research article composition and antioxidant activity of … · 2019. 7. 31. · sipes , melipona...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 801383 5 pageshttpdxdoiorg1011552013801383

Research ArticleComposition and Antioxidant Activity of Geopropolis Collectedby Melipona subnitida (Jandaiacutera) Bees

Silvana Alves de Souza1 Celso Amorim Camara1

Eva Monica Sarmento da Silva2 and Tania Maria Sarmento Silva1

1 Laboratorio de Bioprospeccao Fitoquımica Departamento de Ciencias Moleculares Universidade Federal Rural de Pernambuco52171-900 Recife PE Brazil

2 Colegiado de Zootecnia Universidade Federal do Vale de Sao Francisco 56300-990 Petrolina PE Brazil

Correspondence should be addressed to Tania Maria Sarmento Silva sarmentosilvagmailcom

Received 17 April 2013 Accepted 18 June 2013

Academic Editor Wagner Vilegas

Copyright copy 2013 Silvana Alves de Souza et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

An investigation of the geopropolis collected byMelipona subnitida (jandaıra) stingless bee led to the isolation and characterizationof two phenylpropanoids 6-O-p-coumaroyl-D-galactopyranose (1) and 6-O-cinnamoyl-1-O-p-coumaroyl-120573-D-glucopyranose (2)and seven flavonoids 7-O-methyl-naringenin (3) 7-O-methyl aromadendrin (4) 741015840-di-O-methyl aromadendrin (5) 41015840-O-methylkaempferol (6) 3-O-methyl quercetin (7) 5-O-methyl aromadendrin (8) and 5-O-methyl kaempferol (9)The structure of the newphenylpropanoid (1) was established from IR LC-ESI-MS and NMR spectral data including 2D NMR experiments The extractand fractions demonstrated significant antioxidant activity in DPPH ABTS and 120573-carotenelinoleic acid tests

1 Introduction

Geopropolis is a special type of propolis or bee glue preparedby stingless bees (Meliponinae) As presented [1] geopropolisis a mixture of plant resins and waxes and earth Honeybees(Apis) do not use soil material when preparing traditionalpropolis [2] Previous investigations on tropical propolis haveconcentrated almost exclusively on Apis mellifera bee glueIn tropical South America there are indigenous stinglessbee species (Meliponinae) that collect geopropolis suchas the Melipona subnitida Ducke ldquojandaırardquo bees native tonortheastern Brazil The primary importance of this speciesis associated with environmental conservation and fruitproduction as they pollinate wild plants and cultivated cropsin the semiarid Caatinga (shrub vegetation) and humid pre-Amazonian forest regions [3]

The chemical composition of geopropolis or propolisdepends on the specificity of the local flora at the collectionsite Many constituents have been identified principally frompropolis and phenolic compounds such as flavonoids phe-nolic acids and phenolic acid esters have been reported asmajor constituents of propolis from tropical zones [4]

An investigation of the phenolic constituents of propolisand geopropolis from Venezuela reported the presence ofprenylated benzophenones in all the samples [5] in 1993The chemical compositions of the propolis of five indigenousbee species along with the honeybee did not differ Bankovaet al [6] identified over 50 substances mainly phenoliccompounds in Brazilian geopropolis fromMelipona compres-sipes Melipona quadrifasciata anthidioides and Tetragonaclavipes Twenty-one samples of Brazilian geopropolis from12 different species of stingless bees were analyzed and thepresence of such compounds as di- and triterpenes and gallicacid was detected The same samples showed activity againstStaphylococcus aureus and cytotoxic activity [7] Investi-gations of geopropolis from Melipona fasciculata showedantimicrobial activity against Smutans andC albicans aswellas an immunomodulatory action due to the increase in anti-inflammatory cytokines [8]

Propolis is a powerful antioxidant An antioxidant is amolecule capable of slowing or preventing the oxidation ofother molecules The radical theory in human physiologyclaims that active free radicals are involved in almost all cel-lular degradation processes and lead to cell death Oxidative

2 Evidence-Based Complementary and Alternative Medicine

stress is thought to contribute to the development of chronicand degenerative diseases such as cancer autoimmune disor-ders aging cataract rheumatoid arthritis and cardiovascularand neurodegenerative diseases [9]The antioxidant propertyof propolis is due to its high concentration of phenolics andother antioxidant compounds [10] Propolis or geopropolis isa potential supplement for preventing chronic degenerationdiseases

As a part of our continuing study of the chemical andantioxidant activity of Apis andMelipona products [3 11ndash13]this study undertook the isolation of a new phenylpropanoidgalactose ester from the geopropolis of jandaıra (Meliponasubnitida) in addition to eight phenols This paper describesthe isolation and characterization of the new compound andthe antioxidant activity of this geopropolis

2 Experimental

21 General Melting points were determined using a Koflerhot stage and are uncorrected The infrared absorptionspectra were recorded in KBr pellets using a Varian 640 FT-IR spectrophotometer with a PIKE ATR accessory operatingin the 4000ndash400 cmminus1 range The LC-ESI-MS was obtainedin positive electrospray mode using an Esquire 3000 Plusinstrument (Bruker) TLC plates were run using 60 F

254silica

gel (Merck) Sephadex LH-20 (Sigma) was employed for gelpermeation chromatography 1H and 13C NMR spectra wereobtained using a Bruker DRX 500 (500MHz for 1H 125MHzfor 13C) and Bruker DPX300 (300MHz for 1H 75MHz for13C) in DMSO-119889

6The optical rotation was determined using

a KRUESS Optronic spectrometer All solvents used were ofcommercial HPLC grade

22 Geopropolis Sample The M subnitida ldquojandaırardquo geo-propolis sample was collected in January 2010 at Sıtio Riachowhich is in the municipality of Vieiropolis a semiarid regionin the state of Paraiba Brazil

23 Preparation of Extract and Fractions Geopropolis(550 g)was extractedwith ethanol in an ultrasonicwater bathThe combined ethanolic extract was completely evaporatedunder reduced pressure to afford a brown residue (258 g)A portion of the ethanolic extract (157 g) was suspended inMeOHH

2O and partitioned with hexane and ethyl acetate

to yield the corresponding soluble fractions yielding hexane(48 g) ethyl acetate (45 g) andMeOHH

2O (07 g) fractions

The ethyl acetate fraction that showed the most significantantioxidant activitywas used for the further fractionation andisolation of individual compounds

24 Isolation and Identification of Compounds A portion ofthe EtOAc fraction (45 g) was subjected to chromatographyon a Sephadex LH-20 column with methanol as the mobilephase Compounds 1 (185mg) 2 (85mg) 3 (25mg) 4(70mg) 5 (55mg) 6 (28mg) and 7 (93mg) and themixture of 8 and 9 (45mg) were purified by semipreparativeHPLC on a Luna Phenomenex RP-18 column (21mm times250mm times 5 120583m) and detected at 320 nm at a flow rate of

Table 1 1H and 13C NMR (30075MHz DMSO-1198896) of compound

(1)

Position 120575119862

120575119867

HMBC correlations9 16668 mdash H-8 H-74 15989 mdash H-35 H-267 14495 mdash H-267 14482 77 (119889 119869 = 153) mdash26 13040 75 (119889 119869 = 87) H-71 12505 mdash H-35 H-835 11578 67 (119889 119869 = 87) mdash8 11397 64 (119889 119869 = 159) mdash11015840120572 9232 491 (119889 119869 = 33) 2120572 512057211015840120573 9693 432 (119889 119869 = 78) 212057351015840120573 7644 32 (119898) 4120573 H-312057331015840120573 7472 29 (119905 119869 = 84) 4120573 1120573 512057321015840120573 7360 34 (m) mdash51015840120572 7291 36 (m) H-1101584012057241015840120573 7219 31 (m) mdash41015840120572 7066 32 (m) mdash3120572 7024 31 (119905 119869 = 84) mdash21015840120572 6928 38 (m) mdash61015840a 6397 44 (119889119889 120 15) mdash61015840b 6397 41 (119889 119869 = 111 66) 4120573

15mLmin using a mobile phase of H2O (A) and methanol

(B) as follows 0ndash5min 70ndash80 B 5ndash10min 80ndash90 B 10ndash15min 90ndash100 B and 15-16min 100 B The purity ofthe compounds was assessed via analytical HPLC with diodearray detection The structures of all the isolated compoundswere elucidated based on 1H-NMR 13C-NMR MS IR andUV data

6-O-p-Coumaroyl-D-galactopyranose (1) Amorphouswhite powder MP 165ndash167∘C [120572]

119863= +267 (MeOH c 035

25∘C) LC-ESI-MS (positive mode) mz 327 [M + H]+mz 349 [M + Na]+ mz 309 [M + HminusH

2O]+ mz 165

[Mminusgalactose]+ UV 120582max = 310 nm IR (KBr) ]max 34001690 (C=O) 1607 1513 (C=C from aromatic rings) 1H NMR(DMSO-119889

6 300MHz see Table 1) 13C NMR (DMSO-119889

6

125MHz see Table 1)

6-O-Cinnamoyl-1-O-p-coumaroyl-120573-D-glucopyranose(2) LC-ESI-MS mz 455 [MminusH]+ UV120582max = 310 nm7-O-Methyl-naringenin (3) LC-ESI-MS mz 287[M+ H]+ UV 120582max = 2867-O-Methyl aromadendrin (4) LC-ESI-MS mz 303[M + H]+ UV 120582max = 290741015840-Di-O-methyl aromadendrin (5) LC-ESI-MSmz317 [M + H]+ UV 120582max = 28841015840-O-Methyl kaempferol (6) LC-ESI-MS mz 301[M+ H]+ UV 120582max = 2923-O-Methyl quercetin (7) LC-ESI-MS mz 317 [M +H]+ UV 120582max = 255 367

Evidence-Based Complementary and Alternative Medicine 3

5-O-Methyl aromadendrin (8) LC-ESI-MS mz 303[M+H]+ UV 120582max = 2905-O-Methyl kaempferol (9) LC-ESI-MS mz 301[M+H]+ UV 120582max = 269 365

25 Acid Hydrolysis of 1 Acid hydrolysis was performedusing 1 HCl in MeOH at 100∘C for 2 h from 93mg of 1For aglycone detection the final aqueous acidic mixture wasextracted with EtOAc then the aqueous layer was neutral-ized for determination of the released sugar moieties usingsilica gel plates with CHCl

3MeOH (6 4) The anisaldehyde

reagent was employed as spray for detection of the galactosefromhydrolised samplewhich showed the same greenish graypattern used galactose

26 Determination of Total Phenolic Content The total solu-ble phenolic content of the EtOH extract hexane EtOAc andMeOHH

2O fractions (1mgmL) was determined with the

Folin-Ciocalteu reagent according to the method of Slinkardand Singleton [14] with modification using gallic acid as astandard phenolic compound

27 DPPH∙ Radical Scavenging Assay ABTS+∙ Radical CationDecolorization Assay and Antioxidant Activity in LinoleicAcid Oxidation The free radical scavenger activity DPPH[3] the radical cation decolorization assay ABTS [15] andthe antioxidant activity in linoleic acid oxidation [16] ofEtOH hexane AcOEt MeOHH

2O extract and fractions

were determined

28 Statistical Analysis All samples were analyzed in tripli-cate unless stated otherwise and the results were expressedas the mean plusmn standard deviation All statistical analyseswere carried out using the Microsoft Excel software package(Microsoft Corp Redmond WA USA)

3 Results and Discussion

The ethyl acetate fraction (EtOAc) from jandaıra geopropoliswas chromatographed over Sephadex LH-20 and reverse-phase HPLC yielded compounds 1ndash9 The structures of 1ndash8were established by analysis of the spectral data including 2DNMR and LC-ESI-MS The APT NMR spectra of compound(1) (C

15H18O8 LC-ESI-MS mz 326 [M + H]+) showed

duplicated sugar carbon signal patterns The dual peaks inthe 1HNMR spectra of compound (1) indicated the presenceof both 120572- and 120573-anomers along with two doublets for thearomatic hydrogen system AA1015840BB1015840 at 120575 75 (H-2 6 119889 119869 =87Hz) and120575 67 ppm (H-3 5119889 119869 = 87Hz) and twodoubletsshowing the existence of trans-olefin systems at 120575 77 (H-7 119869 =159Hz) and 120575 64 ppm (H-8 119869 = 159Hz) thus indicatingthe presence of the (E)-p-coumaroyl portion Two anomericprotons were found in a 2 1 ratio at 120575 49 (H-1120572 119889 119869 = 33)and 120575 43 ppm (H-1120573 119889 119869 = 78) with the signals relatedto sugar protons between 120575 44 and 120575 29 ppm The APTspectrum showed signs of a 14-disubstituted benzene patternwith the peaks from methine carbons at 120575 1304 (C-26) and

120575 1158 ppm (C-35) an oxygenated carbon at 120575 1599 (C-4)another quaternary carbon at 120575 1251 ppm (C-1) a carbonester at 120575 1687 (C-9) and two olefinic carbons at 120575 1449 (C-7)and 120575 1137 ppm (C-8) relative to the trans coumaroyl portionIn addition to these signals the APT spectrum also showedthe presence of 11 signals to a hexose in different proportionsThe values for the pyranose agree with galactoseThe locationof the phenylpropanoid group was concluded to be the C-6 position of the galactose moiety based on the evidence ofthe downfield shift of the C-6 methylene signals in the 1HNMR and APT NMR spectra The HMBC spectrum showeda correlation of the methylene hydrogens of galactose to 120575 44(H-6120572) and 120575 41 ppm (H-6120573) with the carbonyl group in 1205751669 showing the ester linkage in the 61015840 position of the sugarThe complete structure assignment of 6-O-p-coumaroyl-D-galactopyranose was performed via a detailed analysis of theCOSY HSQC and HMBC spectra (Table 1) The LC-ESI-MSspectrum revealed the peak atmz 3269 [M +H]+ due to themolecular ion atmz 309 due to the loss of water and atmz165 due to the loss of galactose The peak at mz 147 refers tothe loss of two molecules of water and galactose

The acid hydrolysis of (1) yielded coumaric acid and D-galactose The phenylpropanoid group was concluded to bethe C-6 position of the galactose moiety from the evidenceof the downfield shift of the C-6 methylene signals in the 1Hand 13CNMR spectraTherefore we assigned the structure ofcompound (1) as 6-O-p-coumaroyl-D-galactopyranose Thisidentification of 6-O-p-coumaroyl-D-galactopyranose (1) isfirst reported in the literature The related derivative 6-O-p-coumaroyl-D-glucopyranose has been isolated from severalplant species such as Prunus buergeriana [17] Flacourtiaindica [18] and Petrorhagia velutina [19] The known com-pounds were identified as 6-O-cinnamoyl-1-O-p-coumaroyl-120573-D-glucopyranose (2) [20] 7-O-methyl naringenin (3) 7-O-methyl aromadendrin (4) [21] 741015840-di-O-methyl aromaden-drin (5) 41015840-O-methyl kaempferol (6) 3-O-methyl quercetin(7) [22] 5-O-methyl aromadendrin (8) [23] and 5-O-methylkaempferol (9) [22] as confirmed by comparison of thespectroscopic data (UV IR MS and NMR) with the cor-responding data for the standards or literature values Thechemical structures of the isolated compounds are shown inFigure 1 This is the first report of compounds 1ndash9 from thegeopropolis ofMelipona subnitida (jandaıra) stingless bees

The amount of total phenolics was estimated using theFolin-Ciocalteu reagent ranging from 639 plusmn 86 mgGAEg(gallic acid equivalent per gram of extract) in the EtOHextract and 400plusmn78 256plusmn05 and 1158plusmn08mgGAEg inthe MeOHH

2O hexane and EtOAc fractions respectively

The highest total phenolic level was detected in the EtOAcfraction and the lowest in the hexane fractionThree differentmethods were used to determine the antioxidant propertiesof the geopropolis which allowed us to obtain informationabout the activity of these extracts during the different stagesof the oxidation reaction [24]Themethods used included theinhibition of 120573-carotene cooxidation in a linoleic acid modelsystem and DPPH and ABTS scavenging The extracts andfractions assayed prevented the bleaching of120573-carotene in thecarotenelinoleic acid mixtures (Table 2) However different


Table 2 Total phenolics and antioxidant activity of samples

Extract or fraction Total phenolic content (mgGAEg)a DPPH (EC50)a ABTS (EC50)

a 120573-Carotene bleaching ( OI)b

EtOH 639 plusmn 86 270 plusmn 04 122 plusmn 01 357 plusmn 40

Hexane 256 plusmn 05 2393 plusmn 11 323 plusmn 03 263 plusmn 35

EtOAc 1158 plusmn 08 101 plusmn 00 43 plusmn 00 551 plusmn 19

MeOH H2O 400 plusmn 78 385 plusmn 05 262 plusmn 03 207 plusmn 16

Ascorbic acid 21 plusmn 00 mdashTrolox mdash 30 plusmn 01 813 plusmn 02

Mean value plusmn standard deviation 119899 = 3aAntioxidant concentration required to reduce the original radical population by 50bOxidation inhibition

O

OO HO

OHOH

OH

O

O

OO

OHOH

O

O

O

O

O

1

23

4

56 7

89

123

45

6

3 R1 = R2 = R4 = H R3 = CH3

4 R1 = OH R2 = R4 = H R3 = CH3

5 R1 = OH R2 = H R3 = R4 = CH3

8 R1 = OH R2 = CH3 R3 = R4 = H

6 R1 = R2 = R3 = R4 = H R5 = CH3

7 R2 = R3 = R4 = R5 = H R1 = CH3

9 R1 = R3 = R4 = R5 = H R2 = CH3

HO

HO HO

1

1998400

2998400

3998400

4998400

6998400

2

1998400

2998400 3

998400

4998400

5998400

7998400

8998400

9998400

1998400998400

2998400998400

3998400998400

5998400998400

6998400998400

6998400

7998400998400

8998400998400

9998400998400

5998400

4998400998400

R3O R3O

OR4

OR4

OR2 OR2

R1 OR1

OR5

Figure 1 Structures of phenolic acids and flavonoids found in geopropolis

fractions exhibited varying degrees of antioxidant capacityThe antioxidant activity was determined based on the inhibi-tion of the coupled oxidation of 120573-carotene and linoleic acidat 119905 = 60min (Table 2)The antioxidant activity was 357plusmn40551 plusmn 19 263 plusmn 35 and 207 plusmn 16 for the EtOH EtOAchexane extracts and the MeOHH

2O fraction respectively

In conclusion phenylpropanoids and flavonoids wereisolated from the most active EtOAc fraction (Figure 1) thathad not been tested because they had only been isolated insmall amounts The literature includes a report that showsthat the flavonoid 7 (3-O-methyl quercetin) is active inDPPHradical scavenging and 120573-carotene-linoleic acid bleachingassays [25] The antioxidant activity of the EtOAc fraction

studied in this workmay be related to the antioxidant abilitiesof the flavonoids and phenylpropanoids that were isolated

Conflict of Interests

Theauthors do not have any conflict of interests regarding thecontent of this paper

Acknowledgment

This work was financially supported by grants from FACEPE(Grant no PRONEM APQ-123210610)


References

[1] O M Barth and C Fernandes Pinto da Luz ldquoPalynologicalanalysis of Brazilian geopropolis sedimentsrdquo Grana vol 42 no2 pp 121ndash127 2003

[2] W E Kerr ldquoAbelhas indıgenas brasileiras (Meliponineos) napolinizacao e na producao de mel polen geopropolis e cerardquoInforme Agropecuario vol 13 pp 15ndash27 1987

[3] T M S Silva C A Camara A C da Silva Lins et al ldquoChemicalcomposition and free radical scavenging activity of pollen loadsfrom stingless bee Melipona subnitida Duckerdquo Journal of FoodComposition and Analysis vol 19 no 6-7 pp 507ndash511 2006

[4] V Bankova ldquoChemical diversity of propolis makes it a valu-able source of new biologically active compoundsrdquo Journal ofApiProduct and ApiMedical Science vol 1 pp 23ndash28 2009

[5] F A Tomas-Barberan C Garcıa-Viguera P Vit-Olivier FFerreres and F Tomas-Lorente ldquoPhytochemical evidence forthe botanical origin of tropical propolis from VenezuelardquoPhytochemistry vol 34 no 1 pp 191ndash196 1993

[6] 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

[7] M Velikova V Bankova M C Marcucci I Tsvetkova and AZ Kujumgiev ldquoChemical composition and biological activityof propolis from Brazilian Meliponinaerdquo Zeitschrift fur Natur-forschung C vol 55 no 9-10 pp 785ndash789 2000

[8] S A Liberio A L A Pereira R P Dutra et al ldquoAntimicro-bial activity against oral pathogens and immunomodulatoryeffects and toxicity of geopropolis produced by the stinglessbee Melipona fasciculata Smithrdquo BMC Complementary andAlternative Medicine vol 11 article 108 2011

[9] S Bogdanov ldquoPropolis Composition Health Medicine AReviewrdquo 2012 httpwwwbee-hexagonnetfilesfilefileEHealthPropolisBookReviewpdf

[10] S Bogdanov ldquoPropolis Composition Health MedicineA Reviewrdquo Bee Product Science 2011 httpwwwbee-hexagonnet

[11] T M S Silva C A Camara A C S Lins et al ldquoChemicalcomposition botanical evaluation and screening of radicalscavenging activity of collected pollen by the stingless beesMelipona rufiventris (Urucu-amarela)rdquo Anais da AcademiaBrasileira de Ciencias vol 81 no 2 pp 173ndash178 2009

[12] T M S Silva F P Santos A E Rodrigues et al ldquoPhenolic com-pounds melissopalynological physicochemical analysis andantioxidant activity of jandaira (Melipona subnitida) honeyrdquoJournal of Food Composition and Analysis vol 29 pp 10ndash182013

[13] K R L Freire A C S Lins M C Dorea F A R Santos C ACamara and T M S Silva ldquoPalynological origin phenolic con-tent and antioxidant properties of honeybee-collected pollenfromBahia BrazilrdquoMolecules vol 17 no 2 pp 1652ndash1664 2012

[14] K Slinkard andV L Singleton ldquoTotal phenol analyses automa-tion and comparison with manual methodsrdquo American Journalof Enology Viticulture vol 28 pp 49ndash55 1977

[15] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[16] C L Emmons D M Peterson and G L Paul ldquoAntioxidantcapacity of oat (Avena sativa L) extracts 2 In vitro antioxidantactivity and contents of phenolic and tocol antioxidantsrdquo

Journal of Agricultural and Food Chemistry vol 47 no 12 pp4894ndash4898 1999

[17] H Shimomura Y Sashida and T Adachi ldquoPhenylpropanoidglucose esters from Prunus buergerianardquo Phytochemistry vol27 no 2 pp 641ndash644 1988

[18] N R Amarasinghe L Jayasinghe N Hara and Y FujimotoldquoFlacourside a new 4-oxo-2-cyclopentenylmethyl glucosidefrom the fruit juice of Flacourtia indicardquo Food Chemistry vol102 no 1 pp 95ndash97 2007

[19] B DrsquoAbrosca A Fiorentino A Ricci et al ldquoStructural char-acterization and radical scavenging activity of monomeric anddimeric cinnamoyl glucose esters from Petrorhagia velutinaleavesrdquo Phytochemistry Letters vol 3 no 1 pp 38ndash44 2010

[20] O A Rashwan ldquoNew phenylpropanoid glucosides from Euca-lyptus maculatardquoMolecules vol 7 no 1 pp 75ndash80 2002

[21] E Abdel-Sattar M A Kohiel I A Shihata and H El-AskaryldquoPhenolic compounds from Eucalyptus maculatardquo Pharmazievol 55 no 8 pp 623ndash624 2000

[22] P K Agrawal R S Thakur and M C Bansal Carbon-13 NMRof Flavonoids Elsevier Amsterdam The Netherlands 1989

[23] A R Bilia S Catalano L Pistelli and I Morelli ldquoFlavonoidsfrom Pyracantha coccinea rootsrdquo Phytochemistry vol 33 no 6pp 1449ndash1452 1993

[24] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[25] H Yoo H K Seung J Lee et al ldquoSynthesis and antioxidantactivity of 3-methoxyflavonesrdquo Bulletin of the Korean ChemicalSociety vol 26 no 12 pp 2057ndash2060 2005

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


stress is thought to contribute to the development of chronicand degenerative diseases such as cancer autoimmune disor-ders aging cataract rheumatoid arthritis and cardiovascularand neurodegenerative diseases [9]The antioxidant propertyof propolis is due to its high concentration of phenolics andother antioxidant compounds [10] Propolis or geopropolis isa potential supplement for preventing chronic degenerationdiseases

As a part of our continuing study of the chemical andantioxidant activity of Apis andMelipona products [3 11ndash13]this study undertook the isolation of a new phenylpropanoidgalactose ester from the geopropolis of jandaıra (Meliponasubnitida) in addition to eight phenols This paper describesthe isolation and characterization of the new compound andthe antioxidant activity of this geopropolis

2 Experimental

21 General Melting points were determined using a Koflerhot stage and are uncorrected The infrared absorptionspectra were recorded in KBr pellets using a Varian 640 FT-IR spectrophotometer with a PIKE ATR accessory operatingin the 4000ndash400 cmminus1 range The LC-ESI-MS was obtainedin positive electrospray mode using an Esquire 3000 Plusinstrument (Bruker) TLC plates were run using 60 F

254silica

gel (Merck) Sephadex LH-20 (Sigma) was employed for gelpermeation chromatography 1H and 13C NMR spectra wereobtained using a Bruker DRX 500 (500MHz for 1H 125MHzfor 13C) and Bruker DPX300 (300MHz for 1H 75MHz for13C) in DMSO-119889

6The optical rotation was determined using

a KRUESS Optronic spectrometer All solvents used were ofcommercial HPLC grade

22 Geopropolis Sample The M subnitida ldquojandaırardquo geo-propolis sample was collected in January 2010 at Sıtio Riachowhich is in the municipality of Vieiropolis a semiarid regionin the state of Paraiba Brazil

23 Preparation of Extract and Fractions Geopropolis(550 g)was extractedwith ethanol in an ultrasonicwater bathThe combined ethanolic extract was completely evaporatedunder reduced pressure to afford a brown residue (258 g)A portion of the ethanolic extract (157 g) was suspended inMeOHH

2O and partitioned with hexane and ethyl acetate

to yield the corresponding soluble fractions yielding hexane(48 g) ethyl acetate (45 g) andMeOHH

2O (07 g) fractions

The ethyl acetate fraction that showed the most significantantioxidant activitywas used for the further fractionation andisolation of individual compounds

24 Isolation and Identification of Compounds A portion ofthe EtOAc fraction (45 g) was subjected to chromatographyon a Sephadex LH-20 column with methanol as the mobilephase Compounds 1 (185mg) 2 (85mg) 3 (25mg) 4(70mg) 5 (55mg) 6 (28mg) and 7 (93mg) and themixture of 8 and 9 (45mg) were purified by semipreparativeHPLC on a Luna Phenomenex RP-18 column (21mm times250mm times 5 120583m) and detected at 320 nm at a flow rate of

Table 1 1H and 13C NMR (30075MHz DMSO-1198896) of compound

(1)

Position 120575119862

120575119867

HMBC correlations9 16668 mdash H-8 H-74 15989 mdash H-35 H-267 14495 mdash H-267 14482 77 (119889 119869 = 153) mdash26 13040 75 (119889 119869 = 87) H-71 12505 mdash H-35 H-835 11578 67 (119889 119869 = 87) mdash8 11397 64 (119889 119869 = 159) mdash11015840120572 9232 491 (119889 119869 = 33) 2120572 512057211015840120573 9693 432 (119889 119869 = 78) 212057351015840120573 7644 32 (119898) 4120573 H-312057331015840120573 7472 29 (119905 119869 = 84) 4120573 1120573 512057321015840120573 7360 34 (m) mdash51015840120572 7291 36 (m) H-1101584012057241015840120573 7219 31 (m) mdash41015840120572 7066 32 (m) mdash3120572 7024 31 (119905 119869 = 84) mdash21015840120572 6928 38 (m) mdash61015840a 6397 44 (119889119889 120 15) mdash61015840b 6397 41 (119889 119869 = 111 66) 4120573

15mLmin using a mobile phase of H2O (A) and methanol

(B) as follows 0ndash5min 70ndash80 B 5ndash10min 80ndash90 B 10ndash15min 90ndash100 B and 15-16min 100 B The purity ofthe compounds was assessed via analytical HPLC with diodearray detection The structures of all the isolated compoundswere elucidated based on 1H-NMR 13C-NMR MS IR andUV data

6-O-p-Coumaroyl-D-galactopyranose (1) Amorphouswhite powder MP 165ndash167∘C [120572]

119863= +267 (MeOH c 035

25∘C) LC-ESI-MS (positive mode) mz 327 [M + H]+mz 349 [M + Na]+ mz 309 [M + HminusH

2O]+ mz 165

[Mminusgalactose]+ UV 120582max = 310 nm IR (KBr) ]max 34001690 (C=O) 1607 1513 (C=C from aromatic rings) 1H NMR(DMSO-119889

6 300MHz see Table 1) 13C NMR (DMSO-119889

6

125MHz see Table 1)

6-O-Cinnamoyl-1-O-p-coumaroyl-120573-D-glucopyranose(2) LC-ESI-MS mz 455 [MminusH]+ UV120582max = 310 nm7-O-Methyl-naringenin (3) LC-ESI-MS mz 287[M+ H]+ UV 120582max = 2867-O-Methyl aromadendrin (4) LC-ESI-MS mz 303[M + H]+ UV 120582max = 290741015840-Di-O-methyl aromadendrin (5) LC-ESI-MSmz317 [M + H]+ UV 120582max = 28841015840-O-Methyl kaempferol (6) LC-ESI-MS mz 301[M+ H]+ UV 120582max = 2923-O-Methyl quercetin (7) LC-ESI-MS mz 317 [M +H]+ UV 120582max = 255 367











were determined





















O

OO HO

OHOH

OH

O

O

OO

OHOH

O

O

O

O

O

1

23

4

56 7

89

123

45

6

3 R1 = R2 = R4 = H R3 = CH3

4 R1 = OH R2 = R4 = H R3 = CH3

5 R1 = OH R2 = H R3 = R4 = CH3

8 R1 = OH R2 = CH3 R3 = R4 = H

6 R1 = R2 = R3 = R4 = H R5 = CH3

7 R2 = R3 = R4 = R5 = H R1 = CH3

9 R1 = R3 = R4 = R5 = H R2 = CH3

HO

HO HO

1

1998400

2998400

3998400

4998400

6998400

2

1998400

2998400 3

998400

4998400

5998400

7998400

8998400

9998400

1998400998400

2998400998400

3998400998400

5998400998400

6998400998400

6998400

7998400998400

8998400998400

9998400998400

5998400

4998400998400

R3O R3O

OR4

OR4

OR2 OR2

R1 OR1

OR5








Acknowledgment



References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























were determined





















O

OO HO

OHOH

OH

O

O

OO

OHOH

O

O

O

O

O

1

23

4

56 7

89

123

45

6

3 R1 = R2 = R4 = H R3 = CH3

4 R1 = OH R2 = R4 = H R3 = CH3

5 R1 = OH R2 = H R3 = R4 = CH3

8 R1 = OH R2 = CH3 R3 = R4 = H

6 R1 = R2 = R3 = R4 = H R5 = CH3

7 R2 = R3 = R4 = R5 = H R1 = CH3

9 R1 = R3 = R4 = R5 = H R2 = CH3

HO

HO HO

1

1998400

2998400

3998400

4998400

6998400

2

1998400

2998400 3

998400

4998400

5998400

7998400

8998400

9998400

1998400998400

2998400998400

3998400998400

5998400998400

6998400998400

6998400

7998400998400

8998400998400

9998400998400

5998400

4998400998400

R3O R3O

OR4

OR4

OR2 OR2

R1 OR1

OR5








Acknowledgment



References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























O

OO HO

OHOH

OH

O

O

OO

OHOH

O

O

O

O

O

1

23

4

56 7

89

123

45

6

3 R1 = R2 = R4 = H R3 = CH3

4 R1 = OH R2 = R4 = H R3 = CH3

5 R1 = OH R2 = H R3 = R4 = CH3

8 R1 = OH R2 = CH3 R3 = R4 = H

6 R1 = R2 = R3 = R4 = H R5 = CH3

7 R2 = R3 = R4 = R5 = H R1 = CH3

9 R1 = R3 = R4 = R5 = H R2 = CH3

HO

HO HO

1

1998400

2998400

3998400

4998400

6998400

2

1998400

2998400 3

998400

4998400

5998400

7998400

8998400

9998400

1998400998400

2998400998400

3998400998400

5998400998400

6998400998400

6998400

7998400998400

8998400998400

9998400998400

5998400

4998400998400

R3O R3O

OR4

OR4

OR2 OR2

R1 OR1

OR5








Acknowledgment



References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article composition and antioxidant activity of … · 2019. 7. 31. · sipes , melipona...

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