The Isolation of Antiprotozoal Compounds fromLibyan Propolis

Weam Siheri,1 John O. Igoli,1 Alexander I. Gray,1 Ticiano G. Nasciemento,3 Tong Zhang,1James Fearnley,2 Carol J. Clements,1 Katharine C. Carter,1 John Carruthers,1Ruangelie Edrada EbelQ1

1 and David G. Watson1*1Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK2BeeVital, Whitby, North Yorkshire YO22 5JR, UK3Laboratório de Controle de Qualidade e Análise de Fármacos, Escola de Enfermagem e Farmácia, Universidade Federal de Alagoas,BR 104 Norte, Km 97, Maceió, AL CEP:57072-970, Brazil

Propolis is increasingly being explored as a source of biologically active compounds. Until now, there has been nostudy of Libyan propolis. Two samples were collected in North East Libya and tested for their activity againstTrypanosoma brucei. Extracts from both samples had quite high activity. One of the samples was fractionatedand yielded a number of active fractions. Three of the active fractions contained single compounds, which werefound to be 13-epitorulosal, acetyl-13-epi-cupressic acid and 13-epi-cupressic acid, which have been describedbefore in Mediterranean propolis. Two of the compounds had a minimum inhibitory concentration value of1.56μg/mL against T. brucei. The active fractions were also tested against macrophages infected with Leishmaniadonovani, and again moderate to strong activity was observed with the compounds having IC50 values in therange 5.1–21.9μg/mL. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: Libyan propolis; labdane diterpenes; T. brucei; L. donovani.

INTRODUCTION

Propolis is harvested by honey bees in order to sealcracks of the hives and more importantly eliminatebiological contamination in the colony. It has beenreported to have various biological and pharmacologicalproperties and is potentially a source of new medicinesparticularly for treating infective diseases (Sforcin andBankova, 2011; Salatino et al., 2011; Bankova et al.,2000). Propolis has the advantage as a source of biolog-ically active compounds because it has already beenselected by bees for its biological activity and iscollected from plants in a nondestructive way. Thechemical and bioactive characteristics of propolis arehighly dependent on its geographic origin (Seidel et al.,2008; Watson et al., 2006; Salatino et al., 2005). Althoughit has never been proved, it would seem likely that theplants chosen by the bees in a particular location wouldexhibit activity against the environmental pressuresencountered by the bees, which include protozoal attack(Ruiz-Gonzales and Brown, 2006). In a simulatedexperiment, artificial nectar containing the alkaloidgelsemine, which is also naturally contained in a sourceof nectar favoured by North American bumblebees,was ingested by bumblebees and found to reduce thepathogen load of the protozoan parasite Crithidia bombi(Manson et al., 2010). A number of previous studieshave tested propolis against both Trypansoma andLeishmania species, these studies have largely focusedon the use of extracts rather than isolated compounds.

Extracts from two samples of Portuguese propolis wereinvestigated for their activity against Plasmodiumfalciparum, Leishmania infantum, Trypanosoma bruceiand Trypanosoma cruzi (Falcão et al., 2014). IC50 valueswere mainly <10μg/mL and the greatest activity of1.8μg/mL being against T.brucei. The antileishmanialactivity of Turkish propolis was investigated (Duranet al., 2011), and the two samples tested had IC50 valuesof 175 and 350μg/mL. The effect of an extract ofBrazilian propolis on Trypanosoma evansi was investi-gated (Gressler et al., 2012), and in vitro the IC50 valuefor the extract was found to be 10μg/mL, but thepropolis was ineffective in curing rats infected with T.evansi. Extracts of 18 Cuban propolis extracts ofdifferent types were tested against Leishmaniaamazonenis (Fidalgo et al., 2011). All of the extractsproduced inhibition of L. amazonenis, but they alsodisplayed some toxicity against macrophages. Com-pounds isolated from Baccharis dracunculifolia, whichis the major source of Brazilian green propolis, wereinvestigated for antileishmanial and antiplasmodialactivity (Da Silva et al., 2009). Ursolic acid and hautriwaicacid lactone had IC50 values of 3.7 and 7μg/mL againstLeishmania donovani. The activity of Brazilian greenpropolis against T. cruzi was investigated (Salomão et al.,2011). The extract was most effective against the intra-cellular amastigote stage of the parasite having anIC50 value of 8.5μg/mL. Extracts of Brazilian red andBrazilian green propolis were tested against L.amazonensis (Ayres et al., 2007), and an extract ofred propolis was found to be the most effective havinga minimum inhibitory concentration (MIC) of 25μg/mL.Bulgarian propolis extracts were found to have IC50values of 36–40μg/mL against T. cruzi (Dantas et al.,2006). Thus, it is apparent that antiprotozoal activity of

* Correspondence to: David G. Watson, Strathclyde Institute of Pharmacyand Biomedical Sciences, 161 Cathedral Street, Glasgow G4 0RE, UK.E-mail: d.g.watson@strath.ac.uk

PHYTOTHERAPY RESEARCHPhytother. Res. (2014)Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.5194

Copyright © 2014 John Wiley & Sons, Ltd.

Received 14 March 2014Revised 09 May 2014

Accepted 30 May 2014

Journal Code Article ID Dispatch: 07.06.14 CE: Dumalag, Kristian ArniebelP T R 5 1 9 4 No. of Pages: 5 ME:

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propolis is common to propolis samples from manyregions. Within this context, our study investigated theeffect of Libyan propolis on the parasites Trypanosomabrucei brucei, which is the aetiologic agent of sleepingsickness, and L.donovani, which causes visceral leish-maniasis. The biological properties and chemical profileof Libyan propolis have not been investigated before.Q2

MATERIALS AND METHODS

Materials. Absolute ethanol, HPLC grade acetonitrile,hexane, methanol, formic acid andAcrodisc syringe filterswere obtained from Fisher Scientific (Loughborough,UK). Chloroform, dimethyl sulphoxide (DMSO), deuter-ated chloroform, DMSO-d6, silica gel 60 (0.04–0.06mmmesh size) and Wilmad nuclear magnetic resonance(NMR) tubes were obtained from Sigma Aldrich, Dorset,UK. An ACE C18 column (3mm×150mm, 3μm) wasfrom Hichrom, Reading, UK. T.brucei (ATCC S427blood stream form) was from Fisher Scientific. HPLCgrade Water was produced in house by a Milli Q system(Millipore, UK). RPMI 1640 medium, Dulbecco’smodified Eagle’s medium, penicillin–streptomycin and L-glutamine were obtained from Gibco BRL, Paisley, UK.D-Luciferin potassium salt was obtained fromCaliper LifeScience, MA, USA. Amphotericin B was purchased fromSequoia Research Products (Berkshire, UK).Two propolis samples were collected from Tokra or

Tukra, Al`Aquriyah, Libya, a small village in EasternLibya, located about 70km East of Benghazi city (LBA)and from Qaminis 53km South of Benghazi (LBG). Thebeekeeper scraped the propolis sample off the top of thehive using a spatula and collected it in a clean tray. LBApossessed an intense orange-like odour, was light brownand had a very sticky texture, whereas LBG was darkerbrown and less sticky and had a less intense odour. Asample of the LBA propolis (23 g) was extracted bysonication in 100mL of absolute ethanol for 60min, andthen the extract was filtered and re-extracted twice morewith 100mL of ethanol, filtering each time after that.The extracts were combined, and the solvent was evapo-rated. The crude ethanol solution extract was testedagainst T.brucei.

Animals. Luciferase-expressingL.donovanipromastigoteswere derived from L.donovani strain MHOM/ET/67:MHOM/ET/67:LV82 (XXX). In-house inbred maleBALB/c mice (20–25 g) were used in studies. All animalstudies were carried out in accordance with the Animals(Scientific Procedures) Act 1986 and had UK HomeOffice approved and local ethical approval from theUniversity of Strathclyde.

Open column chromatography and medium pressureliquid chromatography. A sample (2 g) of the ethanolsolution extract of the propolis was dissolved in ethylacetate and mixed with 6 g of silica gel in a beaker,and the solvent was removed slowly under a stream ofnitrogen. Then silica gel (50 g) was mixed with hexane(200mL) and used to pack a glass column. The samplemixed with silica gel was dry loaded onto the top ofthe column, and elution was carried out as followscollecting fractions in 50mL flasks: 200mL of hexane/ethyl acetate, (90:10) F1; then 200mL of hexane/ethylacetate (60:40) F2; then 200mL of hexane /ethyl acetate(40:60) F3; 200mL of ethyl acetate F4; 200mL ofmethanol F5; and finally, 200mL of methanol/water(60:40) F6. All fractions obtained from open columnchromatography were concentrated by rotary evapora-tion and weighed. Fraction F1-3, which had the highestweight, was fractionated by medium pressure liquidchromatography (MPLC) on silica gel using a GraceRevelris Flash Chromatography system (Alltech Ltd.,UK) with evaporative light scattering (ELSD) detectionand UV detection. The sample was loaded onto celite(1.9 g) and packed into a dry loading cartridge. TheRevelris MPLC was set up with a 24 g silica gel columnto run a stepwise gradient at 12mL/min flow rate usinglinear gradients as follows: 100% hexane 0min, hexaneethyl acetate (80:20) 30min and 100% ethyl acetate50min. Fractions were collected automatically whentriggered by the ELSD response. The fractionsassociated with the same peak according to the ELSDchromatogram were combined, and the solvent wasremoved and weighed. The isolated fractions wereprofiled by reversed-phase HPLC with ELSD, GC–MS,LC–MS and NMR.

Instrumental methods. Fractions from the MPLC sepa-ration were profiled using an Agilent 1100 HPLC linkedto a Shodex ELSD. An ACE C18 column (150 × 3mm,3μm)) with a mobile phase of water (A) and acetonitrile(B) with a flow rate of 0.5mL/min and the followinggradient: 0min 70% B, 20min 100% B, and 26min100% B. The samples were prepared at 0.5mg/mL bydissolving in acetonitrile and then adding water to givea solution in water/acetonitrile (30:70).

The crude sample, the fractions obtained fromchromatography and the purified compounds obtainedfrom flash chromatography were dissolved in methanoland analysed by LC–MS in order to confirm theirmasses and molecular formulae. The high-resolutionmass spectra were obtained by using an LTQ Orbitrapmass spectrometer (ThermoFisher, Hemel Hempstead,UK) in negative ion mode with a needle voltage of�4.0 kV. Samples were dissolved in methanol to give1mg/mL, and the sample solution (20μL) was injected.The separation was performed on an ACE C18 column(150 × 3mm, 3μm) from HiChrom UK with 0.1% v/vformic acid inwater asmobile phaseAand 0.1%v/v formicacid in acetonitrile as B at flow rate of 0.300mL/min usingthe gradient described for HPLC–ELSD.

Nuclear magnetic resonance spectroscopy. Samples(5–10mg) of the fractions obtained from the MPLCfractionation, which exhibited good purity, were dissolvedin CDCl3 and transferred to NMR tubes. 1H NMRFigure 1. Structures of compounds isolated from Libyan propolis.

2 W. SIHERI ET AL.

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spectra were measured at a magnetic field strength of400.13MHZ using a JEOL Delta GX 400MHz FTnuclear magnetic resonance instrument. Proton spectrawere referenced to the residual protons in CDCl3. Broad-band decoupled 13C NMR was used to determine thenumber of carbons and their type, and where necessary,

Q3 DEPT experiments were obtained in order to distinguishthe carbons according to the extent of their protonattachments. Correlation spectroscopy, heteronuclearmultiple-bond correlation spectroscopy and heteronuclearmultiple quantum coherence spectra were also obtained.Optical rotation measurements were obtained by

using a Perkin Elmer 341 polarimeter. The sampleswere dissolved in 2mL of chloroform and measuredusing the sodium D line.

Antimicrobial assays.

(a) Trypanosome studies

The in vitro antitrypanosomal tests were carried outby using an AlamarBlue™ assay according to a standardprotocol (Räz et al., 1997; Igoli et al., 2012).

(b) L. donovani studies

Intraperitoneal macrophages were recovered fromthe peritoneal cavity of BALB/c mice 3 days after intra-peritoneal injection with 1mL 3% w/v aqueous sterilestarch solution. The mice were then euthanized, and3mL of incomplete medium (RPMI-1640, 100 μg/mLpenicillin–streptomycin and L-glutamine) was injectedinto the peritoneal cavity. The macrophage-containingmedium was then removed and collected, and theresulting cell suspension centrifuged at 3000 × g for5min and then re-suspended in 10mL completemedium (in complete RPMI-1640 supplemented with10% heat inactivatedQ4 FCS [v/v]). The cells were thenused in antileishmanial assays. Bone marrow was thenharvested from the femurs of each mouse by flushingout the removed bone with 5ml of bone marrowmedium (Dulbecco’s modified Eagle’s medium, 20%heat-inactivated FCS [v/v], 30% L-Cell solution [v/v],100μg/mL penicillin–streptomycin and L-glutamine).The cell suspension was added to sterile petri dishes(one petri dish/mouse) and incubated for 7 days at 37 °Cin an atmosphere of 5% CO2:95% air. The medium wasremoved from the plate, and 7mL TrypLE Express wasadded to detach the bone marrow-derived macrophages.The resulting suspension of bone marrow-derivedmacrophages was collected, pelleted by centrifugationand re-suspended in 10mL of incomplete medium andthen used in antileishmanial assays.The number of live macrophages per millilitre was

determined microscopically using a haemocytometer,by mixing a cell sample with 1:1 Trypan blue (20μL)and viewing at ×10 magnification. In all cases, cellviability was >95%. Cells (0.5 × 105 in 200μL completemedium) were added to the appropriate wells of a 96-well tissue culture plate and incubated for 24 h at 37 °Cin an atmosphere of 5% CO2:95% air. Cells were theninfected with L.donovani luciferase-expressing parasiteusing a 20:1 parasite/host cell ratio. The plate wasincubated as before for 24 h. The medium was removedfrom each well and replaced with 200μL completemedium (control, n= 6) or various concentrations ofthe one of the extracts (diluted in 4% DSMO v/v in

complete medium, n= 3) or amphotericin B solution(4–0.02μg/mL). The plate was incubated as before for72 h, the medium was then removed, and 150μL ofluciferin solution (150μg/mL luciferin in completeRPMI-1640) was added to each well. The Q5BLI emittedper well was determined using the IVIS® imagingsystem (Alsaadi et al., 2012). The suppression in biolu-minescent signal for each test sample was comparedwith the mean control value. The mean IC50 value wasthen calculated for each sample by Probit analysis(Vermeersch et al., 2009).

Statistics.Data were analysed using MINITAB® softwareversion 16.1.1 supplied by Minitab Ltd. Coventry, UK,and an Anderson–Darling test was used to establish ifthe data were normally distributed. Parametric datawere analysed using a Student’s unpaired t-test or byone-way analysis of variance dependent on the numberof treatments/experiments, and significance was con-firmed by a Fisher test. A Mann–Whitney or Kruskal–Wallis test was used to analyse data that did not have anormal distribution. Results were considered statisticallysignificant at a p-value of <0.05.

RESULTS

The results for screening the crude extracts of twopropolis samples and against T.brucei using an Almarblue assay are shown in Table T11. Both extracts were quiteactive, but because LBAwas approximately twofoldmoreactive than LBG, it was selected for fractionation. First, itwas fractionated by open column chromatography, andfraction 1, which was eluted with hexane/ethyl acetate(90:10) and had the highest weight, and fraction 3, whichwas eluted with hexane/ethyl acetate (40:60), wereselected for further fractionation byMPLC. The fractionsobtained from the Grace Revelris system were analysedby HPLC–ELSD, and four of the fractions were foundto be quite pure. The 1H and 13C NMR data for thepure fractions were compared with those in the

Table 1. MIC values for crude LBA and LBG extracts andfractions of LBA tested against T.brucei blood stream form S427

Sample MIC (μg/mL)

LBA1 3.12LBG2 6.25LBAF1 1.56MPLC F1-2 10.00MPLC F1-5 5.00MPLC F1-7 10.00MPLC F1-9 (1) 2.50MPLC F1-10 (2) 1.56MPLC F1-11 1.56MPLC F1-14 5MPLC F1-15 (3) 10MPLC F1-16 2.5MPLC F1-17 2.5MPLC F1-18 5MPLC F1-20 5MPLC F3-7 6.5Suramin 0.178

3ANTIPROTOZOAL COMPOUNDS FROM LIBYAN PROPOLIS

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literature, and the data were found to closely match theNMR data reported previously for the labdane diter-penes 13-epitorulosal (1), acetyl-13-epi-cupressic acid(2), 13-epi-cupressic acid (3) (Abdel-Sattar et al., 2009;Wen-Chiung et al., 1994) and lignin (+) sesamin (4)(Christov et al., 1999). In addition, the following analyt-ical information was obtained: (i) eluted mainly infraction 9 from the MPLC system, which had a weightof 14mg, and it had an optical rotation of +14° (lit +35°);(ii) eluted mainly in fraction 10 from the MPLC system,which had a weight of 7mg, and it had an optical rotationof +92°; (iii) eluted mainly in fraction 15 from the MPLCsystem, which had a weight of 102mg, and it had anoptical rotation of +55.5° (lit +57°); and (iv) eluted infraction 7 when fractionation of open column fraction 3was separated using the MPLC system, and it had aweight of 7mg. The optical rotation of the compoundwas measured in chloroform and gave [α] =+17°.

Biological test results

The results for the fractions obtained from MPLC areshown in Table 1. Most of the fractions contained mix-tures of diterpenes with fractions 9, 10 and 15 containingthe diterpenes 1–3 described earlier. None of thefractions had greater activity than the open columnfraction 1 itself, suggesting that there might be synergis-tic activity between the components within the fraction.Two of the diterpenes, (1) and (2), which elute in frac-tions 9 and 10, have activity equal to the unfractionatedmaterial. The results illustrate that relatively smallstructural changes can have an effect on activity withthe diterpene in fraction 15 being considerably lessactive than the compounds in fractions 9 and 10. Thus,the results point to a potential for carrying out semi-synthetic modifications of active compounds inorder to improve activity. In addition, sesamin, whichwas isolated from F3 by MPLC, gave moderateantitrypanosomal activity.

Antileishmanial activity

The MPLC fractions obtained were also tested againstL.donovani. The results are shown in TableT2 2, and itcan be seen that the IC50 values are somewhat variable,which can be attributed to the difficulty in getting thecompounds to dissolve in the aqueous test medium.

Overall, compound 3, which is in the most polar fractiontested, was the most consistent in terms of activity andthe most active. The fractions were all active, and over-all, their activity was greater in suppressing infection ofperitoneal macrophages than in inhibiting infection ofbone marrow macrophages. Most of the fractions wereactive at concentrations well below the levels at whichthe commonly used stibogluconate treatment is effective(Carter et al., 2001). The levels of DMSO used in thesamples were below the level where it had toxic effectson the leishmania.

DISCUSSION

The composition of the Libyan propolis LBA is typicalof Mediterranean propolis. Profiling of Greek propoliswas carried out by GC-MS (Popova et al., 2010) on sixsamples, three collected from the Greek mainland andthree collected in Crete. All of the samples were richin diterpenes with 37 diterpenes being characterised inthe propolis extracts. It was proposed that the com-pounds were collected from conifer species in theCupressaceae family. Some of these diterpenes, includ-ing compounds 1 and 3, were isolated from Cretan prop-olis (Popova et al., 2009) and tested against a range ofbacteria against which they were found to have moder-ate activity. Compounds 2 and 3 were isolated fromAraucaria heterophylla resin (Abdel-Sattar et al., 2009)and were tested against cancer cell lines. Compounds 2and 3 were found to be quite cytotoxic having IC50values of 2.7 and 9.8μg/mL against MCF7 breast cancercells. Thus, if these compounds were to be effective an-tiparasitic drugs, the therapeutic window might not bethat wide. Propolis proves to consistently have potentbiological activity, and the activity is often specific toparticular microorganisms. In the current case, earliertesting of the constituents of Cretan propolis, two ofwhich were found in the Libyan propolis sample,revealed only moderate activity against bacteria. In thecurrent case, the activity against protozoa is consider-ably higher. Thus, the propolis may reflect the particularenvironmental pressures that the bees are subject towithin the region in which the hive is sited. Propolisremains a fascinating substance, and if in vivo activitycould be demonstrated to match its in vitro activity, itis potentially a cheap readily available antibiotic.

Ackn Q6owledgements

We thank Gavin Bain for making the optical rotation measurementsand the Libyan Government for a scholarship for Weam Siheri.

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Query No. Query Remark

Q1 AUTHOR: Please check author name ‘Ruangelie Edrada Ebel’ if presented correctly.

Q2 AUTHOR: Figure 1 has not been mentioned in the text. Please cite the figure in therelevant place in the text.

Q3 AUTHOR: Please define DEPT.

Q4 AUTHOR: Please define FCS.

Q5 AUTHOR: Please define BLI.

Q6 AUTHOR: Please provide “conflict of interest statement” for this article.

Q7 AUTHOR: Please provide volume number for reference Salomão et al. (2011).

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

Required software to e-Annotate PDFs: Adobe Acrobat Professional or Adobe Reader (version 7.0 or above). (Note that this document uses screenshots from Adobe Reader X) The latest version of Acrobat Reader can be downloaded for free at: http://get.adobe.com/uk/reader/

Once you have Acrobat Reader open on your computer, click on the Comment tab at the right of the toolbar:

1. Replace (Ins) Tool – for replacing text.

Strikes a line through text and opens up a text box where replacement text can be entered.

How to use it

Highlight a word or sentence.

Click on the Replace (Ins) icon in the Annotations section.

Type the replacement text into the blue box that appears.

This will open up a panel down the right side of the document. The majority of tools you will use for annotating your proof will be in the Annotations section, pictured opposite. We’ve picked out some of these tools below:

2. Strikethrough (Del) Tool – for deleting text.

Strikes a red line through text that is to be deleted.

How to use it

Highlight a word or sentence.

Click on the Strikethrough (Del) icon in the Annotations section.

3. Add note to text Tool – for highlighting a section to be changed to bold or italic.

Highlights text in yellow and opens up a text box where comments can be entered.

How to use it

Highlight the relevant section of text.

Click on the Add note to text icon in the Annotations section.

Type instruction on what should be changed regarding the text into the yellow box that appears.

4. Add sticky note Tool – for making notes at specific points in the text.

Marks a point in the proof where a comment needs to be highlighted.

How to use it

Click on the Add sticky note icon in the Annotations section.

Click at the point in the proof where the comment should be inserted.

Type the comment into the yellow box that appears.

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

For further information on how to annotate proofs, click on the Help menu to reveal a list of further options:

5. Attach File Tool – for inserting large amounts of text or replacement figures.

Inserts an icon linking to the attached file in the appropriate pace in the text.

How to use it

Click on the Attach File icon in the Annotations section.

Click on the proof to where you’d like the attached file to be linked.

Select the file to be attached from your computer or network.

Select the colour and type of icon that will appear in the proof. Click OK.

6. Add stamp Tool – for approving a proof if no corrections are required.

Inserts a selected stamp onto an appropriate place in the proof.

How to use it

Click on the Add stamp icon in the Annotations section.

Select the stamp you want to use. (The Approved stamp is usually available directly in the menu that appears).

Click on the proof where you’d like the stamp to appear. (Where a proof is to be approved as it is, this would normally be on the first page).

7. Drawing Markups Tools – for drawing shapes, lines and freeform annotations on proofs and commenting on these marks.

Allows shapes, lines and freeform annotations to be drawn on proofs and for comment to be made on these marks..

How to use it

Click on one of the shapes in the Drawing Markups section.

Click on the proof at the relevant point and draw the selected shape with the cursor.

To add a comment to the drawn shape, move the cursor over the shape until an arrowhead appears.

Double click on the shape and type any text in the red box that appears.