Antiproliferative Activity of Greek Propolis

Harris Pratsinis,1 Dimitris Kletsas,1 Eleni Melliou,2 and Ioanna Chinou2

1Laboratory of Cell Proliferation & Ageing, Institute of Biology, NCSR ‘‘Demokritos’’; and 2Division of Pharmacognosyand Chemistry of Natural Products, Department of Pharmacy, University of Athens, Athens, Greece

ABSTRACT The butanolic extract and the isolated chemical constituents, mainly diterpenes and flavonoids, from Greek

propolis have been tested for their cytostatic activities against human malignant and normal cell strains. The extract and the

diterpenes were found to be the most active against HT-29 human colon adenocarcinoma cells, without affecting normal

human cells. Manool, a diterpene isolated for the first time from Greek propolis, was the most active compound, arresting the

cancer cells at the G2=M phase of the cell cycle.

KEY WORDS: � cancer cells � cell cycle analysis � cytotoxic activity � diterpenes � Greek flora � manool � propolis

INTRODUCTION

Propolis is a resinous natural material produced bybees. It contains sticky compounds coming from several

plants mixed with waxes and other bee excretions. The use ofpropolis in the traditional medicine is known since 3,000 BCin Egypt.1 The word propolis is derived from the Greek‘‘pro’’ (i.e., in front of, or at the entrance to) and ‘‘polis’’ (i.e.,the city or community)—a substance that is thought to beused in the beehive as a protective barrier against their en-emies. It is claimed to improve human health and preventdiseases such as inflammation, heart disease, diabetes, andcancer.2,3 For all these reasons, propolis is extensively usedin folk medicine,4 in cosmetology, and in the food industryfor health foods, beverages, and nutrition supplements.4–6 Inparticular, given the antimicrobial and antioxidant propertiesof propolis, it is gaining increasing interest for inclusion invarious dietary products as a natural preservative.7 Fur-thermore, its use as a nutritional supplement during cancertreatment has been proposed in the context of biologicaltherapy.8 Finally, propolis, as well as honey and royal jelly,fit well in the concept of functional foods, also known asnutraceuticals or medicinal foods.9

The chemical composition of propolis is highly dependedon the flora of the region from which it is collected. Incontrast to propolis of continental Europe, Greek propolishas a different botanical origin due to the unique flora ofGreece that has developed as a result of its geographicalposition. The special character of the nonvolatile constituents

of the Greek propolis (with diterpenes as its major constitu-ents), as well as of the volatile ones, has already been reportedin previous chemical studies.10,11 In the present study, wereport on the biological action of Greek propolis, and morespecifically on the antiproliferative activity of its butanolicextract, as well as nine isolated constituents.

MATERIALS AND METHODS

Material

The sample of propolis was collected at Northwest Greece(the Preveza region) in May 2004. The sample has beensubjected to pollen analysis, which gave a concentration of90% of pollen from Pinaceae, 2.5% from Asteraceae, and 2%from Ericaceae, Cistaceae, and Oleaceae, respectively. Avoucher sample (MEL03) has been deposited in the Herbar-ium of the Laboratory of Pharmacognosy-Chemistry of Nat-ural Products, School of Pharmacy, University of Athens,Athens, Greece.

Extraction and isolation of compounds

Four hundred grams of propolis was cut in small pieces andextracted for 48 hours with CH2Cl2 (3�2 L) and then with n-butanol (3�2 L). After evaporation of the solvent from then-butanol, the residue (4.2 g) was subjected to columnchromatography (4.0 cm) on silica gel 60 (130 g; particlesize, 40–6mm) (Merck, Darmstadt, Germany) with CH2Cl2:methanol (from 100:0 to 95:5 gradient) to afford 35 fractionsof 100 mL: A1–A20, A21–A26, A27–A30, and A31–A35.

Fractions A31–A35 were rechromatographed on mediumpressure liquid chromatography (3�23 cm) with cyclohexane:ethyl acetate (97:3 to 85:15) to afford 13-epi-cupressic acid(2), isocupressic acid (3), and agathadiol (1). Fractions

Manuscript received 17 March 2009. Revision accepted 18 May 2009.

Address correspondence to: Dr. D. Kletsas, Laboratory of Cell Proliferation & Ageing,Institute of Biology, NCSR "Demokritos," 153 10 Athens, Greece, E-mail: dkletsas@bio.demokritos.gr

JOURNAL OF MEDICINAL FOODJ Med Food 13 (2) 2010, 286–290# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2009.0071

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A16–A20 were rechromatographed on medium pressureliquid chromatography to afford 13-epi-torulosal (4) andisoagatholal (5), whereas fractions A1–A4 afforded 7-prenylstrobopinin (9), 7-prenylpinocembrin (8), pinostrobin(6), and manool (7).10 All fractions were analyzed by thin-layer chromatography (silica gel 60F254 [Merck]; CH2Cl2,detection vanillin=H2SO4 reagent). The structure of the iso-lated compounds was determined by spectroscopic methods.Optical rotations were measured with a Perkin-Elmer (Nor-walk, CT, USA) model 341 polarimeter. Ultraviolet spectrawere recorded on a Shimadzu-160A spectrophotometer(Shimadzu, Kyoto, Japan). Nuclear magnetic resonancespectra were recorded on Bruker (Karlsruhe, Germany) DRX400 and AC 200 spectrometers (1H [400 MHz] and 13C[50 MHz)].

Cell culture

The butanolic extract of propolis and the nine isolatedconstituents were tested for their cytotoxic activity againsttwo human solid tumor cell lines—HT-1080 fibrosarcomacells (American Type Culture Collection, Rockville, MD,USA) and HT-29 colorectal adenocarcinoma cells (Euro-pean Collection of Animal Cell Cultures [ECACC], Salisbury,UK)—and on the HL-60 human promyelocytic leukemiacell line (ECACC). Furthermore, the human skin fibroblaststrain AG01523c (Coriell Institute for Medical Research,Camden, NJ, USA) and two other fibroblast strains (DSF9and DSF60), developed in our laboratory from healthy 9-and 60-year-old donors, respectively, as previously de-scribed,12 were also used as normal controls. All cell strains,except HL-60, were routinely cultured in Dulbecco’s min-imal essential medium (Gibco� Invitrogen, Paisley, UK)supplemented with penicillin (100 U=mL), streptomycin(100 mg=mL), and 10% fetal bovine serum (media and an-tibiotics from Biochrom KG, Berlin, Germany) in an envi-ronment of 5% CO2, 85% humidity, and 378C, and theywere subcultured using a trypsin (0.25%)-EDTA (0.02%)solution. HL-60 cells were cultured in RPMI 1640 mediumcontaining antibiotics and 10% fetal bovine serum, as above.

Cytotoxicity assay

The cytotoxicity assay was performed by a modificationof the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method.13,14 In brief, the cells were platedat a density of approximately 5,000 cells per well in 96-wellflat-bottomed microplates, and after 24 hours the test com-pounds were added, appropriately diluted with dimethylsulfoxide. After a 72-hour incubation, the medium was re-placed with MTT (Sigma, St. Louis, MO, USA) dissolved ata final concentration of 1 mg=mL in serum-free, phenol-red-free Dulbecco’s mimimal essential medium for a further4-hour incubation. Then, the MTT formazan was solubilizedin 2-propanol, and the optical density was measured with amicroplate reader at a wavelength of 550 nm (referencewavelength, 690 nm). Daunorubicin hydrochloride was in-cluded in the experiments as a positive control. The resultsrepresent the mean of three independent experiments and are

expressed as IC50, i.e., the concentration that reduced by50% the optical density of treated cells with respect to un-treated controls.

Cell cycle analysis

Cell cycle analysis was performed following incubationof exponentially growing HT-29 cells with the test sub-stances (50mM) for 24 hours or 48 hours. Treated cultureswere then trypsinized, washed in phosphate-buffered saline,fixed in 50% ethanol, and stained with an RNase-containingpropidium iodide solution.15 DNA content was analyzed ona FACSCalibur� (BD Biosciences, San Jose, CA, USA)flow cytometer using ModFit software (Verity SoftwareHouse, Topsham, ME, USA).

RESULTS AND DISCUSSION

The butanolic extract of propolis was cytotoxic for thetwo malignant cell lines tested (IC50 values were 55.5�4.2mg=mL and 92.5� 1.9mg=mL for HT-1080 humanfibrosarcoma and HT-29 colon adenocarcinoma cells, re-spectively [Fig. 1 and Table 1]), whereas it was not as toxicwhen tested on normal human skin fibroblasts (IC50¼242.0� 15.5 mg=mL [Fig. 1 and Table 1]). The IC50 for HT-1080 cells is close to the one reported for the methanolicextract of Brazilian propolis using the same cell line, i.e.,67.3mg=mL,16 whereas the minimal toxicity against humanfibroblasts is in accordance with the results of Al-Shaheret al.17 for concentrations below 1 mg=mL of an ethanolicextract of Brazilian propolis in cultures of human fibroblastsof dental origin.

Subsequently, the cytotoxic activity of nine isolatedconstituents—agathodiol (1), 13-epi-cupressic acid (2),isocupressic acid (3), 13-epi-torulosal (4), isoagatholal (5),pinostrobin (6), manool (7), 7-prenylpinocembrin (8), and 7-prenylstrobopinin (9) (Fig. 2)—were tested on three human

FIG. 1. Cytotoxicity of the butanolic extract of Greek propolis. Thecytotoxic effects of the extract on human fibrosarcoma (HT-1080)cells, human colorectal adenocarcinoma (HT-29) cells, and normalhuman fibroblasts were determined by the MTT assay.

ANTIPROLIFERATIVE ACTIVITY OF GREEK PROPOLIS 287

malignant cell lines (the two previously used and the humanleukemia line HL-60), as well as human skin fibroblasts. Theresults are summarized in Table 1 (values are presented inmM, as well as in mg=mL, to facilitate the comparison withthe activity of the extract). Daunorubicin hydrochloride wasused as a positive control. In general, some of the isolatedconstituents exhibited higher cytotoxicity than the extract,especially against the human colorectal adenocarcinomacell line HT-29, whereas they were inactive against normalcells. Given the fact that propolis is edible, the cytotoxicityof its derivatives against cancer cells of gastrointestinalorigin is of special interest. In previous studies the cytotoxicactivity of propolis against colon cancer cells was mainlyattributed to caffeic acid esters,18 and indeed some syntheticcaffeic acid phenethyl ester analogues have been shown to

possess selective antiproliferative activity against murinecolon cancer cells.19 Nevertheless, we show here that otherconstituents of propolis are also contributing importantly toits activity against human colon adenocarcinoma cells. Inparticular, manool (7) shows the highest activity among thetested constituents (Table 1). It should be noted that manoolhas been previously identified through gas chromatography=mass spectrometry in Brazilian propolis20 and in propolisfrom various regions of Greece11; moreover, in the presentstudy it was isolated from Greek propolis for the first time,and its chemical structure has been determined by com-parison of its nuclear magnetic resonance, mass spectrom-etry, and optical rotation data with literature values.21 Thedifferent chemical character of the Greek propolis is alsonoteworthy: it appears to be a very rich source of diterpenesand not as the great majority of the previous studied Euro-pean propolis samples, which are characterized as Populus-type samples.

Table 2. Percentage Cell Cycle Phase Distribution

Cell cycle phase

Incubation period, compound G0=G1 S G2=M

24 hoursUntreated 54.22 27.17 18.614 53.20 26.86 19.945 55.28 25.68 19.047 53.53 29.14 17.33

48 hoursUntreated 66.35 22.68 10.984 69.21 17.85 12.945 67.41 14.36 18.237 61.28 14.14 24.58

Cell cycle phase distribution was assessed by fluorescence-activated cell

sorting analysis of propidium iodide-stained human colorectal adenocarci-

noma cells (HT-29). Results from one of two similar experiments are given.FIG. 2. Structures of the nine isolated constituents.

Table 1. Cytotoxicity (IC50) of Greek Propolis Constituents

Cytotoxicity for cell strain

HT-29 HT-1080 HL-60 Fibroblasts

mM mg=mL mM mg=mL mM mg=mL mM mg=mL

Butanolic extract Compound 92.5� 1.9 55.5� 4.2 242.0� 15.51 39.1� 1.7 12.0� 0.5 68.7� 2.6 21.0� 0.8 93.1� 9.9 28.5� 3.0 a

2 a a a a

3 100� 15.5 32.0� 5.0 a 90.8� 10.0 29.1� 3.2 a

4 31.1� 1.7 9.5� 0.5 a a a

5 28.7� 5.3 8.7� 1.6 81.9� 3.9 24.9� 1.2 51.2� 3.8 15.6� 1.2 a

6 a a a a

7 22.3� 0.9 6.5� 0.3 66.5� 7.7 19.3� 2.2 a a

8 92.9� 20.3 30.1� 6.6 a a a

9 a a a a

Daunorubicin.HCl 0.028� 0.018 0.022� 0.012 0.391� 0.186 0.255� 0.128

The cytotoxicity of the compounds against human colorectal adenocarcinoma (HT-29), fibrosarcoma (HT-1080), and leukemia (HL-60) cells and normal human

fibroblasts was determined by the MTT assay.a>100mM.

288 PRATSINIS ET AL.

Focusing on the three most active compounds—13-epi-torulosal (4), isoagatholal (5), and manool (7)—we haveperformed cell cycle analysis of HT-29 cells treated with theabove compounds at 50mM for 24 or 48 hours. The resultsare summarized in Table 2, and the effect of compound 7after a 48-hour treatment is illustrated in Figure 3.

It seems that all three compounds exert their cytostaticactivity after the first 24-hour period and that they reduce thepercentage of the malignant cells in the S phase, as expectedalso from their activity in the MTT assay. Furthermore,compounds 4, 5, and 7 all block the cells at the G2=M phase ofthe cell cycle, and, in particular, manool (7) increases ap-proximately 2.5-fold the percentage of cells at G2=M com-pared to the control. In a recent study of ethanolic extractsof Brazilian propolis it was found that their effects on cellcycle vary depending on the botanical origin of propolis;samples collected in southern Brazil tend to act as G2

blockers.22

In conclusion, the sample of Greek propolis we havestudied, as well as some of the isolated chemical com-pounds, presents a different chemical type of Europeanpropolis (closer to the Brazilian one), as all types of Bra-zilian propolis (either green or brown types) are very rich inditerpenic content.23,24 Besides, they have shown cytostaticactivity, especially against human colon cancer cells, con-firming the traditional reputation of propolis as an antitumornatural product.3 Furthermore, the diterpene manool—acompound isolated from Greek propolis and fully structur-ally determined for the first time—has exhibited a promisingprofile as an antiproliferative agent.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. S. Karabournioti (Directorof the Chemical and Analytical Laboratory of the ‘‘Attiki’’Bee-Culturing Company) for the pollen analysis of ourpropolis sample. This study was supported by the General

Secretariat of Research and Technology of Greece (projectPENED 2001), as well as by ‘‘Korres’’ Natural Products, S.A.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

REFERENCES

1. Castaldo S, Capasso F: Propolis, an old remedy used in modern

medicine. Fitoterapia 2002;73(Suppl 1):S1–S6.

2. Banskota AH, Tezuka Y, Kadota S: Recent progress in phar-

macological research of propolis. Phytother Res 2001;15:561–

571.

3. Burdock GA: Review of the biological properties and toxicity of

bee propolis (propolis). Food Chem Toxicol 1998;36:347–363.

4. Ghisalberti EL: Propolis—review. Bee World 1979;60:59–84.

5. Marcucci MC: Propolis—chemical-composition, biological

properties and therapeutic activity. Apidologie 1995;26:83–99.

6. Banskota AH, Tezuka Y, Adnyana IK, et al.: Hepatoprotective

and anti-Helicobacter pylori activities of constituents from Bra-

zilian propolis. Phytomedicine 2001;8:16–23.

7. Ozcan M: Inhibition of Aspergillus parasiticus NRRL 2999 by

pollen and propolis extracts. J Med Food 2004;7:114–116.

8. Galvao J, Abreu JA, Cruz T, et al.: Biological therapy using

propolis as nutritional supplement in cancer treatment. Int J Cancer

Res 2007;3:43–53.

9. Viuda-Martos M, Ruiz-Navajas Y, Fernandez-Lopez J, Perez-

Alvarez JA: Functional properties of honey, propolis, and royal

jelly. J Food Sci 2008;73:R117–R124.

10. Melliou E, Chinou I: Chemical analysis and antimicrobial ac-

tivity of Greek propolis. Planta Med 2004;70:515–519.

11. Melliou E, Stratis E, Chinou I: Volatile constituents of propolis

from various regions of Greece—antimicrobial activity. Food Chem

2007;103:375–380.

12. Pratsinis H, Tsagarakis S, Zervolea I, et al.: Chronic in vivo

exposure to glucocorticoids prolongs cellular lifespan: the case of

Cushing’s syndrome-patients’ fibroblasts. Exp Gerontol 2002;37:

1237–1245.

13. Denizot F, Lang R: Rapid colorimetric assay for cell growth and

survival. Modifications to the tetrazolium dye procedure giving

improved sensitivity and reliability. J Immunol Methods 1986;89:

271–277.

14. Harvala E, Aligiannis N, Skaltsounis AL, et al.: Cytotoxic ger-

macranolides from Inula verbascifolia subsp. methanea. J Nat

Prod 2002;65:1045–1048.

15. Kostakis IK, Tenta R, Pouli N, et al.: Design, synthesis, and an-

tiproliferative activity of some novel aminosubstituted xanthe-

nones, able to overcome multidrug resistance toward MES-SA=Dx5 cells. Bioorg Med Chem Lett 2005;15:5057–5060.

16. Banskota AH, Tezuka Y, Midorikawa K, Matsushige K, Kadota

S: Two novel cytotoxic benzofuran derivatives from Brazilian

propolis. J Nat Prod 2000;63:1277–1279.

17. Al-Shaher A, Wallace J, Agarwal S, Bretz W, Baugh D: Effect

of propolis on human fibroblasts from the pulp and periodontal

ligament. J Endod 2004;30:359–361.

18. Rao CV, Desai D, Kaul B, Amin S, Reddy BS: Effect of caffeic acid

esters on carcinogen-induced mutagenicity and human colon ade-

nocarcinoma cell growth. Chem Biol Interact 1992;84:277–290.

19. Nagaoka T, Banskota AH, Tezuka Y, Saiki I, Kadota S: Selective

antiproliferative activity of caffeic acid phenethyl ester analogues

FIG. 3. Cell cycle effects of (7), shown in the propidium iodide (PI)fluorescence histogram overlay of HT-29 cells treated with (7) for48 hours versus untreated control cells (Cell Quest Software, BDBiosciences).

ANTIPROLIFERATIVE ACTIVITY OF GREEK PROPOLIS 289

on highly liver-metastatic murine colon 26-L5 carcinoma cell

line. Bioorg Med Chem 2002;10:3351–3359.

20. Patricio EF, Cruz-Lopez L, Maile R, Tentschert J, Jones GR,

Morgan ED: The propolis of stingless bees: terpenes from the

tibia of three Frieseomelitta species. J Insect Physiol 2002;48:

249–254.

21. Barrero AF, Sanchez JF, Alvarez-Manzaneda EJ, Dorado MM,

Haidour A: Terpenoids and sterols from the wood of Abies

pinsapo. Phytochemistry 1993;32:1261–1265.

22. Li H, Kapur A, Yang JX, et al.: Antiproliferation of human

prostate cancer cells by ethanolic extracts of Brazilian propolis

and its botanical origin. Int J Oncol 2007;31:601–606.

23. Bankova V, Marcucci MC, Simova S, Nikolova N, Kujumgiev

A, Popov S: Antibacterial diterpenic acids from Brazilian prop-

olis. Z Naturforsch [C] 1996;51:277–280.

24. Midorikawa K, Banskota AH, Tezuka Y, et al.: Liquid

chromatography-mass spectrometry analysis of propolis. Phy-

tochem Anal 2001;12:366–373.

290 PRATSINIS ET AL.