Malignant diseases are responsible for the death of aboutone fifth of the population. The target of much research hasbeen on the discovery of natural and synthetic compoundsthat can be used in the prevention and/or treatment of cancer.Many plants and animal extracts have shown various biologi-cal activities like immunopotentiating and antitumour activi-ties. Honey bee propolis and its components (caffeic acid,caffeic acid phenethyl ester, artepilin C, quercetin, narin-genin, resveratrol, galangin, genistein and other) are of themost promising as antitumour agent.1—8)

Propolis (bee glue) is the generic name for the resinoussubstance collected by honey bees from various plant sourcesand used by bees to seal holes in their honeycombs, smoothout the internal walls, and protect the entrance against intrud-ers.1) It is rich in biochemical constituents, including mostlya mixture of polyphenols, flavonoid aglycones, phenolic acidand their esters, and phenolic aldehydes and ketones, ter-penes, sterols, vitamins, amino acids, etc.2) Healing proper-ties of propolis are known in folk medicine from antiquity.Recently, the interest for propolis as a harmless medicine isincreasing. There have been many attempts to validate bio-logical effects of propolis and elucidate its composition.3—8)

It was shown that propolis and its constituents have strongantimicrobial effect, acting on viruses,9—11) bacteria,12—14)

and fungi.15,16) It was also demonstrated that propolis andsome of its active substances have a pronounced cytostatic,anticarcinogenic and antitumour effect both in “in vitro” and“in vivo” tumor models.6—8,17—22) Immunomodulatory effectsof propolis has also been recorded.23—26) It has been sug-gested that the therapeutic activities of propolis dependmainly on the presence of flavonoids.28) Flavonoids have alsobeen reported to induce the immune system,29,30) and to actas strong oxygen radical scavengers. Dietary intake of an-tioxidant has been associated with a diminished risk of can-cer at various anatomical sites.31) This led us to compare howthe routes (oral and/or systemic) of administration of differ-

ent polyphenolic compounds deriving from propolis and ofpropolis itself influence on Ehrlich ascites tumor growth.Thus, we analyzed not only tumor growth but also parame-ters of immunomodulatory response of animals inoculatedwith test compounds, determining the effect of them on poly-morphonuclear (PMN) and mononuclear (MN) cells, mainlymacrophages.

MATERIALS AND METHODS

Water-Soluble Derivative of Propolis (WSDP) Treat-ment A water-soluble derivative of propolis (WSDP) wasprepared by the method described elsewhere.32) Briefly, Croa-tian propolis from beehives kept at the outskirts of Zagreb,Croatia or Brazilian propolis (CONAP, Belo Horizonte,Minas Gerais, Brazil) was extracted with 96% ethanol, whichwas filtered and evaporated to dryness in vacuum evaporator.The resultant resinous product was added to a stirred solutionof 8% L-lysine (Sigma Chemie, Deisenhofen, Germany) andfreeze-dried to yield WSDP, a yellow-brown powder. WSDPwas stored under sterile conditions at �20 °C. Before useWSDP was dissolved in distilled water. Nikolov et al.32)

demonstrated that WSDP contains: caffeic acid 6.7%, g ,g-di-methylallyl ferulate 1.2%, isopentyl-2-enyl-caffeate 7.4%,pentenyl caffeate 2.2%, g ,g-dimethylallyl caffeate 8.5%,pinobanksin 2.3%, pinocembrin 9.2%, pinobanksin-3-acetate13.6%, benzyl caffeate 0.4%, galangin 7.8%, b-phenyl ethylcaffeate 1.2%, total flavonoids 32.9%, total esters of phenylacid 20.9%.

WSDP was refrigerated under sterile conditions. Beforeuse WSDP was dissolved in distilled water and mice weregiven WSDP per os (p.o.) via gastral tube or intraperitoneally(i.p.). The WSDP was given daily for 7 d, and the daily dosecontained 50 mg kg�1 body weight.

Polyphenolic Compounds For experiments usingpolyphenolic compounds we used Caffeic acid (CA)–3,4-di-

∗ To whom correspondence should be addressed. e-mail: norsolic@yahoo.com © 2005 Pharmaceutical Society of Japan

Synergystic Antitumor Effect of Polyphenolic Components of WaterSoluble Derivative of Propolis against Ehrlich Ascites Tumour

Nada ORSOLIC,*,a Ivan KOSALEC,b and Ivan BASICa

a Department of Animal Physiology, Faculty of Science, University of Zagreb; Rooseveltov trg 6, HR-10 000 Zagreb,Croatia: and b Institute of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb; Schrottova 39, HR-10000 Zagreb, Croatia. Received October 14, 2004; accepted January 21, 2005

Effect of two preparation (Croatian and Brazilian) of water-soluble derivative of propolis (WSDP), caffeicacid, quercetin, chrysin, naringenin (components present in WSDP) on the development of Ehrlich ascites tu-mour (EAT) was evaluated. Test components (50 mg/kg) were given perorally or intraperitoneally 2 h prior theintraperitonel injection of EAT (2�106) cells. It was observed that all test compounds effectively inhibited tu-mour growth and the proliferation of EAT. The volume of ascitic fluid induced by EAT cells and total number ofcells present in the peritoneal cavity was markedly reduced in EAT-bearing mice treated with test components.In treated mice the number of polymorphonuclear (PMN) cells in the peritoneal cavity was increased while thenumber of macrophages was decreased. The macrophage spreading activity revealed that WSDP and all testcompounds affected the functional state of macrophages increasing their tumorcidal activity; the effect of WSDPwas most pronounced indicating synergistic effect of components present in WSDP. Antitumor activity of WSDPmay be the result of different specific mechanism(s) of flavonoids present as compared to individual flavonoidgiven alone. It is likely that the part of antitumor efficacy of test components against EAT cells was the results ofincreased activity of macrophages.

Key words propolis; polyphenolic compound; Ehrlich ascites tumour (EAT) tumor

694 Biol. Pharm. Bull. 28(4) 694—700 (2005) Vol. 28, No. 4

hydroxycinnamic acid (Aldrich-chemie, Milwaukee, WI,U.S.A.), Quercetin dihidydrate (QU) (Fluka, BioChemica,Switzerland), Chrysin and Naringenin (Sigma, Germany).

All polyphenolic compounds were dissolved in ethanoland further dilutions were made in water. The final concen-tration of ethanol was less than or equal to 0.1%. Ethanol(0.1%) was used in the control group. No difference betweenwater as control and 0.1% of ethanol in water was observedin preliminary experiments.

Mice Animal studies were carried out according to theguidelines in force in R. Croatia (Law on the Welfare of Ani-mals, N. N. # 19, 1999) and in compliance to the Guide forthe Care and Use of Laboratory Animals, DHHS Publ. # (NIH) 86—123. Male albino mice of the Swiss strains,weighing from 20—25 g from our conventional mousecolony were used. In any experiment, mice were of the samesex and were approximately 2 months old at the initiation ofeach study. The animals were kept not more then five to acage and were maintained on a pellet diet and water ad libi-tum. Experimental groups comprised of 7 or 15 mice each.

Tumor Cells of Ehrlich ascites tumor were used andmaintained in male Swiss albino mice through serial in-traperitoneal inoculation at 7- or 9-d intervals in an ascitisform. After harvesting and preparation of cells, their totalnumber and viability were determined by counting inBürker–Türk chamber using trypan blue dye. The desiredconcentration of tumor cells (2�106 cells per 0.5 ml) was ob-tained by dilution with saline (0.9% sodium chloride solu-tion). Viability of tumor cells was always higher than 90%.Below this percentage, the cells were discarded and the entireprocedure was repeated.

Count of the Total Number of Cells Present in the Peri-toneal Cavity The total number of cells present in the peri-toneal cavity was determined by counting in Bürker–Türkchamber.

Differential Count of the Cells Present in the PeritonealCavity The cells in the peritoneal cavity of mice were har-vested and stained with May Grünwald and water solution ofGiemsa (1 part Giemsa: 2 part water) and later differentiatedinto MN, PMN and tumor cells. To differentiate MN fromPMN cells, classic morphological patterns33) were used. Toidentify tumor cells, the paper by Fecchio34) was used asbasic reference. Differential cell counts were determined bycounting at least 800 cells in each sample. Wet cell mono-layer was photographed at �400 magnification with a phase-contrast microscope.

Determination of Functional Activity of MacrophagesFunctional activity of macrophages in the peritoneal cavitywas determined by the spreading technique adapted from Ra-binovitch et al.35) Thus, 103 cells in 0.1 ml of the cellular sus-pension obtained in the peritoneal cavity was placed overglass cover slips at room temperature for 15 min. The non-adherent cells were removed by washing with phosphatebuffer saline, and the adherent cells were incubated in culturemedium 199 containing 10 nM Hepes at 37 °C for 1 h. Fol-lowing this, the culture medium was removed and the cellswere fixed with 2.5% glutaraldehyde. Then, the cells werestained with a 5% solution of Giemsa and examined undermicroscopy where the percentage of spread cells were deter-mined under 40� magnification. Spread cells were those thatpresented cytoplasmic elongation, while the non-spread cells

were rounded,33—36) see Fig. 4. Wet adherent cells were pho-tographed at �400 magnification with a phase-contrast mi-croscope.

Experimental Procedure Seven groups of 15 mice wereformed for p.o. treatment and seven groups of 7 mice for i.p.treatment, as follows in Table 1.

Seven mice of each group were sacrificed in ether chamberon the 14th day after tumor cell inoculation. After desinfec-tion of external abdominal region, each animal was inocu-lated with 3 ml of saline solution and after gentle agitation ofabdominal well, the solution containing peritoneal cells wasremoved for cellular evaluation. The following variables wereanalyzed: the total number of cells, differential count of thecells present in the peritoneal cavity, and determination offunctional activity of macrophages. The remaining animals,i.e., 8 animals of each group for the survival analysis wereused.

Survival Analysis For the survival analysis CBA micewere given test components p.o. at doses of 50 mg kg�1 for7 d starting 2 h prior tumor inoculation. Endpoint of experi-ments was determined by spontaneous death of animals. Results are expressed as percent of mean survival time oftreated animals over mean survival time of the control group(treated vs. control, T/C %). The percentage of increasedlifespan (ILS%) was calculated according the formula:ILS%�(T�C)/C�100 where T represents mean survivaltime of treated animals; C represents mean survival time ofthe control group. By NCI criteria, T/C exceeding 125% andILS exceeding 25% indicate that the drug has significant an-titumour activity.37)

Statistics Results are expressed as means�S.D. obtainedfrom 2 experiments. Statistical significance was evaluatedusing the Student’s t test.

RESULTS

Determination of Total Number of Cells in the Peri-toneal Cavity The effect of Croatian and Brazilian WSDPand its polyphenolic compounds on volume of ascitic fluidinduced by EAT cells, total number of cells and cell viabilityin mice-bearing Ehrlich ascites tumour were examined bycounting total cells in peritoneal cavity in Bürker–Türkchamber using trypan blue. The volumes of ascites fluid andtotal number of EAT cells were significantly reduced in

April 2005 695

Table 1. Experimental Design

Groups

Tumor control (EAT)a) Tumor control (EAT)a)

Test componentsb) were given to mice Test componentsb) were given to mice orally: intraperitoneally:

EAT�50 mg kg�1 of Brazilian WSDP EAT�50 mg kg�1 of Brazilian WSDPEAT�50 mg kg�1 of Croatian WSDP EAT�50 mg kg�1 of Croatian WSDPEAT�50 mg kg�1 of caffeic acid EAT�50 mg kg�1 of caffeic acidEAT�50 mg kg�1 of chrysin EAT�50 mg kg�1 of chrysinEAT�50 mg kg�1 of naringenin EAT�50 mg kg�1 of naringeninEAT�50 mg kg�1 of quercetin EAT�50 mg kg�1 of quercetin

a) 2�106 tumor cells/mouse injected i.p.; groups comprised of 7 or 15 mice each.b) The test components were given daily for 7 d starting 2 h prior tumor inoculation,and the daily dose contained 50 mg kg�1 body weight.

mice-bearing AET protected with WSDP or its polyphenoliccompounds (Table 2, Fig. 1). Among six individual test com-ponents, Croatian WSDP was the most potent, inhibiting pro-liferation of EAT cell for 79%. Brazilian WSDP and narin-genin were intermediate in their inhibitory ability. Systemictreatment with all components (without Croatian WSDP andnaringenin) was more effective than perorally treatment in re-duction of total number of cells for 1—25%.

The effect of test components on the survival rate of mice-bearing EAT cells is shown in Table 3. The results demon-strate that ILS for WSDP was 27% or 32.4% in p.o. treatedgroup. Polyphenolyc compounds inhibited tumor growth andincreased life span (ILS) of mice for 7 to 27%. Survival rateof mice-bearing EAT after i.p. treatment with test compo-nents were not done.

Determination of the Percentage of the Mononuclear,Polymorfonuclear Cells and Tumour Cells in PeritonealCavity Figure 2 and Table 4 show the effects of two prepa-rations of WSDP or its polyphenolic compounds on the per-centage of MN, PMN and tumour cells. All tumour-bearinggroups treated with test components revealed significantlyhigher percentage of PMN and less percentage of MN cellscomparing to control. This effect was more pronounced inmice treated with test components intraperitoneally.

Determination of the Percentage of Macrophage Har-vested in the Peritoneal Cavity The results obtained for

the variable percentage of spreading of macrophages har-vested in peritoneal cavity are shown in Figs. 3 and 4. Thetreatment with test components yielded an increase in thefunctional activity of macrophages in mice bearing-EATcompared with control group. It should also be pointed outthat for the tumour-bearing group treated with test compo-nents, there was an increase in the median values of spread-ing percentage accompanying with an increase in the testcomponents cytotoxicity to EAT cells and in their effect oninduction of apoptosis in tumour cells (Fig. 2). Similar datawere given in mice treated i.p. with preparations of WSDPand its polyphenolic compounds.

DISCUSSION

The analysis of the total number of cells present in theperitoneal cavity revealed that all the experimental groups in-oculated with tumor cells in the presence of test components,exhibited a significant reduction of tumour size as well as thetotal number of cells in peritoneal cavity (Table 2, Fig. 1).Moreover, the survival rates of EAT-bearing mice were in-creased after treatment with test components (Table 3). Thesedata suggest that test components might interfere with thegrowth of Ehrlich ascites tumour cells directly during earlyphase of treatment leading to a considerable elimination ofthese cells. Treatment with test components yielded an in-crease in the percentage of PMN cells when compared with

696 Vol. 28, No. 4

Table 2. Effect of WSDP and Its Polyphenolic Compounds on Viability and Tumor Volume in Mice-Bearing Ehrlich Ascites Tumor after Perorally or In-traperitoneally Treatment

Test components were given p.o.a) Test components were given i.p.a)

ExperimentalVolume of Total number of Viability of Mortality Volume of Total number of Viability of Mortality

groupsb)

ascitic fluid cells (N�106) cells of cells ascitic fluid cells (N�106) cells of cells (ml) X�S.D. (%) (%) (ml) X�S.D. (%) (%)

Control 7.1 882.49�63.70 99.07 0.93 7.9 867.35�72.37 97.49 2.51Cr. WSDP 2.65 186.49�55.9* 94.43 5.57 2.85 167.25�52.7* 94.89 5.11Br. WSDP 4.75 285.82�87.13* 97.56 2.43 6 123.04�30.08* 88.71 11.29Caffeic acid 5.5 436.62�86.5* 98.34 1.66 4 270.93�40.93* 98.18 1.82Naringenin 4.05 233.625�56.9* 95.7 4.3 5 304.68�57.03* 97.49 2.51Chrysin 7.15 773.00�48.75 95.64 4.36 5.9 533.65�53.4* 99.57 0.43Quercetin 5.8 472.47�86.0* 96.21 3.79 8 289.45�87.3* 96.08 3.92

a) The test components were given p.o. or i.p. daily for 7 d starting 2 h prior tumor inoculation, and the daily dose contained 50 mg kg�1 body weight. b) Mice (n�7) ofeach groups were sacrrificed on the 14th day after i.p. tumor cell inoculation (2�106/tumor cells/mouse). The value was significantly different (∗ p�0.001) from the correspondingvalue of untreated animals analyzed by Student’s t test.

Table 3. Antitumor Activity WSDP and Its Polyphenolic Components onErlich Ascites Tumora)

Range of Mean ofGroupb) survival survival ILS%c) T/C%d) Effectivenesse)

time (d) time (d)

Control 11—14 12.5Croatian WSDP 12—26 15.8 32.36 132.36 �Brasilian WSDP 12—26 14.36 26.4 126.4 �Caffeic acid 12—17 14.75 14.75 114.75 �Naringenin 13—19 15.83 26.66 126.66 �Chrysin 12—17 16.54 14.88 114.88 �Quercetin 11—17 13.3 6.66 106.66 �

a) Tumor cells (2�106) were injected to mice i.p.; group comprises 8 mice each.b) The test components were given p.o. daily for 7 d starting 2 h prior tumor inocula-tion, and the daily dose contained 50 mg kg�1 body weight. c) ILS% (increased lifespan %)�(T�C)/C�100. T: mean survival days of treated group. C: mean survivaldays of control group. d) T/C, treated vs. control. e) �: ILS% �25; �: ILS%�25.

Fig. 1. The Total Number of Cells in the Peritoneal Cavity of Mice-Bear-ing Ehrlich Ascites Tumor after Perorally or Intraperitoneally Treatmentwith WSDP and Its Polyphenolic Compounds

The test components were given p.o. or i.p. daily for 7 d starting 2 h prior tumor inoc-ulation, and the daily dose contained 50 mg kg�1 body weight. The total number of cellsin the peritoneal cavity were determined on the 14th day after i.p. tumor cell inocula-tion (2�106/tumor cells/mouse). Group comprises 7 mice each. The data are expressedas the mean percentage of the the experiments of the untreated control.

April 2005 697

Table 4. Percentage of the Cells Present in the Peritoneal Cavity of the Animals on the 14th Day of Ehrlich Ascites Tumor Growth after Perorally or In-traperitonealy Treatment with WSDP and Its Polyphenolic Compounds

Test components were given p.o.a) Test components were given i.p.a)

Experimentalgroupb) Lymphocytes Neutrophils Macrophages Tumor cells Lymphocytes Neutrophils Macrophages Tumor cells

X�S.D. X�S.D. X�S.D. X�S.D. X�S.D. X�S.D. X�S.D. X�S.D.

Control 0.75�0.88 9.00�6.69 22.62�8.86 67.72�8.45 1.37�0.91 12.62�4.50 27.37�16.86 58.64�16.97Cr. WSDP 1.50�2.13 31.00�21.13 17.62�5.09 49.87�18.57 2.14�2.41 27.14�18.05 17.85�12.01* 52.85�26.72Br. WSDP 1.50�1.19 19.25�6.62** 18.12�11.51 61.12�13.18 1.25�1.58 47.75�13.87*** 15.25�8.74* 35.75�10.34Caffeic acid 1.40�1.78 23.00�16.30 20.25�14.63 54.25�12.03 1.37�1.84 38.50�8.81*** 14.00�6.88** 46.12�10.11Chrysin 0.50�1.21 18.50�17.56 33.25�26.20 47.5�29.13 2.25�2.31 22.75�4.62** 14.25�11.01* 60.75�10.29Naringenin 1.37�2.08 30.37�15.87** 19.00�4.40 49.25�13.87** 2.12�1.72 29.37�13.78* 17.12�6.81* 51.37�15.64Quercetin 2.00�2.72 32.62�15.04** 14.00�9.54** 51.37�10.91 1.75�2.86 34.00�12.40** 9.12�3.44** 55.37�13.09

a) The test components were given p.o. or i.p. daily for 7 d starting 2 h prior tumor inoculation, and the daily dose contained 50 mg kg�1 body weight. b) Mice (n�7) ofeach groups were sacrrificed on the 14th day after i.p. tumor cell inoculation (2�106/tumor cells/mouse). The value was significantly different (∗ p�0.05; ∗∗ p�0.01; ∗∗∗ p�0.001)from the corresponding value of untreated animals analyzed by Student’s t test.

Fig. 2. The Cells Obtained from Ascites Fluid in the Peritoneal Cavity of the Animals on the 14th Day of Ehrlich Ascites Tumor Growth after p.o. or i.p.Treatment with WSDP and Its Polyphenolic Compounds

The test components were given p.o. or i.p. daily for 7 d starting 2 h prior tumor inoculation, and the daily dose contained 50 mg kg�1 body weight. Cells were stained with MayGrünwald and water solution of Giemsa (1 part Giemsa: 2 part water) and later differentiated into MN, PMN and tumor cells. Treatment with WSDP and its polyphenolic com-pounds increased the fuctional activity of macrophages to tumor cells and induced the apoptotic process in them (a, b, e).

control group. According to Freire,38) PMN cells can lead tothe elimination of cells that are strange to the host by oxida-tive and non-oxidative mechanisms. However, in spite ofnoticing a quantitative increase of PMN cells in test compo-nent treated groups, a marked decrease in the percentage oftumour cells were not seen. This suggests that PMN cellsalone are not capable of inhibiting tumour growth. The num-ber of MN cells was decreased to the presence of test compo-nents. Most of the MN cells in the peritoneal cavity weremacrophages that are considered the major cells involved in tumour rejection.21,34,39) However, in spite of the non-occurrence of an increase in the number of MN cells, a significant increase was observed in the activity of peritonealmacrophages in test component-treated groups. It should alsobe pointed out that for the tumor-bearing group treated withtest components, there was an increase in the median valuesof spreading percentage accompanying the increase in thetest components cytotoxicity on EAT cells and in their effect

698 Vol. 28, No. 4

Fig. 3. The Percentage of Macrophage Spreading in the Peritoneal Cavityof the Animals on the 14th Day of Ehrlich Ascites Tumor Growth after Perorally Treatment with WSDP and Its Polyphenolic Compounds

The test components were given p.o. daily for 7 d starting 2 h prior tumor inoculation, and the daily dose contained 50 mg kg�1 body weight. Mice (n�7) of each groups were sacrrificed on the 14th day after i.p. tumor cell inoculation(2�106/tumor cells/mouse). The value was significantly different (∗ p�0.05;∗∗ p�0.01; ∗∗∗ p�0.001) from the corresponding value of untreated animals analyzedby Student’s t test.

Fig. 4. Macrophage Spreading Obtained from Ascites Fluid in the Peritoneal Cavity of the Animals on the 14th Day of Ehrlich Ascites Tumor Growthfrom Control Group (a, b) or after p.o. or i.p. treatment with WSDP and Its Polyphenolic Compounds (c—f)

The test components were given p.o. or i.p. daily for 7 d starting 2 h prior tumor inoculation, and the daily dose contained 50 mg kg�1 body weight. Macrophages were stainedwith a 5% solution of Giemsa.

on induction of apoptosis in tumor cells (Fig. 2). The in-crease of macrophage activity, possibly due to the test com-ponents, might have been responsible for the slower growthof tumour cells. It is well known that MN cells, mainlymacrophages, are the major cells involved in tumour rejec-tion. The variable macrophage spreading revealed that thetreatment with test components affects the functional state ofmacrophages. Figure 4 shows macrophage spreading with anincrease in size and content large cytoplasmic vacuoles. Thebest results were achieved with preparations of WSDP (Croa-tian and Brazilian). It is likely that the antitumor activity ofWSDP is the result of synergistic activities of its polypheno-lic compounds. Thus, the antitumor effect observed might bedue to an increase of peritoneal macrophage activity and notto an increase in macrophage number. Reason of the decreaseof the percentage of macrophages in test component-treatedgroups might be due to an increase of phagocytic activity ofmacrophages. The other possible mechanisms of antitumourinfluence of test components, as we described previously, in-clude immunomodulatory activity of these products30,40,41)

their cytotoxic activity to tumour cells,41,42) their capability ofinducing changes in the cellular level of glutathione,42) andtheir capability to induce apoptosis and/or necrosis.41) Thustest components may have direct and/or indirect action on tumour cells by stimulating the host cells, mainlymacrophages. Such stimulation might induce production andrelease of several cytokines such as IL-1, IL-6, IL-8, TNF-aand NO.21,25,26,41) Some of these cytokines have direct cyto-toxic effect on tumour cells while others act on other cellsand activate them: natural killer cells-NK and cytotoxic Tlymphocytes. In addition, these cytokines might stimulateproduction of C-reactive protein and complement factor C3that would act as opsonins on tumor cells.43) The combina-tion of these effects might impede tumour growth and lead toelimination of tumour cells. It is likely that for antitumor ef-fect close contact of test components (especially flavonoids)and tumor cells are more important for cytotoxic effect totumor cells and induction of apoptosis.41,44,45) Since the anti-tumor effect of WSDP in concentration of 50 mg/kg (mixtureof different flavonoids) inhibited proliferation of Ehrlich as-cites tumor cells better than individual compounds givenalone in the same concentration we presumed that combina-tion of flavonoids in WSDP could have synergistic effect. Itwas shown that the combination of different flavonoids addedto culture of human breast cell line (MDA-MB-435 cells)were more effective in inhibition of proliferation of tumorcells than individual flavonoid alone.18) In addition, when thecitrus flavonoids were combined individually with quercetin,they suppressed the proliferation of the cells at much lowerconcentration than either compound given alone. Further-more, if the combination of flavonoids was given to rats induced with mammary tumors by 7,12-dimethylbenz(a)an-thracene (DMBA) it was shown that combination of flavonoidswas the most effective at delaying the onset of tumors as wellas in reducing tumor burden comparing to the individualtreatments.18) Our observation is in agreement with thesefindings, implying that more different mechanisms are in-cluded in inhibition of proliferation of EAT cells with combi-nation of flavonoid (WSDP) treatments as compared tomechanism(s) involved with action of an individual com-pound. These findings and the results of the influence of

WSDP on formation of metastasis published recently41) sug-gest that flavonoids present in WSDP exert synergistic effectto the proliferation of tumor cells.

In conclusion, we have shown that WSDP and/or itspolyphenolic compounds are effective at reducing tumoursize as the total number of cells in peritoneal cavity in mice-bearing EAT. The increase in the animals’ survival time andthe important macrophage stimulation after treatment withtest components are significant results which certainly de-serve further studies. It is also important to emphasize thatp.o. route of administration influences the tumor growth, giv-ing priority to oral use of tested compounds in further inves-tigation either in experimental or clinical trials.

REFERENCES

1) Ghisalberti E., Bee World, 60, 59—84 (1979).2) Walker P., Crane E., Apidologie, 18, 327—334 (1987).3) Bankova V., Christov R., Stoev G., Popov S., J. Chromatogr., 607,

150—153 (1992).4) Bankova V., Christov R., Popov S., Z. Naturforsch. C, 49, 6—10

(1994).5) Marcucci M. C., Apidologie, 26, 83—99 (1995).6) Matsuno T., Matsumoto Y., Saito M., Morikawa J., Z. Naturforsch C,

52, 702—704 (1997).7) Kimoto T., Aga M., Hino K., Koya-Miyata S., Yamamoto Y., Micallef

M. J., Hanaya T., Arai S., Ikeda M., Kurimoto M., Anticancer Res., 21,221—228 (2001).

8) Akao Y., Maruyama H., Matsumoto K., Ohguchi K., Nishizawa K.,Sakamoto T., Araki Y., Mishima S., Nozawa Y., Biol. Pharm. Bull., 26,1057—1059 (2003).

9) Amoros M., Sauvager F., Girre L., Cormier M., Apidologie, 23, 231—245 (1992).

10) Debiaggi M., Tateo F., Pagani L., Luini M., Romero E., Microbiolog-ica, 13, 207—213 (1990).

11) Amoros M., Lurton E., Boustie J., Girre L., Sauvager F., Cormier M.,J. Nat. Prod., 57, 644—647 (1994).

12) Brumfitt W., Hamillton-Miller J. T. M., Franklin I., Microbios, 62,19—22 (1990).

13) Grange J. M., Davey R. W., J. R. Soc. Med., 83, 159—160 (1990).14) Focht J., Hansen S. H., Nielsen J. V., Arzneim-Forsch/Drug Res., 43,

921—924 (1993).15) La Torre A., Guccione M., Imbroglini G., Apicoltura, 6, 169—177

(1990).16) Lori G. A., Ind. Apic., 1, 38—43 (1990).17) Scheller S., Krol W., Swiacik J., Owczarek S., Gabrys J., Shani J., Z.

Naturforsch., 44c, 1063—1065 (1989).18) So F. V., Guthrie N., Chambers A. F., Moussa M., Carroll K. K., Nutr.

Cancer, 26, 167—181 (1996).19) Hayashi A., Gillen A. C., Lott J. R., Altern. Med. Rev., 5, 546—552

(2000).20) Chiao C., Carothers A. M., Grunberger D., Solomon G., Preston A. G.,

Barrett C. J., Cancer Res., 55, 3576—3583 (1995).21) Rao C. V., Desai D., Rivenson A., Simi B., Amin S., Reddy S. B., Can-

cer Res., 55, 2310—2315 (1995).22) Orsolic N., Basic I., J. Ethnopharmacol., 84, 265—273 (2003).23) Basic I., Orsolic N., Tadic Z., Macedo Fereire Alcici N., Brbot Sara-

novic A., Bendelja K., Krsnik B., Rabatic S., “17th International Can-cer Congress,” ed. by Moraes M., Brentani R., Bevilaqua R., Rio deJeneiro, Brazil, 1988, pp. 63—75.

24) Sun Z. J., Pan C. E., Liu H. S., Wang G. J., World J. Gastroenterol., 8,79—81 (2002).

25) Manolova N., Maximova V., Manolova Z., Stoilova I., Korczak E.,Denis A., Acta Microbiol. Bulg., 21, 76—81 (1987).

26) Dimov V., Ivanovska N., Bankova V., Popov S., Vaccine, 10, 817—823(1992).

27) Dimov V., Ivanovska N., Manolova N., Bankova V., Nikolov N., PopovS., Apidologie, 22, 155—162 (1991).

28) Havsteen B., Biochem. Pharmacol., 32, 1141—1148 (1983).29) Wleklik M., Zahorska R., Luczak V., Acta Microbiol. Pol., 32, 1141—

April 2005 699

1148 (1997).30) Orsolic N., Basic I., Mellifera, 3, 56—64 (2003).31) Moreno M., Isla M., Sampietro A., Vattuone M., J. Ethnopharmacol.,

71, 109—114 (2000).32) Nikolov N., Marekov N., Bankova V., Popov S., Ignatova R.,

Vladimirova I., Bul. Pat. Appl. 79903/28, 05, (1987).33) Bergman R. A., Afifi A. K., Heidger P. M., “Histology,” Saunders

Publishing, Philadelphia, Pa, 1995.34) Fecchio D., Sirois P., Russo M., Jancar S., Inflammation, 14, 125—132

(1990).35) Rabinovitch M., Manejias R. E., Russo M., Abbey E. E., Cell Im-

munol., 29, 86—95 (1977).36) Rabinovitch M., DeStefano M. J., Exp. Cell Res., 77, 323—334

(1973).37) Plowman J., Dykes D. J., Hollingshead M., Simpson-Herren L., Alley

M. C., “Anticancer Drug Development Guide: Preclinical Screening,Clinical Trials, and Approval,” ed. by Teicher B., Humana, Totowa, NJ,

1995, p. 101.38) Freire B. F. A., “O Neutrófilo: Morfologia, Cinética e Função,” Botu-

catu: Universidade Estadual Paulista, Faculdade de Medicina, SaoPaulo, 1994, p. 46 [Monografia].

39) Sezzi M. L., Bellelli L., Nista A., Oncology, 26, 529—539 (1972).40) Orsolic N., Sver L., Terzic S., Tadic Z., Basic I., Nutr. Cancer, 47,

156—163 (2003).41) Orsolic N., Horvat Knezevic A., Sver L., Terzic S., Basic I., J.

Ethnopharmacol., 94, 307—315 (2004).42) Orsolic N., Bendelja K., Brbot-Saranovic A., Basic I., Period Biol.,

106, 367—372 (2004).43) Bellelli L., Sezzi M. L., Oncology, 33, 215—218 (1976).44) Kimoto T., Arai S., Kohguchi M., Aga M., Nomura Y., Micallef M. J.,

Kurimoto M., Mito K., Cancer Detect. Prev., 22, 506—515 (1998).45) Csokay B., Prajda N., Weber G., Olah E., Life Sci., 60, 2157—2163

(1997).

700 Vol. 28, No. 4