at

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Results obtained by RPHPLC-DAD-ESI-MSMS analyses of geopropolis from Scaptotrigona postica retention times (Rt) MS datafor positive ion mode and structure proposed

Rt [M + Na]+ [M + H]+ Structure proposed

1 39 mdash [M + H]+ 443MSMSmdash291 Catechin-3-O-gallate

2 56 [M + Na]+mdash365 [M + H]+mdash343 Caffeoyl glucoside

3 65 [M + H]+mdash415MSMSmdash397 (100) 379 (40) 361 (30) 331 (50)

7-Methoxy-5-hydroxy-8-C-flavonerhamnoside

4 155 [M + H]+ 430MSMSmdash412 (100) 385 (70) 367 (10) 315 (20)

7(3-Methoxy-2-methylbutyryl)-9-echimidinylretronecine or methoxyechimidine derivative

5 160 [M + H]+ 444MSMSmdash426 (100) 399 (20)

7(3-Ethoxy-2-methylbutyryl)-9-echimidinylretronecine or ethoxyechimidine derivative

6 165 [M + H]+ 619MSMSmdash601 (80) 583 (20) 565 (20) 535 (100) Acacetin-di-C-acetyl dirhamnoside

7 179 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (40) 789 (20) 771 (30)723 (60) 705 (50)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

8 192 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (80) 807 (40) 789 (30) 771 (20)723 (70) 705 (100) 687 (40)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

9 202 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (20) 771 (30)723 (60) 705 (70) 687 (40)

Apigenin-di-C-malonyl trihexosideisomer

10 204 [M + H]+ 857MSMSmdash839 (100) 803 (30) 737 (40) 719 (30) Acacetin-di-C-malonyl trihexoside

11 211 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (90) 807 (50) 789 (30) 771 (25)723 (100) 705 (60) 687 (10)

Apigenin-di-C-malonyl trihexosideisomer

12 226 [M + H]+ 563MSMSmdash417

Acacetin-8-C-arabinoside-7-O-rhamnoside

13 245 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (10) 771 (20)723 (70) 705 (60)

apigenin-di-C-malonyl trihexosideisomer

14 270[M + Na]+ 617MSMSmdash599(100) 581 (20)

[M + H]+ 595MSMSmdash577 (100) 559 (30) 541 (20) 529 (40)511 (50) 499 (30) 475 (30) 457 (60)

Vicenin-2

15 275[M + Na]+mdash445MSMSmdash427(100) 409 (18)

[M + H]+mdash423MSMSmdash405 (100) 387 (30) 369 (30) 357 (60)327 (30)

Catechin arabinoside

16 280 [M + Na]+ 459MSMSmdash441

[M + H]+mdash437MSMSmdash419 (100) 401 (20) 383 (10) 371 (40)341 (20)

Catechin rhamnoside

17 285 [M + H]+ 565MSMSmdash547 (100) 529 (70) 511 (50) 427 (60)

Apigenin-68-di-C-arabinosideglucoside

18 294 [M + H]+ 547MSMSmdash401

Chrysin-8-C-rhamnoside-7-O-rhamnoside

19 298 [M + H]+ 517MSMSmdash355 (80) 337 (100) 35-Dicaffeoyl quinic acid

20 310 [M + H]+ 595MSMSmdash577 (100) 567 (70) 551 (90) Luteolin-8-C-caffeoyl rhamnoside

21 334 [M + H]+ 487 Caffeoylquinic acid-O-arabinoside

Evidence-Based Complementary and Alternative Medicine 7

119898119911 619 which afterMSMS experiments produced fragmentions at 119898119911 601 583 565 and 535 Compound 10 exhibitedprotonated molecule at 119898119911 857 which after MSMS exper-iments produced fragment ions at 119898119911 839 803 737 and719 Compound 20 showed protonated molecule at 119898119911 595which after MSMS experiments produced fragment ions at119898119911 577 567 and 551 Compounds 6 10 and 20 were ten-tatively characterized as acacetin-di-C-acetyl dirhamnosideacacetin-di-C-malonyl trihexoside and luteolin-8-C-caffeoylrhamnoside respectively

Flavones di-CO-glycosides were also found in low con-tent The ESI-MS spectrum of compound 12 exhibited proto-nated molecule at 119898119911 563 which after MSMS experimentsproduced high fragment ion at119898119911 417 [MndashHminus 146]minus whichwas attributed to the loss of the O-deoxyhexosyl radical(rhamnose moiety) probably linked to phenolic hydroxylgroup in 7-position of aglycone [27ndash32] In the same waycompound 18 exhibited protonated molecule at 119898119911 547which afterMSMS experiments produced high fragment ionat119898119911 401 [M ndash H minus 146]minus Compounds 12 and 18 presentedO-glycosylation on the phenolic hydroxyl group of aglyconewhich was corroborated by literature data [29ndash32] and weresuggested as acacetin-8-C-arabinoside-7-O-rhamnoside andchrysin-8-C-rhamnoside-7-O-rhamnoside respectively

The trans-cinnamic acids are esterified at one or moreof the hydroxyls at positions 1 3 4 and 5 of quinic acidoriginating a series of positional isomers [33ndash35] For com-pound 19 the ESI-MS spectrum showed protonatedmoleculeat 119898119911 517 which after MSMS experiments gave fragmentions at 119898119911 355 (caffeoylquinic acid moiety) and 119898119911 337suggesting di-O-caffeoylquinic acid positional isomers TheESI-MS spectrum of compound 21 exhibited protonatedmolecule at119898119911 487 According to literature data [28 33ndash35]compound 19 was characterized as 35-di-O-caffeoylquinicacid and compound 21 as caffeoylquinic acid-O-arabinosiderespectively

Compound 1 was characterized as catechin-3-O-gallatebased on ESI-MS data that showed protonated molecule at119898119911 443 which after MSMS experiments produced frag-ment ion at 119898119911 291 (protonated catechin) attributed to theloss of galloyl moiety (152 u) [36 37] Other catechin deriva-tives compounds 15 and 16 exhibited sodiated moleculesat 119898119911 445 and 119898119911 459 and protonated molecules at 119898119911423 and 119898119911 437 respectively After MSMS experimentsboth compounds exhibited fragment ions generated by theloss of water molecules indicating the presence of the sugarmolecules linked to the catechin The main fragmentationstake place in the sugar which possess the weakest bondsand the cross-ring cleavages of the saccharide residue areobserved through the successive loss of molecules of wateras shown in Table 1 According to literature data [36 37]compounds 15 and 16 are tentatively characterized as catechinarabinoside and catechin rhamnoside

The presence of 12-dihydropyrrolizidine alkaloids hadbeen observed in bee products and echimidine is the mainalkaloid reported in honey [15ndash18 38] According to Boppreet al [15ndash17] these compounds are transferred to the honeyby bees that visit the flowers to forage nectar and pollenHowever there are no reports about human poisoning

from honey by 12-dihydropyrrolizidine alkaloids [15ndash18 38]Pyrrolizidine alkaloids similar to echimidine are detected forthe first time in this geopropolis from Scaptotrigona postica

TheESI-MS spectrumof compound4 showed protonatedmolecule at 119898119911 430 which after fragmentation producedfragment ions at119898119911 412 attributed to the loss of water (18 u)and at 119898119911 385 attributed to the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid The fragment ion [M +H minus 115]+ at 119898119911 315 was attributed to the loss of methoxydihydro angelic acid moiety that is present on the C-7 ofopen chain diester type of pyrrolizidine alkaloid The lossof angelic acid moiety from echimidine was observed byBoppre et al [15ndash17] For compound 5 the ESI-MS spectrumexhibited protonated molecule at 119898119911 444 while its ESI-MSMS spectrum exhibited the same fragmentation patternas 4 with fragment ions at 119898119911 426 and 119898119911 399 attributedto the loss of water (18 u) and the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid According to literatureMS data [15ndash18 38] the echimidine derivatives 4 and 5were tentatively identified as 7(3-methoxy-2-methylbutyryl)-9-echimidinylretronecine and 7(3-ethoxy-2-methylbutyryl)-9-echimidinylretronecine respectively

4 Discussion

Herpes simplex virus type 1 (HSV-1) is an important pathogenfor humans and the discovery of novel effective antiherpeticdrugs without adverse effects is of great interest [39] Acy-clovir is the first-line treatment for themanagement of herpessimplex virus 1 (HSV-1) and virus 2 (HSV-2) diseases How-ever long-term administration of this drug for the treatmentof chronic infections in the immunocompromised host canlead to the development of acyclovir resistance [19 20 39 40]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about biological activities In order to examinethe antiviral activity of HMG against antiherpes simplexvirus the cells were treated with HMG 1 hour before infec-tion 3 hours after infection and virucidal condition andit was observed that in all conditions and concentrationstested the HMG showed great potential in inhibition ofreplication of HSV-1 Virucida test results suggest that HMGinterfered with virion envelope structures or are maskingviral compounds which are necessary for adsorption or entryinto host cells Apparently free herpes virus is very sensitiveto HMG and the inhibition of HSV-1 appears to occurbefore entering into the cell In our study in all conditionsand concentrations tested EMG showed great potential inreducing the number of copies of viral mRNA Recently ithas been reported that essential oils demonstrated a similarantiviral effect on herpes viruses [41 42]

Besides this HMG showed the highest antiviral activitywhen added during the intracellular replication period Ourresults are in accordance with that observed by Amoros et al[43] during intracellular replication Huleihel and Isanu(2002) [44] observed strong interaction between the propolisextracts and the surface of Vero cells but no direct interactionwith herpes virus particles The studies about the antiviral

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

(a) (b)

GC

V

(c)

Figure 2 Vero cells were cultivated on Aclar film and were inoculated with HSV-1 or virucida sample after 48 hrs (a) Vero cells wereinoculated with HSV-1 and processed by DEM Note particle viral (b) Vero cells were inoculated with HSV-1 and processed by TEM Notethe presence of a typical particle viral (c) Vero cells were inoculated with virucida sample and processed by TEM Note Complex the Golgivesicles Not found the HSV like particles

different positions (di-O-glycosides di-C-glycosides and di-CO-glycosides) or at the same position (O-diglycosidesCO-diglycosides) [27ndash32] Sodium adduct ions [M + Na]+at 22 u are often detected in first-order mass spectra obtainedwith ESI in the positive ion mode [27]

The main flavonoids found in HMG were flavones di-C-glycosides (Table 1) The ESI-MSMS spectra of protonatedmolecules of flavones di-C-glycosides 6ndash11 13 14 and 17displayed neutral losses of water (18 u) as fragments [27ndash32]The main flavone di-C-glycoside compound 14 exhibitedprotonated molecule at119898119911 595 and sodiated molecule [M +Na]+ at 119898119911 617 respectively Its ESI-MSMS spectrum inpositive ion mode produced fragment ions at 577 559 541529 511 499 475 and 457 obtained through the loss of water(Table 1) Compound 17 exhibited protonated molecule at119898119911 565 which afterMSMS experiments exhibited fragmentions at 119898119911 547 529 511 and 427 According to literature

data [29ndash32] compound 14 was characterized as apigenin-68-di-C-glucoside also known as vicenin-2 and compound17was characterized as apigenin-68-di-C-arabinoside gluco-side respectively

Apigenin-di-C-malonyl trihexoside isomers (compounds7ndash9 11 and 13) showed different retention times but the samesodiated molecule at 119898119911 865 and protonated molecule at119898119911 843 (Table 1) respectively The malonyl moiety corre-sponds to 86 u Besides this the ESI-MSMS spectra of pro-tonated molecule showed the same fragmentation pattern at119898119911 825 807 789 771 723 705 and 687 attributed to the lossof water from glucose andor galactose as saccharides [29ndash32] which possess different intensities for each compoundas can be seen in Table 1 what could be used to discriminatebetween these apigenin-di-C-malonyl trihexoside isomers

Other flavones-di-C-glycosides are acacetin and luteolinderivatives Compound 6 showed protonated molecule at

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Results obtained by RPHPLC-DAD-ESI-MSMS analyses of geopropolis from Scaptotrigona postica retention times (Rt) MS datafor positive ion mode and structure proposed

Rt [M + Na]+ [M + H]+ Structure proposed

1 39 mdash [M + H]+ 443MSMSmdash291 Catechin-3-O-gallate

2 56 [M + Na]+mdash365 [M + H]+mdash343 Caffeoyl glucoside

3 65 [M + H]+mdash415MSMSmdash397 (100) 379 (40) 361 (30) 331 (50)

7-Methoxy-5-hydroxy-8-C-flavonerhamnoside

4 155 [M + H]+ 430MSMSmdash412 (100) 385 (70) 367 (10) 315 (20)

7(3-Methoxy-2-methylbutyryl)-9-echimidinylretronecine or methoxyechimidine derivative

5 160 [M + H]+ 444MSMSmdash426 (100) 399 (20)

7(3-Ethoxy-2-methylbutyryl)-9-echimidinylretronecine or ethoxyechimidine derivative

6 165 [M + H]+ 619MSMSmdash601 (80) 583 (20) 565 (20) 535 (100) Acacetin-di-C-acetyl dirhamnoside

7 179 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (40) 789 (20) 771 (30)723 (60) 705 (50)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

8 192 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (80) 807 (40) 789 (30) 771 (20)723 (70) 705 (100) 687 (40)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

9 202 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (20) 771 (30)723 (60) 705 (70) 687 (40)

Apigenin-di-C-malonyl trihexosideisomer

10 204 [M + H]+ 857MSMSmdash839 (100) 803 (30) 737 (40) 719 (30) Acacetin-di-C-malonyl trihexoside

11 211 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (90) 807 (50) 789 (30) 771 (25)723 (100) 705 (60) 687 (10)

Apigenin-di-C-malonyl trihexosideisomer

12 226 [M + H]+ 563MSMSmdash417

Acacetin-8-C-arabinoside-7-O-rhamnoside

13 245 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (10) 771 (20)723 (70) 705 (60)

apigenin-di-C-malonyl trihexosideisomer

14 270[M + Na]+ 617MSMSmdash599(100) 581 (20)

[M + H]+ 595MSMSmdash577 (100) 559 (30) 541 (20) 529 (40)511 (50) 499 (30) 475 (30) 457 (60)

Vicenin-2

15 275[M + Na]+mdash445MSMSmdash427(100) 409 (18)

[M + H]+mdash423MSMSmdash405 (100) 387 (30) 369 (30) 357 (60)327 (30)

Catechin arabinoside

16 280 [M + Na]+ 459MSMSmdash441

[M + H]+mdash437MSMSmdash419 (100) 401 (20) 383 (10) 371 (40)341 (20)

Catechin rhamnoside

17 285 [M + H]+ 565MSMSmdash547 (100) 529 (70) 511 (50) 427 (60)

Apigenin-68-di-C-arabinosideglucoside

18 294 [M + H]+ 547MSMSmdash401

Chrysin-8-C-rhamnoside-7-O-rhamnoside

19 298 [M + H]+ 517MSMSmdash355 (80) 337 (100) 35-Dicaffeoyl quinic acid

20 310 [M + H]+ 595MSMSmdash577 (100) 567 (70) 551 (90) Luteolin-8-C-caffeoyl rhamnoside

21 334 [M + H]+ 487 Caffeoylquinic acid-O-arabinoside

Evidence-Based Complementary and Alternative Medicine 7

119898119911 619 which afterMSMS experiments produced fragmentions at 119898119911 601 583 565 and 535 Compound 10 exhibitedprotonated molecule at 119898119911 857 which after MSMS exper-iments produced fragment ions at 119898119911 839 803 737 and719 Compound 20 showed protonated molecule at 119898119911 595which after MSMS experiments produced fragment ions at119898119911 577 567 and 551 Compounds 6 10 and 20 were ten-tatively characterized as acacetin-di-C-acetyl dirhamnosideacacetin-di-C-malonyl trihexoside and luteolin-8-C-caffeoylrhamnoside respectively

Flavones di-CO-glycosides were also found in low con-tent The ESI-MS spectrum of compound 12 exhibited proto-nated molecule at 119898119911 563 which after MSMS experimentsproduced high fragment ion at119898119911 417 [MndashHminus 146]minus whichwas attributed to the loss of the O-deoxyhexosyl radical(rhamnose moiety) probably linked to phenolic hydroxylgroup in 7-position of aglycone [27ndash32] In the same waycompound 18 exhibited protonated molecule at 119898119911 547which afterMSMS experiments produced high fragment ionat119898119911 401 [M ndash H minus 146]minus Compounds 12 and 18 presentedO-glycosylation on the phenolic hydroxyl group of aglyconewhich was corroborated by literature data [29ndash32] and weresuggested as acacetin-8-C-arabinoside-7-O-rhamnoside andchrysin-8-C-rhamnoside-7-O-rhamnoside respectively

The trans-cinnamic acids are esterified at one or moreof the hydroxyls at positions 1 3 4 and 5 of quinic acidoriginating a series of positional isomers [33ndash35] For com-pound 19 the ESI-MS spectrum showed protonatedmoleculeat 119898119911 517 which after MSMS experiments gave fragmentions at 119898119911 355 (caffeoylquinic acid moiety) and 119898119911 337suggesting di-O-caffeoylquinic acid positional isomers TheESI-MS spectrum of compound 21 exhibited protonatedmolecule at119898119911 487 According to literature data [28 33ndash35]compound 19 was characterized as 35-di-O-caffeoylquinicacid and compound 21 as caffeoylquinic acid-O-arabinosiderespectively

Compound 1 was characterized as catechin-3-O-gallatebased on ESI-MS data that showed protonated molecule at119898119911 443 which after MSMS experiments produced frag-ment ion at 119898119911 291 (protonated catechin) attributed to theloss of galloyl moiety (152 u) [36 37] Other catechin deriva-tives compounds 15 and 16 exhibited sodiated moleculesat 119898119911 445 and 119898119911 459 and protonated molecules at 119898119911423 and 119898119911 437 respectively After MSMS experimentsboth compounds exhibited fragment ions generated by theloss of water molecules indicating the presence of the sugarmolecules linked to the catechin The main fragmentationstake place in the sugar which possess the weakest bondsand the cross-ring cleavages of the saccharide residue areobserved through the successive loss of molecules of wateras shown in Table 1 According to literature data [36 37]compounds 15 and 16 are tentatively characterized as catechinarabinoside and catechin rhamnoside

The presence of 12-dihydropyrrolizidine alkaloids hadbeen observed in bee products and echimidine is the mainalkaloid reported in honey [15ndash18 38] According to Boppreet al [15ndash17] these compounds are transferred to the honeyby bees that visit the flowers to forage nectar and pollenHowever there are no reports about human poisoning

from honey by 12-dihydropyrrolizidine alkaloids [15ndash18 38]Pyrrolizidine alkaloids similar to echimidine are detected forthe first time in this geopropolis from Scaptotrigona postica

TheESI-MS spectrumof compound4 showed protonatedmolecule at 119898119911 430 which after fragmentation producedfragment ions at119898119911 412 attributed to the loss of water (18 u)and at 119898119911 385 attributed to the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid The fragment ion [M +H minus 115]+ at 119898119911 315 was attributed to the loss of methoxydihydro angelic acid moiety that is present on the C-7 ofopen chain diester type of pyrrolizidine alkaloid The lossof angelic acid moiety from echimidine was observed byBoppre et al [15ndash17] For compound 5 the ESI-MS spectrumexhibited protonated molecule at 119898119911 444 while its ESI-MSMS spectrum exhibited the same fragmentation patternas 4 with fragment ions at 119898119911 426 and 119898119911 399 attributedto the loss of water (18 u) and the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid According to literatureMS data [15ndash18 38] the echimidine derivatives 4 and 5were tentatively identified as 7(3-methoxy-2-methylbutyryl)-9-echimidinylretronecine and 7(3-ethoxy-2-methylbutyryl)-9-echimidinylretronecine respectively

4 Discussion

Herpes simplex virus type 1 (HSV-1) is an important pathogenfor humans and the discovery of novel effective antiherpeticdrugs without adverse effects is of great interest [39] Acy-clovir is the first-line treatment for themanagement of herpessimplex virus 1 (HSV-1) and virus 2 (HSV-2) diseases How-ever long-term administration of this drug for the treatmentof chronic infections in the immunocompromised host canlead to the development of acyclovir resistance [19 20 39 40]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about biological activities In order to examinethe antiviral activity of HMG against antiherpes simplexvirus the cells were treated with HMG 1 hour before infec-tion 3 hours after infection and virucidal condition andit was observed that in all conditions and concentrationstested the HMG showed great potential in inhibition ofreplication of HSV-1 Virucida test results suggest that HMGinterfered with virion envelope structures or are maskingviral compounds which are necessary for adsorption or entryinto host cells Apparently free herpes virus is very sensitiveto HMG and the inhibition of HSV-1 appears to occurbefore entering into the cell In our study in all conditionsand concentrations tested EMG showed great potential inreducing the number of copies of viral mRNA Recently ithas been reported that essential oils demonstrated a similarantiviral effect on herpes viruses [41 42]

Besides this HMG showed the highest antiviral activitywhen added during the intracellular replication period Ourresults are in accordance with that observed by Amoros et al[43] during intracellular replication Huleihel and Isanu(2002) [44] observed strong interaction between the propolisextracts and the surface of Vero cells but no direct interactionwith herpes virus particles The studies about the antiviral

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Results obtained by RPHPLC-DAD-ESI-MSMS analyses of geopropolis from Scaptotrigona postica retention times (Rt) MS datafor positive ion mode and structure proposed

Rt [M + Na]+ [M + H]+ Structure proposed

1 39 mdash [M + H]+ 443MSMSmdash291 Catechin-3-O-gallate

2 56 [M + Na]+mdash365 [M + H]+mdash343 Caffeoyl glucoside

3 65 [M + H]+mdash415MSMSmdash397 (100) 379 (40) 361 (30) 331 (50)

7-Methoxy-5-hydroxy-8-C-flavonerhamnoside

4 155 [M + H]+ 430MSMSmdash412 (100) 385 (70) 367 (10) 315 (20)

7(3-Methoxy-2-methylbutyryl)-9-echimidinylretronecine or methoxyechimidine derivative

5 160 [M + H]+ 444MSMSmdash426 (100) 399 (20)

7(3-Ethoxy-2-methylbutyryl)-9-echimidinylretronecine or ethoxyechimidine derivative

6 165 [M + H]+ 619MSMSmdash601 (80) 583 (20) 565 (20) 535 (100) Acacetin-di-C-acetyl dirhamnoside

7 179 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (40) 789 (20) 771 (30)723 (60) 705 (50)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

8 192 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (80) 807 (40) 789 (30) 771 (20)723 (70) 705 (100) 687 (40)

Apigenin-68-di-C-malonyl glucosidedihexoside isomer

9 202 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (20) 771 (30)723 (60) 705 (70) 687 (40)

Apigenin-di-C-malonyl trihexosideisomer

10 204 [M + H]+ 857MSMSmdash839 (100) 803 (30) 737 (40) 719 (30) Acacetin-di-C-malonyl trihexoside

11 211 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (90) 807 (50) 789 (30) 771 (25)723 (100) 705 (60) 687 (10)

Apigenin-di-C-malonyl trihexosideisomer

12 226 [M + H]+ 563MSMSmdash417

Acacetin-8-C-arabinoside-7-O-rhamnoside

13 245 [M + Na]+ 865[M + H]+ 843MSMSmdash825 (100) 807 (50) 789 (10) 771 (20)723 (70) 705 (60)

apigenin-di-C-malonyl trihexosideisomer

14 270[M + Na]+ 617MSMSmdash599(100) 581 (20)

[M + H]+ 595MSMSmdash577 (100) 559 (30) 541 (20) 529 (40)511 (50) 499 (30) 475 (30) 457 (60)

Vicenin-2

15 275[M + Na]+mdash445MSMSmdash427(100) 409 (18)

[M + H]+mdash423MSMSmdash405 (100) 387 (30) 369 (30) 357 (60)327 (30)

Catechin arabinoside

16 280 [M + Na]+ 459MSMSmdash441

[M + H]+mdash437MSMSmdash419 (100) 401 (20) 383 (10) 371 (40)341 (20)

Catechin rhamnoside

17 285 [M + H]+ 565MSMSmdash547 (100) 529 (70) 511 (50) 427 (60)

Apigenin-68-di-C-arabinosideglucoside

18 294 [M + H]+ 547MSMSmdash401

Chrysin-8-C-rhamnoside-7-O-rhamnoside

19 298 [M + H]+ 517MSMSmdash355 (80) 337 (100) 35-Dicaffeoyl quinic acid

20 310 [M + H]+ 595MSMSmdash577 (100) 567 (70) 551 (90) Luteolin-8-C-caffeoyl rhamnoside

21 334 [M + H]+ 487 Caffeoylquinic acid-O-arabinoside

Evidence-Based Complementary and Alternative Medicine 7

119898119911 619 which afterMSMS experiments produced fragmentions at 119898119911 601 583 565 and 535 Compound 10 exhibitedprotonated molecule at 119898119911 857 which after MSMS exper-iments produced fragment ions at 119898119911 839 803 737 and719 Compound 20 showed protonated molecule at 119898119911 595which after MSMS experiments produced fragment ions at119898119911 577 567 and 551 Compounds 6 10 and 20 were ten-tatively characterized as acacetin-di-C-acetyl dirhamnosideacacetin-di-C-malonyl trihexoside and luteolin-8-C-caffeoylrhamnoside respectively

Flavones di-CO-glycosides were also found in low con-tent The ESI-MS spectrum of compound 12 exhibited proto-nated molecule at 119898119911 563 which after MSMS experimentsproduced high fragment ion at119898119911 417 [MndashHminus 146]minus whichwas attributed to the loss of the O-deoxyhexosyl radical(rhamnose moiety) probably linked to phenolic hydroxylgroup in 7-position of aglycone [27ndash32] In the same waycompound 18 exhibited protonated molecule at 119898119911 547which afterMSMS experiments produced high fragment ionat119898119911 401 [M ndash H minus 146]minus Compounds 12 and 18 presentedO-glycosylation on the phenolic hydroxyl group of aglyconewhich was corroborated by literature data [29ndash32] and weresuggested as acacetin-8-C-arabinoside-7-O-rhamnoside andchrysin-8-C-rhamnoside-7-O-rhamnoside respectively

The trans-cinnamic acids are esterified at one or moreof the hydroxyls at positions 1 3 4 and 5 of quinic acidoriginating a series of positional isomers [33ndash35] For com-pound 19 the ESI-MS spectrum showed protonatedmoleculeat 119898119911 517 which after MSMS experiments gave fragmentions at 119898119911 355 (caffeoylquinic acid moiety) and 119898119911 337suggesting di-O-caffeoylquinic acid positional isomers TheESI-MS spectrum of compound 21 exhibited protonatedmolecule at119898119911 487 According to literature data [28 33ndash35]compound 19 was characterized as 35-di-O-caffeoylquinicacid and compound 21 as caffeoylquinic acid-O-arabinosiderespectively

Compound 1 was characterized as catechin-3-O-gallatebased on ESI-MS data that showed protonated molecule at119898119911 443 which after MSMS experiments produced frag-ment ion at 119898119911 291 (protonated catechin) attributed to theloss of galloyl moiety (152 u) [36 37] Other catechin deriva-tives compounds 15 and 16 exhibited sodiated moleculesat 119898119911 445 and 119898119911 459 and protonated molecules at 119898119911423 and 119898119911 437 respectively After MSMS experimentsboth compounds exhibited fragment ions generated by theloss of water molecules indicating the presence of the sugarmolecules linked to the catechin The main fragmentationstake place in the sugar which possess the weakest bondsand the cross-ring cleavages of the saccharide residue areobserved through the successive loss of molecules of wateras shown in Table 1 According to literature data [36 37]compounds 15 and 16 are tentatively characterized as catechinarabinoside and catechin rhamnoside

The presence of 12-dihydropyrrolizidine alkaloids hadbeen observed in bee products and echimidine is the mainalkaloid reported in honey [15ndash18 38] According to Boppreet al [15ndash17] these compounds are transferred to the honeyby bees that visit the flowers to forage nectar and pollenHowever there are no reports about human poisoning

from honey by 12-dihydropyrrolizidine alkaloids [15ndash18 38]Pyrrolizidine alkaloids similar to echimidine are detected forthe first time in this geopropolis from Scaptotrigona postica

TheESI-MS spectrumof compound4 showed protonatedmolecule at 119898119911 430 which after fragmentation producedfragment ions at119898119911 412 attributed to the loss of water (18 u)and at 119898119911 385 attributed to the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid The fragment ion [M +H minus 115]+ at 119898119911 315 was attributed to the loss of methoxydihydro angelic acid moiety that is present on the C-7 ofopen chain diester type of pyrrolizidine alkaloid The lossof angelic acid moiety from echimidine was observed byBoppre et al [15ndash17] For compound 5 the ESI-MS spectrumexhibited protonated molecule at 119898119911 444 while its ESI-MSMS spectrum exhibited the same fragmentation patternas 4 with fragment ions at 119898119911 426 and 119898119911 399 attributedto the loss of water (18 u) and the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid According to literatureMS data [15ndash18 38] the echimidine derivatives 4 and 5were tentatively identified as 7(3-methoxy-2-methylbutyryl)-9-echimidinylretronecine and 7(3-ethoxy-2-methylbutyryl)-9-echimidinylretronecine respectively

4 Discussion

Herpes simplex virus type 1 (HSV-1) is an important pathogenfor humans and the discovery of novel effective antiherpeticdrugs without adverse effects is of great interest [39] Acy-clovir is the first-line treatment for themanagement of herpessimplex virus 1 (HSV-1) and virus 2 (HSV-2) diseases How-ever long-term administration of this drug for the treatmentof chronic infections in the immunocompromised host canlead to the development of acyclovir resistance [19 20 39 40]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about biological activities In order to examinethe antiviral activity of HMG against antiherpes simplexvirus the cells were treated with HMG 1 hour before infec-tion 3 hours after infection and virucidal condition andit was observed that in all conditions and concentrationstested the HMG showed great potential in inhibition ofreplication of HSV-1 Virucida test results suggest that HMGinterfered with virion envelope structures or are maskingviral compounds which are necessary for adsorption or entryinto host cells Apparently free herpes virus is very sensitiveto HMG and the inhibition of HSV-1 appears to occurbefore entering into the cell In our study in all conditionsand concentrations tested EMG showed great potential inreducing the number of copies of viral mRNA Recently ithas been reported that essential oils demonstrated a similarantiviral effect on herpes viruses [41 42]

Besides this HMG showed the highest antiviral activitywhen added during the intracellular replication period Ourresults are in accordance with that observed by Amoros et al[43] during intracellular replication Huleihel and Isanu(2002) [44] observed strong interaction between the propolisextracts and the surface of Vero cells but no direct interactionwith herpes virus particles The studies about the antiviral

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

119898119911 619 which afterMSMS experiments produced fragmentions at 119898119911 601 583 565 and 535 Compound 10 exhibitedprotonated molecule at 119898119911 857 which after MSMS exper-iments produced fragment ions at 119898119911 839 803 737 and719 Compound 20 showed protonated molecule at 119898119911 595which after MSMS experiments produced fragment ions at119898119911 577 567 and 551 Compounds 6 10 and 20 were ten-tatively characterized as acacetin-di-C-acetyl dirhamnosideacacetin-di-C-malonyl trihexoside and luteolin-8-C-caffeoylrhamnoside respectively

Flavones di-CO-glycosides were also found in low con-tent The ESI-MS spectrum of compound 12 exhibited proto-nated molecule at 119898119911 563 which after MSMS experimentsproduced high fragment ion at119898119911 417 [MndashHminus 146]minus whichwas attributed to the loss of the O-deoxyhexosyl radical(rhamnose moiety) probably linked to phenolic hydroxylgroup in 7-position of aglycone [27ndash32] In the same waycompound 18 exhibited protonated molecule at 119898119911 547which afterMSMS experiments produced high fragment ionat119898119911 401 [M ndash H minus 146]minus Compounds 12 and 18 presentedO-glycosylation on the phenolic hydroxyl group of aglyconewhich was corroborated by literature data [29ndash32] and weresuggested as acacetin-8-C-arabinoside-7-O-rhamnoside andchrysin-8-C-rhamnoside-7-O-rhamnoside respectively

The trans-cinnamic acids are esterified at one or moreof the hydroxyls at positions 1 3 4 and 5 of quinic acidoriginating a series of positional isomers [33ndash35] For com-pound 19 the ESI-MS spectrum showed protonatedmoleculeat 119898119911 517 which after MSMS experiments gave fragmentions at 119898119911 355 (caffeoylquinic acid moiety) and 119898119911 337suggesting di-O-caffeoylquinic acid positional isomers TheESI-MS spectrum of compound 21 exhibited protonatedmolecule at119898119911 487 According to literature data [28 33ndash35]compound 19 was characterized as 35-di-O-caffeoylquinicacid and compound 21 as caffeoylquinic acid-O-arabinosiderespectively

Compound 1 was characterized as catechin-3-O-gallatebased on ESI-MS data that showed protonated molecule at119898119911 443 which after MSMS experiments produced frag-ment ion at 119898119911 291 (protonated catechin) attributed to theloss of galloyl moiety (152 u) [36 37] Other catechin deriva-tives compounds 15 and 16 exhibited sodiated moleculesat 119898119911 445 and 119898119911 459 and protonated molecules at 119898119911423 and 119898119911 437 respectively After MSMS experimentsboth compounds exhibited fragment ions generated by theloss of water molecules indicating the presence of the sugarmolecules linked to the catechin The main fragmentationstake place in the sugar which possess the weakest bondsand the cross-ring cleavages of the saccharide residue areobserved through the successive loss of molecules of wateras shown in Table 1 According to literature data [36 37]compounds 15 and 16 are tentatively characterized as catechinarabinoside and catechin rhamnoside

The presence of 12-dihydropyrrolizidine alkaloids hadbeen observed in bee products and echimidine is the mainalkaloid reported in honey [15ndash18 38] According to Boppreet al [15ndash17] these compounds are transferred to the honeyby bees that visit the flowers to forage nectar and pollenHowever there are no reports about human poisoning

from honey by 12-dihydropyrrolizidine alkaloids [15ndash18 38]Pyrrolizidine alkaloids similar to echimidine are detected forthe first time in this geopropolis from Scaptotrigona postica

TheESI-MS spectrumof compound4 showed protonatedmolecule at 119898119911 430 which after fragmentation producedfragment ions at119898119911 412 attributed to the loss of water (18 u)and at 119898119911 385 attributed to the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid The fragment ion [M +H minus 115]+ at 119898119911 315 was attributed to the loss of methoxydihydro angelic acid moiety that is present on the C-7 ofopen chain diester type of pyrrolizidine alkaloid The lossof angelic acid moiety from echimidine was observed byBoppre et al [15ndash17] For compound 5 the ESI-MS spectrumexhibited protonated molecule at 119898119911 444 while its ESI-MSMS spectrum exhibited the same fragmentation patternas 4 with fragment ions at 119898119911 426 and 119898119911 399 attributedto the loss of water (18 u) and the loss of (CH

3CHOH) group

of echimidinylretronecinemoiety linked at C-9 of open chaindiester type of pyrrolizidine alkaloid According to literatureMS data [15ndash18 38] the echimidine derivatives 4 and 5were tentatively identified as 7(3-methoxy-2-methylbutyryl)-9-echimidinylretronecine and 7(3-ethoxy-2-methylbutyryl)-9-echimidinylretronecine respectively

4 Discussion

Herpes simplex virus type 1 (HSV-1) is an important pathogenfor humans and the discovery of novel effective antiherpeticdrugs without adverse effects is of great interest [39] Acy-clovir is the first-line treatment for themanagement of herpessimplex virus 1 (HSV-1) and virus 2 (HSV-2) diseases How-ever long-term administration of this drug for the treatmentof chronic infections in the immunocompromised host canlead to the development of acyclovir resistance [19 20 39 40]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about biological activities In order to examinethe antiviral activity of HMG against antiherpes simplexvirus the cells were treated with HMG 1 hour before infec-tion 3 hours after infection and virucidal condition andit was observed that in all conditions and concentrationstested the HMG showed great potential in inhibition ofreplication of HSV-1 Virucida test results suggest that HMGinterfered with virion envelope structures or are maskingviral compounds which are necessary for adsorption or entryinto host cells Apparently free herpes virus is very sensitiveto HMG and the inhibition of HSV-1 appears to occurbefore entering into the cell In our study in all conditionsand concentrations tested EMG showed great potential inreducing the number of copies of viral mRNA Recently ithas been reported that essential oils demonstrated a similarantiviral effect on herpes viruses [41 42]

Besides this HMG showed the highest antiviral activitywhen added during the intracellular replication period Ourresults are in accordance with that observed by Amoros et al[43] during intracellular replication Huleihel and Isanu(2002) [44] observed strong interaction between the propolisextracts and the surface of Vero cells but no direct interactionwith herpes virus particles The studies about the antiviral

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

activity of propolis had revealed different modes of antiviralaction [43 44] It remains to be determined whether theinhibitory effect of HMG is due to binding of some con-stituents of the propolis to viral proteins involved in hostcell adsorption and penetration or is due to damage of thevirions possibly in their envelopes form thereby impairingtheir ability to infect host cells

On the other hand the administration of HMG beforethe time of infection yielded the most significant inhibitoryeffectThus the inhibitory action of EMG on viral replicationcould be through the IFN pathways Recently Melchjorsen etal [45] showed that the initial response of the cells infectedby HSV was the secretion of antiviral substances such asdefensins and nitric oxide and mainly the production ofcytokines including IFNs and chemokines The type IFNs(IFN-120572120573) are key cytokines produced during the very firsthours after HSV infection [45] The outcome of HSV infec-tions is dependent on the balance between virus propagationand effective immune response Appropriate expression ofIFNs cytokines and chemokines is essential for efficient hostdefense against infection [45] Our study could suggest thatHMG stimulated Vero cells in the production of interferon

All the results obtained in our study are consistent withthe images obtained by transmission electron microscopy(TEM) in which the presence of virions was not observedinto the cytoplasm of cells inoculated with virucida sampleBesides this no changes were observed in the nuclear mem-brane which could suggest the formation of nucleocapsids[46] into the nucleus (Figure 2)

Propolis from different localities exhibited antiviral activ-ity at different points in the viral replication [43 44] Asynergism was demonstrated when binary flavones-flavonolcombinations of extracts from propolis were tested againstHSV The results demonstrated that extracts from propoliswere more active than its isolated constituents The extractscontaining many different components exhibited significanthigher antiherpetic effects as well as higher selectivity indicesthan single isolate constituents [47]

For geopropolis from Scaptotrigona postica [5 6] thereare no studies about chemical composition In our study themain constituents found in HMG were flavones di-C-glyco-sides showing vicenin-2 as the major constituent togetherwith pyrrolizidine alkaloids as 7(3-methoxy-2-methylbuty-ryl)-9-echimidinylretronecine and caffeoylquinic acid-O-arabinoside The geopropolis of Melipona fasciculata Smithfrom Baixada Maranhense also in Maranhao state Brazilshowed a different chemical composition containing pheno-lic acids and hydrolyzable tannins (gallotannins and ellagi-tannins) as main constituents [4]

The antiviral activity of compounds identified in EMGwith the exception of echimidine derivatives is known Thegreat potential of HMG in inhibition of HSV-1 indicated asignificant effect on the stages of replication of the virus whatcould be attributed to the high content of C-glycosylflavonesThe C-glycosylflavonoids isolated from crude aqueousextract obtained from Cecropia glaziovii leaves exhibitedantiherpes activity against human herpes virus types 1and 2 by plaque reduction assay [48 49] Several flavone6-C-monoglycosides showed potent in vitro antiviral effect

[50] Apigenin and not similar compounds like luteolinnaringenin and quercetin was active against enterovirus71 infection [51] providing an evidence that one hydroxylgroup difference in the B ring between apigenin and luteolinresulted in the distinct antiviral mechanisms [52] Flavonoidsshowed highly effective antiviral activity against HSV-1 andHSV-2 [53]

Propolis extracts which are rich in flavonoids and phe-nylcarboxylic acids exhibited high levels of antiviral activ-ity against HSV-2 in viral suspension tests [54] Flavon-oid and catechin derivatives showed inhibitory activity againstherpes simplex virus type 1 and herpes simplex virus type2 tested in vitro on RC-37 cells using a plaque reductionassay and exhibited high antiviral activity against both herpesviruses in viral suspension tests [55] A Brazilian propolis thatcontained artepillin C as a main prenylated phenylpropanoidand chrysin as a main flavonoid showed not only direct anti-HSV-1 activity but also immunological activity against intra-dermal HSV-1 infection in mice [56] Catechin-3-O-gallate[57 58] and 34-dicaffeoylquinic acid exhibited significantantiviral action [59]

Thus the great potential of HMG in reducing the copiesof the viral DNA at low concentrations can be explained bythe known antiviral activity of C-glycosylflavones catechin-3-O-gallate and 34-dicaffeoylquinic acid The HMG actedinhibiting the viral replication as also inhibiting the entry ofthe virus into cells This extract inhibited the replication ofa virus of great importance to public health however morestudies are necessary to elucidate the mechanisms of actionand identify the molecules responsible for these effects

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This study was kindly supported by FAPESP (Fundacao deApoio a Pesquisa do Estado de Sao Paulo) and CNPqWilsonMelo gently provided a geopropolis sample

References

[1] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[2] S Huang C-P Zhang K Wang G Li and F-L Hu ldquoRecentadvances in the chemical composition of propolisrdquo Moleculesvol 19 no 12 pp 19610ndash19632 2014

[3] R P Dutra A M C Nogueira R R D O Marques M CP Costa and M N S Ribeiro ldquoPharmacognostic evaluationof geopropolis of Melipona fasciculata Smith from BaixadaMaranhense Brazilrdquo Brazilian Journal of Pharmacognosy vol18 no 4 pp 557ndash562 2008

[4] R P Dutra B V D Abreu M S Cunha et al ldquoPhenolic acidshydrolyzable tannins and antioxidant activity of geopropolisfrom the stingless bee Melipona fasciculata smithrdquo Journal ofAgricultural and Food Chemistry vol 62 no 12 pp 2549ndash25572014

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

[5] W Kerr ldquoAbelhas indıgenas brasileiras (meliponıneos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 no 1 pp 15ndash27 1987

[6] P Nogueira-Neto A vida e criacao de abelhas indıgenas semferrao (Meliponinae) Nogueirapes Sao Paulo Brazil 1997

[7] M G da Cunha M Franchin L C D Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary and AlternativeMedicine vol 13 article 23 2013

[8] E C C da Silva M P Muniz R D C S Nunomura SM Nunomura and G A C Zilse ldquoPhenolic constituentsand antioxidant activity of geopropolis from two species ofamazonian stingless beesrdquoQuimicaNova vol 36 no 5 pp 628ndash633 2013

[9] S Alves De Souza C A Camara E Monica Sarmento Da Silvaand T M S Silva ldquoComposition and antioxidant activity ofgeopropolis collected by Melipona subnitida (jandaıra) beesrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 801383 5 pages 2013

[10] J F Campos U P dos Santos L F B Macorini et alldquoAntimicrobial antioxidant and cytotoxic activities of propolisfrom Melipona orbignyi (Hymenoptera Apidae)rdquo Food andChemical Toxicology vol 65 no 1 pp 374ndash380 2014

[11] A A Righi G Negri and A Salatino ldquoComparative chemistryof propolis from eight brazilian localitiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID267878 14 pages 2013

[12] A Salatino C C Fernandes-Silva A A Righi and M LF Salatino ldquoPropolis research and the chemistry of plantproductsrdquo Natural Product Reports vol 28 no 5 pp 925ndash9362011

[13] V F de Castro Ishida G Negri A Salatino and M F C LBandeira ldquoA new type of Brazilian propolis prenylated benzo-phenones in propolis from Amazon and effects against cario-genic bacteriardquo Food Chemistry vol 125 no 3 pp 966ndash9722011

[14] C FMassaroM Katouli T Grkovic et al ldquoAnti-staphylococcalactivity of C-methyl flavanones from propolis of Australianstingless bees (Tetragonula carbonaria) and fruit resins ofCorymbia torelliana (Myrtaceae)rdquo Fitoterapia vol 95 no 1 pp247ndash257 2014

[15] M Boppre S M Colegate and J A Edgar ldquoPyrrolizidinealkaloids ofEchiumvulgarehoney found in pure pollenrdquo Journalof Agricultural and Food Chemistry vol 53 no 3 pp 594ndash6002005

[16] M Boppre S M Colegate J A Edgar and O W FischerldquoHepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollenrdquoJournal of Agricultural and Food Chemistry vol 56 no 14 pp5662ndash5672 2008

[17] M Boppre ldquoThe ecological context of pyrrolizidine alkaloidsin food feed and forage an overviewrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 28 no 3 pp 260ndash281 2011

[18] L Cramer H-M Schiebel L Ernst and T BeuerleldquoPyrrolizidine alkaloids in the food chain developmentvalidation and application of a new HPLC-ESI-MSMS sumparametermethodrdquo Journal of Agricultural and Food Chemistryvol 61 no 47 pp 11382ndash11391 2013

[19] A Azwa and S E Barton ldquoAspects of herpes simplex virusa clinical reviewrdquo Journal of Family Planning and ReproductiveHealth Care vol 35 no 4 pp 237ndash242 2009

[20] F Morfin and D Thouvenot ldquoHerpes simplex virus resistanceto antiviral drugsrdquo Journal of Clinical Virology vol 26 no 1 pp29ndash37 2003

[21] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[22] T K Nagasse-Sugahara J J Kisielius M Ueda-Ito et alldquoHuman vaccinia-like virus outbreaks in Sao Paulo and GoiasStates Brazil virus detection isolation and identificationrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 46no 6 pp 315ndash322 2004

[23] S J Read and J BKurtz ldquoLaboratory diagnosis of commonviralinfections of the central nervous system by using a single multi-plex PCR screening assayrdquo Journal of Clinical Microbiology vol37 no 5 pp 1352ndash1355 1999

[24] R E Kingsley and N L Cole ldquoPreparation of cultured mam-malian cells for transmission and scanning electronmicroscopyusing Aclar filmrdquo Journal of Electron Microscopy Technique vol10 no 1 pp 77ndash85 1988

[25] G Mariappan N Sundaraganesan and S Manoharan ldquoThespectroscopic properties of anticancer drug Apigenin inves-tigated by using DFT calculations FT-IR FT-Raman andNMR analysisrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 95 no 1 pp 86ndash99 2012

[26] J A Manthey ldquoFourier transform infrared spectroscopic anal-ysis of the polymethoxylated flavone content of orange oilresiduesrdquo Journal of Agricultural and Food Chemistry vol 54no 9 pp 3215ndash3218 2006

[27] B Abad-Garcıa L A Berrueta S Garmon-Lobato B Galloand F Vicente ldquoA general analytical strategy for the char-acterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detectioncoupled to electrospray ionization and triple quadrupole massspectrometryrdquo Journal of Chromatography A vol 1216 no 28pp 5398ndash5415 2009

[28] S C Gouveia and P C Castilho ldquoCharacterization of phenoliccompounds in Helichrysum melaleucum by high-performanceliquid chromatography with on-line ultraviolet and mass spec-trometry detectionrdquo Rapid Communications in Mass Spectrom-etry vol 24 no 13 pp 1851ndash1868 2010

[29] F Ferreres R F Goncalves A Gil-Izquierdo et al ldquoFurtherknowledge on the phenolic profile of Colocasia esculenta (L)Shottrdquo Journal of Agricultural and Food Chemistry vol 60 no28 pp 7005ndash7015 2012

[30] D Barreca C Bisignano G Ginestra et al ldquoPolymethoxylatedC- and O-glycosyl flavonoids in tangelo (Citrus reticulatatimes Citrus paradisi) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 141 no 2 pp 1481ndash1488 2013

[31] D Barreca E Bellocco U Leuzzi and G Gattuso ldquoFirstevidence of C- and O-glycosyl flavone in blood orange (Citrussinensis (L) Osbeck) juice and their influence on antioxidantpropertiesrdquo Food Chemistry vol 149 pp 244ndash252 2014

[32] G Negri R Mattei and F R Mendes ldquoAntinociceptive activityof the HPLC- and MS-standardized hydroethanolic extract ofPterodon emarginatus Vogel leavesrdquo Phytomedicine vol 21 no4 pp 1062ndash1069 2014

[33] U Gasic S Keckes D Dabic et al ldquoPhenolic profile and antiox-idant activity of Serbian polyfloral honeysrdquo Food Chemistry vol145 no 3 pp 599ndash607 2014

[34] Y Sapozhnikova ldquoDevelopment of liquid chromatography-tandemmass spectrometrymethod for analysis of polyphenolic

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

compounds in liquid samples of grape juice green tea andcoffeerdquo Food Chemistry vol 150 no 1 pp 87ndash93 2014

[35] K S Robbins Y Ma M L Wells P Greenspan and R BPegg ldquoSeparation and characterization of phenolic compoundsfrom US pecans by liquid chromatography-tandem massspectrometryrdquo Journal of Agricultural and Food Chemistry vol62 no 19 pp 4332ndash4341 2014

[36] A Delcambre and C Saucier ldquoIdentification of new flavan-3-ol monoglycosides by UHPLC-ESI-Q-TOF in grapes and winerdquoJournal of Mass Spectrometry vol 47 no 6 pp 727ndash736 2012

[37] MBUcarGUcarA Pizzi andOGonultas ldquoCharacterizationof Pinus brutia bark tannin byMALDI-TOFMS and 13CNMRrdquoIndustrial Crops and Products vol 49 pp 697ndash704 2013

[38] C T Griffin M Danaher C T Elliott D Glenn Kennedy andA Furey ldquoDetection of pyrrolizidine alkaloids in commercialhoney using liquid chromatography-ion trap mass spectrome-tryrdquo Food Chemistry vol 136 no 3-4 pp 1577ndash1583 2013

[39] F Xu J A Schillinger M R Sternberg et al ldquoSeroprevalenceand coinfection with herpes simplex virus type 1 and type 2 inthe United States 1988ndash1994rdquo Journal of Infectious Diseases vol185 no 8 pp 1019ndash1024 2002

[40] G Andrei and R Snoeck ldquoHerpes simplex virus drug-resistance new mutations and insightsrdquo Current Opinion inInfectious Diseases vol 26 no 6 pp 551ndash560 2013

[41] M T H Khan A Ather K D Thompson and R GambarildquoExtracts and molecules from medicinal plants against herpessimplex virusesrdquo Antiviral Research vol 67 no 2 pp 107ndash1192005

[42] C Koch J Reichling J Schneele and P Schnitzler ldquoInhibitoryeffect of essential oils against herpes simplex virus type 2rdquoPhytomedicine vol 15 no 1-2 pp 71ndash78 2008

[43] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[44] MHuleihel andV Isanu ldquoAnti-herpes simplex virus effect of anaqueous extract of propolisrdquo Israel Medical Association Journalvol 4 no 11 pp 923ndash927 2002

[45] J Melchjorsen S Matikainen and S R Paludan ldquoActivationand evasion of innate antiviral immunity by herpes simplexvirusrdquo Viruses vol 1 no 3 pp 737ndash759 2009

[46] T C IMettenleiter B G Klupp andH Granzow ldquoHerpesvirusassembly an updaterdquo Virus Research vol 143 no 2 pp 222ndash234 2009

[47] P Schnitzler A Neuner S Nolkemper et al ldquoAntiviral activityand mode of action of propolis extracts and selected com-poundsrdquoPhytotherapyResearch vol 24 no 1 pp S20ndashS28 2010

[48] I T Silva G M Costa P H Stoco E P Schenkel F HReginatto and C M O Simoes ldquoIn vitro antiherpes effectsof a C-glycosylflavonoid-enriched fraction of Cecropia glazioviiSnethrdquo Letters in Applied Microbiology vol 51 no 2 pp 143ndash148 2010

[49] D D Orhan B Ozcelik S Ozgen and F Ergun ldquoAntibacterialantifungal and antiviral activities of some flavonoidsrdquoMicrobi-ological Research vol 165 no 6 pp 496ndash504 2010

[50] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[51] P Ji C M Chen Y A Hu et al ldquoAntiviral activity of Paulowniatomentosa against enterovirus 71 of hand foot and mouthdiseaserdquo Biological amp Pharmaceutical Bulletin vol 38 no 1 pp1ndash6 2015

[52] X W Lv M Qiu D Y Chen N Zheng Y Jin and ZW Wu ldquoApigenin inhibits enterovirus 71 replication throughsuppressing viral IRES activity and modulating cellular JNKpathwayrdquo Antiviral Research vol 109 no 1 pp 30ndash41 2014

[53] L Yarmolinsky M Huleihel M Zaccai and S Ben-ShabatldquoPotent antiviral flavone glycosides from Ficus benjaminaleavesrdquo Fitoterapia vol 83 no 2 pp 362ndash367 2012

[54] S Nolkemper J Reichling K H Sensch and P SchnitzlerldquoMechanism of herpes simplex virus type 2 suppression bypropolis extractsrdquo Phytomedicine vol 17 no 2 pp 132ndash1382010

[55] P Schnitzler S Schneider F C Stintzing R Carle and JReichling ldquoEfficacy of an aqueous Pelargonium sidoides extractagainst herpesvirusrdquo Phytomedicine vol 15 no 12 pp 1108ndash1116 2008

[56] M Kurokawa T Shimizu Y Takeshita et al ldquoEfficacy of Brazil-ian propolis against herpes simplex virus type 1 infection inmice and theirmodes of antiherpetic efficaciesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID976196 9 pages 2011

[57] F Jiang W Chen K Yi et al ldquoThe evaluation of catechins thatcontain a galloylmoiety as potential HIV-1 integrase inhibitorsrdquoClinical Immunology vol 137 no 3 pp 347ndash356 2010

[58] H-Y HoM-L Cheng S-FWeng Y-L Leu andD T-Y ChiuldquoAntiviral effect of epigallocatechin gallate on enterovirus 71rdquoJournal of Agricultural and Food Chemistry vol 57 no 14 pp6140ndash6147 2009

[59] K Kuwata T Takemura T Urushisaki et al ldquo34-dicaffe-oylquinic acid a major constituent of Brazilian propolisincreases TRAIL expression and extends the lifetimes of miceinfected with the influenza a virusrdquo Evidence-based Comple-mentary and Alternative Medicine vol 2012 Article ID 9468677 pages 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom