Research ArticleReceived: 11 December 2014 Revised: 4 May 2015 Accepted article published: 3 July 2015 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7330

Chemical profiling and antioxidant activityof Bolivian propolisNélida Nina,a,b Cristina Quispe,a,c Felipe Jiménez-Aspee,a CristinaTheoduloz,d Alberto Giméneze and Guillermo Schmeda-Hirschmanna*

Abstract

BACKGROUND: Propolis is a relevant research subject worldwide. However, there is no information so far on Bolivian propolis.Ten propolis samples were collected from regions with high biodiversity in the main honey production places in Bolivia andwere analyzed for their total phenolics (TP), flavonoids (TF) and antioxidant activity. The chemical profiles of the samples wereassessed by TLC, HPLC-DAD, HPLC-DAD-MS/MSn and NMR analysis.

RESULTS: TP, TF, TLC and NMR analysis showed significant chemical differences between the samples. Isolation of the mainconstituents by chromatography and identification by HPLC-DAD-MS/MSn achieved more than 35 constituents. According totheir profiles, the Bolivian propolis can be classified into phenolic-rich and triterpene-rich samples. Propolis from the valleys(Cochabamba, Chuquisaca and Tarija) contained mainly prenylated phenylpropanoids, while samples from La Paz and SantaCruz contained cycloartane and pentacyclic triterpenes. Phenolic-rich samples presented moderate to strong antioxidantactivity while the triterpene-rich propolis were weakly active.

CONCLUSION: High chemical diversity and differential antioxidant effects were found in Bolivian propolis. Our results provideadditional evidence on the chemical composition and bioactivity of South American propolis.© 2015 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: propolis; Bolivia; triterpenes; phenylpropanoids; artepillin C; antioxidant

INTRODUCTIONGathering of honey and bee products was a relevant activityof hunther–gatherers and societies of agricultural Amerindianssince pre-hispanic times. Before the European conquest, honey,wax and other products were obtained from native stinglessbees (Hymenoptera: Meliponini). In the Chaco region and easternBolivia, indigenous groups practiced meliponiculture in historicaltimes. This traditional practice was lost over time with the intro-duction of the high honey-producing European bee Apis melliferaL. Over 30 species of native bees can be found in Bolivia. The mostprized bees for its honey among native groups from the AmboróNational Park are: Melipona brachychaeta, M. grandis, Scaptotrigonadepilis, S. polysticta, S. aff. xanthotricha and Tetragonisca fiebrigi.1

In the north of La Paz, Tacana indigenous communities movedwild colonies of the native bees Trigona (Tetragosnisca) angus-tula and Melipona rufiventris to beehives to raise native bees.2 Atpresent, most of the honey as well as other bee products are fromA. mellifera.

Bolivia is a megadiverse country located in the center of SouthAmerica (latitude 9∘ 32′ and 22∘ 53′ S and longitude 69∘ 35′

and 57∘ 27′ W; Fig. 1). The country comprises a transition zonebetween humid tropical and dry subtropical climates, includinglowlands from less than 300 m.a.s.l. as well as high mountainsof almost 7000 m.a.s.l. This geographic diversity is responsiblefor the great biodiversity found in Bolivia.3 Bolivia comprises 12eco-regions, namely: Chiquitano dry forest, Tucumano–Bolivian

forest, Inter-Andean dry forest, Cerrado, Chaco Serrano, GranChaco, Prepuna, Northern Puna, Southern Puna, flooded savannas,south-west Amazon forest and Yungas.4 Despite this remarkablebiodiversity, little is known about the use and composition of beeproducts, including propolis.

Propolis is a bee product of worldwide interest due to its bio-logical properties.5,6 The variability in its composition is due to thebotanical origin of the plant resins gathered by the bees as well asthe climate and geographic location of the hives.

∗ Correspondence to: Guillermo Schmeda-Hirschmann, Laboratorio de Químicade Productos Naturales, Instituto de Química de Recursos Naturales, Universi-dad de Talca, casilla 747, 3460000 Talca, Chile. E-mail: schmeda@utalca.cl

a Laboratorio de Química de Productos Naturales, Instituto de Química de Recur-sos Naturales, Universidad de Talca, Casilla 747, 3460000, Talca, Chile

b Facultad de Ciencias de la Salud, Programa de Magister en Ciencias Biomédi-cas, Universidad de Talca, 3460000, Talca, Chile

c Facultad de Ciencias de la Salud, Universidad Arturo Prat, Casilla 121, Iquique,1110939, Chile

d Laboratorio de Cultivo Celular, Facultad de Ciencias de la Salud, Universidad deTalca, Casilla 747, 3460000, Talca, Chile

e Instituto de Investigaciones Fármaco-Bioquímicas (IIFB), Facultad de CienciasFarmacéuticas y Bioquímicas, Universidad Mayor de San Andrés, La Paz, Bolivia

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Figure 1. Map showing the location of Bolivia in South America and the propolis collection places. La Paz (A: El Sillar; B: Villa Coroico); Cochabamba(C: Chapare; D: Valle Alto); Chuquisaca (E: Sucre); Tarija (F, San Andrés); Santa Cruz (G: Camiri; H: Okinawa). (The image file is licensed under CreativeCommons, Attribution 3.0 Unported, with some modifications.)

The chemical composition and bioactivity of propolis fromseveral South American countries, including Argentina, Brazil,Chile and Venezuela, has been described. In Brazil, the greenpropolis contains mainly phenylpropanoids,7 while other propo-lis types yielded triterpenes,8 diterpenes,9 benzophenones10

and isoflavonoids.11 Propolis from Venezuela is rich inbenzophenones.12 Propolis from Argentina,13,14 Chile15 – 18

and Uruguay19 contains phenolic compounds such as lignans,flavonoids and caffeic acid derivatives. However, there is no sci-entific information about the chemical composition of Bolivianpropolis. The collection and commercialization of propolis isassociated with honey production. The main centers for bee-keeping in Bolivia are the Departamentos of La Paz, Santa Cruz,Cochabamba, Chuquisaca and Tarija. The aim of the present workwas to provide a first insight into the chemical composition andantioxidant effect of Bolivian propolis. In order to achieve thisgoal, Bolivian propolis samples were analyzed for phenolic andflavonoid content, antioxidant activity and identity of the mainconstituents by hyphenated methods.

MATERIALS AND METHODSChemicalsFolin–Ciocalteu phenol reagent, 2,4,6-tri(2-piridyl)-1,3,5-triazine(TPTZ), sodium acetate, 1,1-diphenyl-2-picrylhydrazyl radical(DPPH), quercetin, catechin, gallic acid and aluminium trichlo-ride (AlCl3) were purchased from Sigma–Aldrich (St. Louis,

MO, USA). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Calbiochem(San Diego, CA, USA). HPLC-grade methanol, formic acid, ferrictrichloride (FeCl3) and thin-layer chromatography plates (TLC,Kieselgel F254), were from Merck (Darmstadt, Germany). Ultra-pure water was obtained using a Barnsted EasyPure water filter(Thermo Scientific, Marietta, OH, USA).

Propolis samples and processingThe propolis samples investigated were collected in the main cen-ters of honey production in Bolivia during the years 2013–2014.Propolis was collected in the Yungas from La Paz (A: El Sillar 15∘24′ S, 67∘ 09′ W; B: Villa Coroico 15∘ 27′ S, 67∘ 15′ W); Cochabamba(C: Chapare 17∘ 00′ S, 65∘ 40′ W; D: Valle Alto, 17∘ 33′ S, 65∘50′ W); Chuquisaca (E: Sucre, 19∘ 03′ S, 65∘ 16′ W); Tarija (F: SanAndres, 21∘ 38′ S, 64∘ 51′ W, two samples); and Santa Cruz (G:Camiri, 20∘ 03′ S, 63∘ 31′ W, two samples, and H: Okinawa, 17∘12′ S, 62∘ 53′ W). The collection places are shown in Fig. 1 andFig. 2. Samples of crude propolis are stored at the IIFB, La Paz,Bolivia.

The crude propolis samples were extracted three times withMeOH at a 1:10 w/v ratio at room temperature. After filtrationand concentration at reduced pressure, extracts were lyophilizedfor analyses and chemical profiling. All samples were analyzed byHPLC and the composition of the crude propolis was compared byHPLC and 1H NMR (400 MHz).

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Figure 2. Landscape of some of the propolis collection places in Bolivia. A: Chuquisaca, B: Cochabamba, C: Tarija, D: Yungas.

Total phenolic and total flavonoid contentsThe total phenolic (TP) and total flavonoid (TF) content was deter-mined as previously described.20,21 Stock solutions of 1 mg mL−1

were prepared in MeOH. For TP, the results are expressed as gramsof gallic acid equivalents per kilogram methanolic extract (g GAEkg−1). For TF, results are expressed as grams of catechin equivalentsper kilogram of methanolic extract (g CE kg−1).

The 2,2-diphenyl-1-picryl hydrazyl assayThe free radical scavenging activity of the samples was determinedby the discoloration of the free radical DPPH (20 mg L−1).20 Themethanolic extracts were dissolved in MeOH to a final concen-tration of 300 mg L−1. The stock solution was diluted in 96-wellplates to final concentrations of 100, 33 and 11 mg L−1. Quercetinwas used as the reference compound. Absorbance was mea-sured at 517 nm in a universal microplate reader (ELx 800; BiotekInstruments Inc., Winooski, VT, USA). Scavenging of DPPH radicalwas calculated as % of discoloration. When the inhibition valueswere more than 50%, the scavenging concentration (SC50) wasdetermined using the OriginPro 8.0 software (OriginLab Corpora-tion, Northampton, MA, USA) and are expressed as miligrams ofmethanolic extract per liter of solution (mg L−1).

Ferric reducing antioxidant power assayThe FRAP assay was performed as previously described.20,22 TheFRAP working solution was prepared mixing 300 mmol L−1 acetatebuffer, 10 mmol L−1 TPTZ and 20 mmol L−1 FeCl3 solution in a10:1:1 v/v/v ratio. A 300 μg mL−1 extract aliquot (150 μL) was mixedwith 2.85 mL of FRAP solution and left to stand in darkness for30 min. Absorbance was read at 593 nm using a Thermo Spec-tronic Helios Alfa spectrometer (Cambridge, UK). Quantificationwas performed using a standard curve of the antioxidant Trolox.

Results are expressed as millimole Trolox equivalents per kilogramof methanolic extract (mmol TE kg−1).

Trolox equivalent antioxidant activity assayABTS radical-scavenging activity was determined according toJiménez-Aspee et al.20 and Nenadis et al.23 The ABTS•+ was gen-erated by mixing ABTS solution with potassium persulfate. Thesolution was stored in the dark for 16 h, and diluted with methanolto a final absorbance of 0.700± 0.050 at 734 nm. Determinationswere carried out by mixing ABTS•+ with fresh standard (1 mmol L−1

Trolox) or extract (100, 150, 200, 250 and 300 mg L−1). Absorbanceswere read at 734 nm after 6 min. Results are expressed asμmol Trolox equivalents per gram of methanolic extract(μmol L−1 TE g−1).

TLC and NMR analysisTLC analyses were performed on silica gel, using petroleum ether(PE) and ethyl acetate (EtOAc) 7:3 or 8:2 v/v as eluent. Spots werevisualized under UV light (254 and 365 nm) and after treatmentwith p-anisaldehyde–sulfuric acid reagent and/or a methanolicsolution of diphenylboric acid ethanolamine complex (naturalproducts reagent, NPR).24 The composition of the crude propolissamples was assessed by 1H NMR (400 MHz) analysis.

Isolated compounds were identified by spectroscopic and spec-trometric means. The NMR spectra were recorded on a BrukerAvance 400 (Bruker, Rheinstetten, Germany) spectrometer at400 MHz for 1H and 100 MHz for 13C in CDCl3 or MeOH-d4. Chem-ical shifts are given in ppm and the coupling constant in Hz.Fourier transform infrared (FT-IR) spectroscopy was performedusing a Nicolet Nexus 470 FT-IR (Thermo Electron Corporation,Ramsey, MN, USA). QTOF-MS were carried out using a MicromassQ-Tof micro (Waters, Elstree, UK). GC-MS was performed on a GStrace 1300 equipment (Fisher Scientific, Milan, Italy). Data werecompared with the NIST 2011 computer libraries.

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HPLC-DAD analysisThe system used was a Shimadzu equipment (Shimadzu Corpora-tion, Kyoto, Japan) consisting of a LC-20AT pump, a SPD-M20A UVdiode array detector, CTO-20 AC column oven and a LabSolutionsoftware. A MultoHigh 100 RP 18–5 μm (250× 4.6 mm) column(Cs-Chromatographie Service GmbH, Langerwehe, Germany)maintained at 25 ∘C. The HPLC analyses were performed using alinear gradient solvent system consisting of 0.1% formic acid (A)and acetonitrile (B) as follows: 0–15 min, 40–60% B; 15–30 min,60% B; 30–40 min, 60–80% B; 40–50 min, 80–100% B; 50–60 min,back to 40% B. The flow rate was 1 mL min−1. The volume injectedwas 20 μL. The compounds were monitored at 250 nm and UVspectra from 200 to 600 nm were recorded for peak characteri-zation. The conditions for the HPLC-DAD analysis were used forHPLC-ESI-MS/MS measurements.

HPLC-ESI-MS/MS analysisData were recorded on a HPLC-ESI-MS/MS system which consistedof the HPLC HP1100 (Agilent Technologies Inc., Santa Clara, CA,USA) connected through a split to the mass spectrometer Esquire4000 Ion Trap LC/MS(n) system (Bruker Daltonik GmbH, Bremen,Germany). Ionization was performed at 3000 V assisted by nitro-gen as nebulizing gas at 24 psi and as drying gas at 365 ∘C and aflow rate of 6 L min −1. Negative ions were detected using full scan(m/z 20–2200) and normal resolution [scan speed 10 300 m/z s− 1;peak with 0.6 FWHM (m/z)−1. The trap parameters were set in ioncharge control (ICC) using manufacturer default parameters, andmaximum accumulation time of 200 ms. Collision-induced disso-ciation (CID) was performed by collisions with helium backgroundgas present in the trap and automatically controlled through theSmartFrag option.

Isolation of compounds by high speed counter-currentchromatographyThe separation was performed using a Mk5 QuikPrep 500 HSCCCinstrument (AECS, South Wales, UK) equipped with four poly-tetrafluoroethylene multilayer coils of 5 mm× 2.16 i.d. (SS)tubing of approximately 120 mL of volume capacity each.Several solvent systems were tested including: PE/acetonitrile(ACN)/dichloromethane (DCM) (10:7:3 v/v/v) (A), PE/ACN (1:1 v/v)(B) and PE/MeOH (2:1 v/v) (C). The upper (U) to lower (L) phase ratiowas as follows: A: 53.4% U, 46.6% L; B: 45.9% U, 54.1% L; C: 40.2%U, 59.8% L. To select the solvent system more suitable for isola-tion, crude extract samples were applied on TLC developed withthe upper phase and lower phase of the mixtures as the elutingsolvent. Spots were visualized under UV light and developed byspraying with p-anisaldehyde and NPR. Best results were achievedusing as solvent system PE/ACN 1:1. A TLC comparison of the LaPaz (El Sillar) and Santa Cruz (Camiri 1) samples in the differentsolvent systems is shown as supplementary material. The twophases were separated and sonicated prior to use. Separation wascarried out by the head-to-tail mode in the following conditions.Stationary phase: PE; mobile phase: ACN, speed: 500 rpm; flowrate: 4 mL min−1. The solvent was pumped into the column witha SSi Series II HPLC pump using a constant flow of 4 mL min−1.A manual sample injection valve with 10 mL loop was used tointroduce the sample into the column. The fraction collector usedwas a Gilson Inc. (Middleton, WI, USA) model FC 203B.

After the mobile phase front emerged and hydrodynamic equi-librium was established in the column, 500 mg of crude extractwas dissolved in 4 mL each of upper and lower phase. The sam-ple was injected via a glass syringe to a 10 mL sample loop. The

HSCCC separation was performed at 20 ∘C. Some 270 fractions of4 mL each were collected. After stopping rotation, an additional110 fractions of 4 mL each were collected by extrusion. Fractionswere monitored by TLC using as mobile phase PE/EtOAc 9:1 andthose with similar TLC pattern were combined and taken to dry-ness under reduced pressure. From the La Paz (El Sillar) sample,fractions 48–51 afforded the triterpene 25 (2 mg), 79–97 yieldedlupeol 31 (40 mg) and 73–80 contained 26 (4 mg). The other frac-tions (mixtures in very low amount) were not further investigated.The Santa Cruz (Camiri 1) sample yielded after HSCCC a mixtureof mangiferonic acid methyl ester 30 (fractions 40–45, 22 mg),24(E)-cycloart-24-en-26-ol-3-one 28 and mangiferonic acid 29(fractions 50–56, 40 mg). Fractions 77–85 afforded a mixture ofunsaturated fatty acids.

Isolation by column chromatographyThe main compounds from La Paz, Santa Cruz and Cochabambapropolis were isolated by column chromatography (CC) on silicagel and Sephadex LH-20. About 3.8 g of crude propolis extractfrom Santa Cruz (Camiri 1) was dissolved in 120 mL CHCl3, filteredand taken to dryness under reduced pressure. Some 3.5 g of theCHCl3 extract was chromatographed in a silica gel column [300 g,column length: 40 cm; internal diameter (i.d.): 5 cm] column usinga PE/EtOAc gradient as eluent as follows: PE/EtOAc 8:2 (500 mL),PE/EtOAc 7:3 (300 mL), PE/EtOAc 6:4 (300 mL), 1:1 (500 mL) andEtOAc (300 mL). Fractions with similar TLC patterns were combinedand pooled into three groups of interest. Pool 1 (fractions 2–7,480 mg) contained a mixture of lup-20(29)-en-3-one 32, lupeol31, 𝛼-amyrin 33 and benzyl benzoate 35. Pool 2 (fractions 10–13,200 mg) contained lupeol 31 and cycloartenol 24 while pool 3(fractions 16–20, 147 mg) yielded a mixture of geranylgeraniol andother terpenes. All were identified by NMR and comparison withthe GC-MS spectral database. Some 3.5 g of the Okinawa (SantaCruz) crude propolis extract was dissolved in 120 mL CHCl3 toobtain the less polar constituents. The CHCl3 extract (2.7 g) wasfractionated by CC (column length: 31 cm; i.d. 5 cm) on silica gel(273.8 g, Merck). The column was eluted first with 300 mL of PE, fol-lowed by a PE/EtOAc solvent gradient: PE/EtOAc 95:5 (300 mL); 9:1(500 mL); 8:2 (500 mL); 75:25 (500 mL); 7:3 (500 mL); 6:4 (1000 mL)5:5 (600 mL) and 4:6 (600 mL); EtOAc (500 mL); EtOAc/MeOH 1:1(300 mL) and MeOH (500 mL). The PE fraction did not contain com-pounds of interest and was discarded. Fractions were collectedstarting with the PE/EtOAc 95:5 eluate, compared by TLC (silicagel, PE/EtOAc 7:3) and pooled according to the chromatographicbehavior. The fractions were analysed by NMR and GC-MS. Frac-tion pool 1 yielded the cycloartenone 27 while pools 5, 8 and 10afforded cinnamic acid 34 and a mixture of triterpenes bearing acyclopropan ring. The constituents of the very complex mixturewere not identified.

The propolis sample from Villa Coroico (La Paz) contained largeamounts of unsaturated fatty acids/waxes as determined by TLCand NMR analysis. One gram of the sample was treated withMeOH/KOH 0.5 mol L−1 (50 mL) and BF4 (1 mL) under reflux toobtain the corresponding methyl esters. After heating the mixturefor 15 min, hexane (10 mL) was added. The organic phase was driedand submitted to GC-MS, palmitic acid and oleic acid were iden-tified as the main constituents. The unsaponificable fraction wasneutralized with diluted HCl (0.1 mol L−1), extracted with CHCl3

(50 mL), washed with water and dried under reduced pressure.The chloroform soluble portion (130 mg) was applied onto a sil-ica gel (90 g, Merck) column (column length 28 cm; i.d. 2.5 cm) andeluted with petroleum ether PE/EtOAc (9:1 v/v), isocratic to yield

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Table 1. Extraction yield, content of phenolics and flavonoids, and antioxidant activity of MeOH-soluble extract of Bolivian propolis

SampleExtraction yields

(% w/w)TP

(g GAE kg−1)TF

(g CE kg−1)DPPH (SC50 in mg L−1

or % inhibition at 100 mg L−1)FRAP

(mmol TE kg−1)TEAC

(μmol L−1 TE g−1)

Tarija-San Andrés 1 54.97 155.4± 2.4 32.9± 4.3 30.94± 4.18 745.72± 36.72 2666.56Tarija-San Andrés 2 41.10 154.7± 5.8 34.6± 5.1 43.37± 0.65 672.73± 37.48 2145.31Cochabamba, Valle Alto 59.68 161.0± 3.8 44.2± 2.8 40.46± 1.15 740.41± 34.39 2264.09Cochabamba, Chapare 45.76 66.3± 1.2 16.4± 1.7 48.27± 6.06% 246.26± 5.52 1067.53Chuquisaca, Sucre 53.28 176.0± 4.8 57.1± 2.8 27.08± 0.50 852.13± 54.76 2151.96Santa Cruz, Camiri 1 19.88 43.0± 0.3 5.5± 0.6 37.20± 2.30% 232.81± 14.95 536.01Santa Cruz, Camiri 2 56.25 153.3± 2.8 29.7± 1.2 58.71± 1.87 905.95± 46.74 2210.83Santa Cruz, Okinawa 9.48 46.1± 0.3 7.5± 0.6 25.69± 1.26% 246.67± 26.13 701.36La Paz, El Sillar 11.53 ND ND 5.31± 0.33% ND 225.43La Paz, Villa Coroico 13.22 ND ND 4.54± 0.19% ND *

Quercetin – – – 7.82± 0.30 10 769.85± 164.33 8157.90

*Denotes Pearson’s coefficient P> 0.05.TP, total phenolics; GAE, gallic acid equivalents; TF, total flavonoids; CE, catechin equivalents; DPPH, diphenyl picryl hydrazyl radical; SC, scavengingcapacity; FRAP, ferric reducing antioxidant power; TE, Trolox equivalent; TEAC, Trolox-equivalent antioxidant capacity; ND, not detected.

70 fractions. After TLC comparison, fractions with similar TLC pat-terns were pooled into three groups: 1 (fractions 17–23, 21 mg), 2(fractions 34–51, 39 mg) and 3 (fractions 52–62, 8 mg). The poolscontained mixtures of cycloartanes triterpenes 24, 31, 27 and 32.Some 5.0 g of the Cochabamba (Valle Alto) sample was dissolvedin 120 mL CHCl3, filtered and taken to dryness to afford of CHCl3

extract. The CHCl3 extract (4.8 g) was fractionated by CC (columnlength 45 cm; i.d. 5 cm) on silica gel (320.0 g, Merck). The columnwas eluted with a PE and EtOAc gradient as follows: PE/EtOAc7:3 (1000 mL); PE/EtOAc 6:4 (500 mL); PE/EtOAc 1:1 (300 mL) andEtOAc 100% (300 mL). The fractions (65) were monitored by TLC(PE/EtOAc 6:4) and pooled into 12 groups. Fractions 27–43 werecombined and permeated on a Sephadex LH-20 (column length25 cm, i.d. 2.5 cm) using MeOH/DCM 1:1 as mobile phase to affordkaempferol 7-O-methyl ether 13 (110 mg, fractions 48–70), inagreement with UV and QTOF data. The fractions 52–60 were per-meated on a Sephadex LH-20 (column length 25 cm, i.d. 2.5 cm)column using MeOH as eluent to afford 3-prenyl-p-coumaric acid10 (118 mg, fractions 34–46) and kaempferol 3-methyl ether 9(42 mg, fractions 64–92).

Statistical analysisDeterminations of TP, TF, DPPH and FRAP were performed intriplicate and results are expressed as mean values± SD. For theTEAC assay, a curve was plotted for each sample and a correlationcoefficient with 95% confidence limit was established. To assessthe relationship between the antioxidant activities and the TP andTF content, Pearson’s correlation coefficients were calculated with95% confidence. All statistical analyses were carried out using thesoftware SPSS 14.0 for Windows.

RESULTS AND DISCUSSIONUse of propolis in BoliviaPropolis is an apicultural product that is becoming relevant forproducers in Bolivia due to an increase in commercial demand. Areview on the gathering of honey and its by-products by the ChacoIndians was reported recently.25 The propolis in Bolivia is used aspart of the traditional medicine mainly for respiratory ailments.Some 10–15 drops of the ethanolic extract are added to one glass

of water and taken orally. Another application is as a spray for coldsand sore throat. Propolis is also made into candies that are chewedas antiseptic. The uses are very similar to those in other places ofthe world, following the general marketing trends affecting offerand demand in natural products. We did not know of previousstudies on Bolivian propolis.

Total phenolic and flavonoid contentExtraction yields of the different propolis ranged from low, for LaPaz (11.53 and 13.22%) and two out of three Santa Cruz samples(9.48 and 19.88%), to high for the propolis of Tarija (41.10 and54.97%), Cochabamba (45.76 and 59.68%), Chuquisaca (53.28%)and one of the Santa Cruz samples (56.25%) (Table 1). The TPcontent of the MeOH–propolis extract was variable, with lowervalues ranging from 43.0 and 46.1 g GAE kg−1 for two Santa Cruzsamples. Most of the samples presented TP values in the rangeof 153.3 to 176.0 GAE kg−1, with similar content for the Tarija,Cochabamba, Chuquisaca and one of the Santa Cruz samples(Table 1). A similar trend was observed for the TF content, wherecollection with lower TP also showed the less concentration ofTF (5.5 to 7.5 CE kg−1). The samples from La Paz (El Sillar andVilla Coroico) did not contain detectable levels of phenolic orflavonoids. The low solubility of the samples in methanol and theundetectable levels of TP and TF suggest that the constituents ofthe samples are of different chemical nature than the phenolicsusually expected in propolis.

Antioxidant activityThe antioxidant effect of the samples was compared using threedifferent methods, namely: discoloration of the free radical DPPH,TEAC using the ABTS radical and FRAP reduction. Results arepresented in Table 1. A clear picture is obtained when the TP,TF and antioxidant assays are compared. Samples with lowestTP and TF content were also the less active towards DPPH, withvalues ranging from 4.54 to 48.27% discoloration at 100 mg L−1,FRAP from inactive to 246.67 mmol TE kg−1 and TEAC frominactive to 1067.53 μmol L−1 TE g−1, respectively. Best in vitroantioxidant activity was observed for the Chuquisaca and Tarijasamples.

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Cochabamba - Valle Alto A

Cochabamba - Chapare

Chuquisaca

Tarija San Andres-1

Tarija San Andres-2

Figure 3. HPLC profiles of Bolivian propolis: (A) phenolic-type propolis; (B) triterpene-type propolis.

TLC and NMR analysisTLC analysis of the crude propolis samples showed a commonchromatographic behavior for the Cochabamba, Tarija andChuquisaca samples, while the Santa Cruz and La Paz sampleswere very different. No spots were observed for the La Paz samples(silica gel, PE/EtOAc 9:1) under UV light. However, when sprayedwith p-anisaldehyde and heating, violet spots developed withRf values of 0.98 and 0.68. TLC analysis allowed a differentiation

of the samples in two groups. One of them consisted mainly ofterpenes (La Paz and Santa Cruz) and the second one had morephenolic constituents (Cochabamba, Chuquisaca and Tarija). Thecomposition pattern of the different samples was also assessed by1H NMR. Spectra were analyzed for diagnostic signals for phenolicsand terpenes. Different 1H NMR patterns were observed for thesamples. A clear differentiation was found for the Santa Cruz,La Paz and Cochabamba (Chapare) samples, where triterpenes

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La Paz - El Sillar B

La Paz - Villa Coroico

Santa Cruz - Okinawa

Santa Cruz Camiri-1

Santa Cruz Camiri-2

Figure 3. Continued.

were observed instead of phenolics. The Chuquisaca, Tarija andCochabamba (Valle Alto) samples contained mainly phenolics.The observation is in agreement with the results of TP, TF contentand antioxidant assays. The TLC and NMR analysis support theresults on TP and TF content of the La Paz samples (El Sillar andVilla Coroico) which contained mainly triterpenes.

HPLC analysisAll MeOH propolis extracts were compared by HPLC to get aninsight into the chemical diversity of Bolivian propolis and todisclose similarities and differences in the chemical compositionaccording to the collection place. The HPLC chromatograms are

presented in Fig. 3A and B. The Bolivian propolis showed distinc-tive profiles, according to the collection area. Two main types ofpropolis were found, differing in the identity and chromatographicpattern of constituents. The HPLC profiles from Cochabamba,Chuquisaca and Tarija have a similar fingerprint, with UV absorb-ing compounds and are shown in Fig. 3A. The HPLC profiles of theLa Paz and Santa Cruz propolis showed few UV-absorbing con-stituents and are presented in Fig. 3B. Therefore, selected sam-ples were worked up to isolate and identify the main compoundsby spectroscopic means, mainly NMR analysis. The Cochabamba(Valle Alto) sample, with a similar HPLC pattern, UV spectra and Rt

as the Cochabamba (Chapare), Tarija and Chuquisaca propolis was

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Compound R1 R2 R3

1

2

Caffeoyl Caffeoyl Caffeoyl

Caffeoyl Caffeoyl H

4

8

10

11

16

18

19

20

21

22

34

3 Caffeoyl H H

R1 R2

7 H CH3

12 Acetate H

R1 R2 R3 R4

9 CH3 H H OH

13 H CH3 H OH

14 CH3 H OH OCH3

15 CH3 CH3 H OH

17 CH3 CH3 H H

R1 R2 R3 R4

Prenyl OBenzoate H H

OPrenyl OH H H

Prenyl OH H H

OH OH H Prenyl

OCinnamoyl OH H H

H OPhenethyl H H

Prenyl OCH3 H H

H OCinnamoyl H H

Prenyl OH Prenyl H

Prenyl ODihydrocinnamoyl H H

H H H H

OO

23 35

Figure 4. Phenolic compounds tentatively identified in Bolivian propolis.

analyzed by HPLC-ESI-MS/MS to identify the constituents. The dif-ferences in the above mentioned propolis is mainly in the relativeproportion of constituents.

HPLC-ESI-MS/MS analysisThe identification of the main compounds of the Bolivian propo-lis by HPLC-ESI-MS/MS was carried out comparing the Rt, UV andMS fragmentations patterns with literature.13,26 – 28 A total of 23phenolic compounds were identified in the phenolic-rich samples.The compounds profile of the different phenolic-rich samples wascompared using the Rt and UV data as well as the MS/MS frag-mentation patterns. Representative chromatograms are presentedin Fig. 3A. The structures of the compounds 1–4 and 7–23 are

presented in Fig. 4. The compounds 9, 10 and 13 were identifiedby NMR analysis after isolation, the 1H and 13C NMR data were infull agreement with literature. The references associated with thecompounds identified are included in Table 2.

Bolivian propolis typesPhenolic-rich propolisThe Bolivian propolis with high phenolic content, showed threemain groups of compounds, namely: caffeoylquinic acids (1–3),flavonoids including dihydroflavonols (7 and 12) and flavonols(9, 13–17) as well as phenylpropanoids (4, 8, 10, 11, 16, 18–23).Phenylpropanoids were the most abundant constituents andincluded p-coumaric and caffeic acid derivatives. Esters included

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Table 2. Identification of phenolic compounds in Bolivian propolis by HPLC-ESI-MS/MS data

Compound Peak Rt (min) 𝜆max (nm) [M−H]− MS/MS ions (m/z) Reference

Tricaffeoyl quinic acid 1 2.5 326, 290sh, 234 677 515, 353, 191 27Dicaffeoyl quinic acid 2 3.8 322, 295sh 515 353 27Chlorogenic acid 3 4.0 326, 290sh 353 190, 178, 134 27Benzyl prenyl p-coumaric acid 4 4.7 310, 287sh, 224 335 291 –p-Coumaric acid derivative 5 5.2 314, 297sh, 232sh, 215 345 330, 315, 261, 217 –Ellagic acid 6 7.4 370 301 257 26Pinobanksin methyl ether 7 10.7 371sh, 326sh, 291 285 285, 267, 256, 243, 227 27Caffeic acid isoprenyl ether 8 10.7 326, 295sh 247 203 –Kaempferol 3-methyl ether* 9 11.4 349, 321sh, 293sh, 267 299 284, 273, 255, 245, 227, 213, 198 –Drupanin* 10 13.5 314, 295sh, 232, 216 231 187, 131 27Caffeic acid isoprenyl ester 11 15.6 319, 298sh 247 178, 134 27, 28Pinobanksin acetate 12 16.8 290 313 271, 253 28Kaempferol 7-O-methyl ether* 13 17.9 364, 318sh, 292sh, 266 299 284 26Quercetin dimethyl ether 14 18.6 363, 335sh, 289sh, 269 329 314, 271, 215 26, 28Kaempferol dimethyl ether 15 19.0 347, 325sh, 293sh, 267 313 298, 283, 269, 255 264-Cinnamoyloxy caffeic acid 16 19.0 322, 297sh 295 211, 177, 133 –3,7-Dimethoxy-5-hydroxyflavone 17 19.2 353, 325sh, 293sh, 267 297 282, 267, 255, 241 134-Phenethyloxy cinnamic acid 18 20.0 311, 296sh, 239 267 162, 118 –3-Prenyl-4-methoxy cinnamic acid 19 22.4 315, 295sh, 237 245 213, 201, 189, 145 –4-Cinnamoyloxy cinnamic acid 20 23.8 311, 295sh, 238 279 235, 195, 161, 118 –Artepillin C 21 25.3 313, 237 299 255, 199 27Baccharin 22 35.1 280, 229 363 319, 187 272,2-Dimethyl-6-carboxy ethenyl-8-prenyl-2H-benzopyran 23 36.5 305sh, 277, 241 297 253, 198 27

Rt, retention time.*Identified by NMR analysis.

benzoyl, phenethyl, cinnamoyl, prenyl and dihydrocinnamylmoieties. The main compounds in all phenolic-rich propoliswere drupanin (3-prenyl-p-coumaric acid 10) and artepillin C(3,5-diprenyl-p-coumaric acid 21). Drupanin and artepillin C werereported as constituents of Brazilian propolis,9,29 Brazilian greenpropolis30 and green propolis from Baccharis dracunculifolia.7

Drupanin and artepillin C were also described as constituents ofBaccharis grisebachii.31 This is the second time that both com-pounds are reported from South American propolis and the firsttime from Bolivian samples. The occurrence of the prenyl cinnamicacid derivatives suggests that at least one of the botanical sourcesof the Bolivian propolis from Cochabamba, Chuquisaca and Tarijamight be Baccharis species.30 – 32 A propylene glycol extract ofthe main botanical source of Brazilian green propolis, the shrubBaccharis dracunculifolia, was investigated for antioxidant activityusing isolated rat liver mitochondria. The study confirmed theantioxidant effect and the mechanisms of action involved as wellas the occurrence of artepillin C as one of the main constituents.32

The exudate and aerial parts of B. grisebachii were assessed forantioxidant effect using the DPPH, superoxide anion, xanthineoxidase inhibition and lipoperoxidation in human erythrocytes aswell as for cytotoxicity. The extracts and the isolated compoundsartepillin C and drupanin displayed antioxidant effect in vitro withlow cytotoxicity.31 Artepillin C and drupanin were shown to modu-late the protein hypoxia-inducible factor (HIF-1), with implicationsin angiogenesis and ischemic cardiovascular diseases.33 Szliszkaet al. investigated the anti-inflammatory effect of an ethanolextract of Brazilian green propolis on activated macrophages.30

The authors found that the extract inhibited the production ofROS and RNS, modulating the chemokine-mediated inflamma-tion. The cytotoxic effect of Brazilian propolis was described

by Banskota et al.,9 including the effect of artepillin C towardsfibrosarcoma HT-1080 and colon L5-26 cell lines. The ED50 valuesof 45.47 and 59.32 mg L−1, respectively, can be regarded as verylow compared with the reference compound doxorubicin, withED50 values of 0.034 and 0.015 mg L−1, respectively. The flavonoidsoccurring in Bolivian propolis are of widespread occurrence andincluded mainly methyl ethers of kaempferol and quercetin.Several kaempferol methyl ethers were reported from Portuguesepropolis.26,34,35 However, we cannot associate these constituentswith a botanical source of propolis in Bolivia.

Triterpene-rich propolisPropolis from La Paz and Santa Cruz showed a very differentprofile compared to other South American propolis. In the HPLCchromatograms at 250 nm (Fig. 3B), few signals were observed, inagreement with the TP and TF content of the samples (Table 1).After repeated column chromatography, HSCCC and prepar-ative TLC on silica gel, 12 compounds were isolated and/oridentified by spectroscopic and spectrometric means (Fig. 5).Seven of the compounds showed in the 1H NMR spectra char-acteristic signals for a cyclopropane ring, an unsaturated sidechain with different oxidation degrees and either an hydroxylor a ketone function at C-3, compatible with cycloartane triter-penes. The compounds were identified as cycloart-24-en-3𝛽-ol24,36 cycloart-24-en-3𝛽 ,26-diol 25,37 cycloart-24-en-3𝛽 ,26-al26, cycloart-24-en-3-one 27,38 cycloart-24-en-26-ol-3-one 28,39

mangiferonic acid 29 and mangiferonic acid methyl ester 30.40

The 𝛽-hydroxy function at C-3 of compound 26 differs in the stere-ochemistry from the 𝛼-hydroxy compound described by Januarioet al.41 The main triterpene 31 showed an isopropenyl side chain,

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24 β- 3

25 β- 2OH

26 β-

27 3

28 2OH

29

30

OH, H CH

OH, H CH

OH, H CHO

O CH

O CH

O COOH

O COOCH3

Compound X RCompound X

31 H, β-OH

32 O

33

Figure 5. Triterpenes identified in Bolivian propolis.

six quaternary methyl groups and a 𝛽-hydroxy function at C-3, inagreement with the pentacyclic triterpene lupeol. The compound32 was identified as lup-20(29)-en-3-one. The spectral data are inagreement with literature.42 The compound 33 was identified as𝛼-amyrin by GC-MS analysis.

The occurrence of triterpenes in propolis was reported forsamples from Brazil, Myanmar, Saudi Arabia and Cameroon.Cycloartane triterpenes have been described as constituentsfrom a propolis sample collected in north-eastern Brazil.8 Thecompounds found in the Brazilian study included mangifer-onic acid 29 and other derivatives as well as C-31 triterpenes.The Bolivian propolis showed a more complex pattern of C-30triterpenes and did not contain C-31 constituents. Mangiferonicacid 29 and compound 26 were identified in Myanmar propolispresenting mainly cycloartenol triterpenes as constituents.43

Jerz et al. described several diterpenes and triterpenes froma Saudi Arabian propolis sample, including cycloartenol 24.44

While the Saudi Arabian propolis contain diterpenes as maincompounds, acetates from pentacyclic triterpenes and twocycloartanes, the Bolivian samples did not afford diterpenes.Triterpenes and alk(en)ylresorcinols were isolated and identifiedin Cameroonian propolis, including the triterpenes 24, 29, 31 and33 occurring in Bolivian propolis.40 Our work reports for the firsttime the occurrence of the triterpenes 25, 27, 28, 30 and 32 inpropolis.

Due to the scarcity of phytochemical studies on the Bolivianflora, it is difficult to clearly associate the compounds occurringin propolis with the possible botanical sources. Triterpenes arethe main constituents in the Santa Cruz and La Paz (Yungas) sam-ples. Lupeol 31 was the main compound in all samples and iswidespread in plants. Lupeol is a known and safe bioactive triter-pene with several described effects.45 This compound presentsanti-inflammatory and antiproliferative effects and occurs in foodand medicinal plants.46

The compound 29, with a more restricted occurrence, wasreported from Mangifera indica.37,47 This tree is cultivated in SantaCruz and Yungas for its fruits and might be one of the sourcesof this compound. Other cycloartane triterpenes have beendescribed from Rapanea,41 Euphorbia,36 Gardenia38 and Aglaiaspecies,39 among others.

There was a positive correlation between the TP content andFRAP (r = 0.964, P < 0.01) as well as between TP content andTEAC (r = 0.961, P < 0.01). In addition, a positive correlation wasfound between TF content and FRAP (r = 0.856, P < 0.01) as well asbetween TF content and TEAC (r = 0.829, P < 0.05). These resultsare in agreement with the report from Kalogeropoulos et al.who studied propolis from Greece describing a positive correla-tion between FRAP and the content of ferulic acid, chrysin andapigenin.48 Although we did not find these compounds, otherphenolic acids were identified in the Bolivian propolis such ascoumaric, quinic, caffeic and chlorogenic acid derivatives. Theantioxidant activity of Venetian propolis was attributed to caf-feic acid and its derivatives.49 The positive correlation observedbetween the antioxidant activity and the TP content may suggestthat phenolic acids are responsible, at least in part, for the totalantioxidant activity of our samples. In contrast, a recent study ofChilean propolis reports lack of correlation between TP and antiox-idant effect.18

CONCLUSIONSome 35 constituents, including 25 phenolics and 10 triterpeneswere identified from 10 different samples of Bolivian propolisby spectroscopic and/or spectrometric means. According to thechemical composition, Bolivian propolis can be classified into twomain groups, namely phenolic-rich and triterpene-rich samples.The phenolic-rich propolis contains prenylated phenylpropanoids,flavonoids and caffeic acid derivatives, while the triterpene-richsamples showed mixtures of cycloartane and pentacyclic triter-penes. The antioxidant activity of the samples can be explainedby its composition. The most active as antioxidant were those withhigher phenolic and flavonoid content while propolis containingmainly triterpenes were devoid of antioxidant activity. The highvariability in sample composition according to geographic originopens the possibility to develop selective products from Bolivianpropolis and to differentiate origin according to the constituentprofile. More work should be undertaken to disclose the poten-tial of Bolivian propolis as a source of medicinal and nutraceuticalproducts. The identification of the botanical sources of this beeproduct remains to be established.

ACKNOWLEDGEMENTN.N thanks AGCI for a Magister grant, and F.J-A thanks CONICYTfor a doctoral grant (2013–21130048). We are grateful to theUniversidad de Talca for financial support (PIEI-QUIM-BIO).

SUPPORTING INFORMATIONSupporting information may be found in the online version of thisarticle.

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