Water-soluble derivative of propolis and its polyphenolic compounds enhance tumoricidal activity of macrophages

Journal of Ethnopharmacology 102 (2005) 37–45

Water-soluble derivative of propolis and its polyphenolic compounds enhance tumoricidal activity of macrophages Nada Orˇsoli´c ∗ , Ivan Baˇsi´c Department of Animal Physiology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10 000 Zagreb, Croatia Received 23 September 2004; received in revised form 24 April 2005; accepted 20 May 2005 Available online 27 July 2005

Abstract Many plants and the plant-derived honeybee propolis have shown biological activities like immunomodulation and antitumor effect. The effect of two water-soluble propolis derivatives (WSDP) from Croatia and Brazil, caffeic acid, quercetin, chrysin and naringenin which are present in WSDP was assessed on the development of Ehrlich ascites tumor (EAT). The compounds (50 mg kg−1 ) were given by gastric intubations (po) 2 h prior to the intraperitoneal injection of EAT (2 × 106 ) cells. It was observed that WSDP and its compounds effectively inhibited tumor growth and proliferation of EAT. The volume of ascitic fluid induced by EAT cells and total number of cells present in the peritoneal cavity was markedly reduced in EAT-bearing mice treated with test components. Treatment with test components increased the number of polymorphonuclear (PMN) cells and decreased the number of macrophages in the peritoneal cavity of treated animals. The macrophage spreading activity revealed that WSDP and all test compounds affected the functional state of macrophages increasing their tumoricidal activity. The effect of WSDP was most pronounced suggesting synergistic effect of components present in WSDP. It is likely that part of the antitumor efficacy of the assayed components against EAT cells was the results of increased macrophage activity. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Propolis; Polyphenolic compounds; EAT tumor; Macrophages

1. Introduction Dietary factors play an important role in human health and in the development of certain chronic diseases including cancer (Doll, 1992; Rogers et al., 1993). Some foods contain antitumor compounds as well as mutagens and/or carcinogens (Ames and Gold, 1997). The target of much research has been on discovery of natural and synthetic compounds that can be used in the prevention and/or treatment of cancer. Many plants have been shown to produce various biological effects like immunomodulating and antitumor activities. Propolis (bee glue) is the generic name for the resinous substance collected by honeybees from various plant sources Abbreviations: CA, caffeic acid; EAT, Ehrlich ascites tumor; IL-1, interleukin 1; IL-6, interleukin 6; IL-8, interleukin 8; MN, mononuclerar cells; NO, nitric oxide; PMN, polymorphonuclear cells; QU, quercetin; TNF, tumor necrosis factor; WSDP, water-soluble derivative of propolis ∗ Corresponding author. Tel.: +385 1 4826 266; fax: +385 1 4826260. E-mail address: [email protected] (N. Orˇsoli´c). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.05.036

and used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance against intruders (Ghisalberti, 1979). It is rich in biochemical constituents, including mostly a mixture of polyphenols, flavonoid aglycones, phenolic acid and their esters, phenolic aldehydes and ketones, terpenes, sterols, vitamins, amino acids, etc. (Walker and Crane, 1987). Healing properties of propolis are known in folk medicine from antiquity. Recently, the interest for propolis as a harmless medicine is increasing. There have been many attempts to validate biological effects of propolis and elucidate its composition (Scheller and Stojko, 1977; Stojko and Scheller, 1987; Scheller et al., 1990; Bankova et al., 1992, 1994; Marcucci, 1995). It was shown that propolis and its constituents have strong antimicrobial effect, acting on viruses (Debiaggi et al., 1990; Amoros et al., 1992, 1994), bacteria (Brumfitt et al., 1990; Grange and Davey, 1990; Focht et al., 1993), and fungi (La Torre et al., 1990; Lori, 1990). It was also demonstrated that propolis and some of its active substances have a pronounced cytostatic,

38

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45

anticarcinogenic and antitumor effect both in “in vitro” and “in vivo” tumor models (Ban et al., 1983; Scheller et al., 1989; Chiao et al., 1995; Rao et al., 1995; Baˇsi´c et al., 1998; Orˇsoli´c and Baˇsi´c, 2003; Orˇsoli´c et al., 2004a,b). Immunomodulatory effects of propolis has also been recorded (Manolova et al., 1987; Neychev et al., 1988; Dimov et al., 1991, 1992; Orˇsoli´c and Baˇsi´c, 2003; Orˇsoli´c et al., 2004b). Our studies and those by others (Manolova et al., 1987; Neychev et al., 1988; Scheller et al., 1988; Baˇsi´c et al., 1998; Orˇsoli´c and Baˇsi´c, 2003) suggested that WSDP stimulated macrophages and influenced specific and non-specific immune defense mechanism, through the release of migration inhibitory factor, macrophage phagocytosis, elevation of number of rosetteforming and antibody producing cells, respectively. Activated macrophages were shown to be a major component of host defense against neoplastic growth in experimental tumor systems (Baˇsi´c et al., 1998; Orˇsoli´c and Baˇsi´c, 2003). It was also demonstrated that increased levels of IL-1 (Dimov et al., 1991) and TNF (Dimov et al., 1992) produced by activated macrophages correlated directly with two other criteria for macrophage activation: enhanced in vitro responsiveness to chemotactic stimuli (Meltzer and Oppenheim, 1977; Meltzer et al., 1975) and macrophage-induced tumor cytotoxicity (Monacada et al., 1991; Natan and Hibbs, 1991; Orˇsoli´c et al., 2004b). It has been suggested that the therapeutic activities of propolis depend mainly on the presence of flavonoids (Havsteen, 1983). Flavonoids have also been reported to induce the immune system (Wleklik et al., 1997; Orˇsoli´c and Baˇsi´c, 2003), and to act as strong oxygen radical scavengers. Dietary intake of antioxidant has been associated with a diminished risk of cancer at various anatomical sites (Moreno et al., 2000; Orˇsoli´c and Baˇsi´c, 2003). This led us to compare the effects of the treatment of animals bearing Ehrlich ascites tumor using WSDP and/or its polyphenolic compounds. Thus, we analyzed not only tumor growth but also parameters of immunomodulatory response of animals administered perorally with test compounds, determining the effect of them on polymorphonuclear (PMN) and mononuclear (MN) cells, mainly macrophages. Evaluation of the biological activities of ingredients in propolis and elucidation of the mechanism of their functions provide substantial clues for the development of new drug candidates.

to dryness under reduced presure. The resultant resinous product was added to a stirred solution of 8% l-lysine (Sigma Chemie, Deisenhofen, Germany) and freeze-dried to yield WSDP, a yellow-brown powder of Croatian or green powder of Brazilian propolis. Nikolov et al. (1987) demonstrated that European WSDP contains caffeic acid (6.7%), ␥,␥-dimethylallyl ferulate (1.2%), isopentyl-2-enylcaffeate (7.4%), pentenyl caffeate (2.2%), ␥,␥-dimethylallyl caffeate (8.5%), pinobanksin (2.3%), pinocembrin (9.2%), pinobanksin-3-acetate (13.6%), benzyl caffeate (0.4), galangin (7.8%), ␤-phenyl ethyl caffeate (1.2%), flavonoids (32.9%), esters of phenyl acid (20.9%). Park et al. (2002) reported that green Brazilian propolis in addition to the same components of the European samples, contains coumaric and ferulic acid, isosakuretin and kaempferide and the quantity of pinobanksin was much higher than that in European propolis. Recent analyses by high performance liquid chromatography (HPLC) from our laboratory have shown that our preparations of WSDP speciments from either Croatian or Brazilian propolis contained m/V total polyphenols 14.78% (caffeic acid 2.02%, naringenin 2.41%; chrysin 2.45%, pinocembrin 3.06%, galangin 2.12%) and 15.79% (caffeic acid 2.2%, naringenin 0.54%; chrysin 3.38%, pinocembrin 5.44%, galangin 3.07%), respectively (unpublished). WSDP was refrigerated under sterile conditions. Before use WSDP was dissolved in distilled water and mice were given WSDP per os (po) via gastral tube. The WSDP was given daily for 7 days, and the daily dose contained 50 mg kg−1 body weight. 2.2. Polyphenolic compounds The following polyphenolic compounds were used: caffeic acid (CA)—3,4-dihydroxycinnamic acid (Aldrich– Chemie, Milwaukee, WI, USA), quercetin dihidydrate (QU) (Fluka, BioChemica, Switzerland), chrysin and naringenin (Sigma, Germany). All polyphenolic compounds were dissolved in ethanol and further dilutions were made in water. The final concentration of ethanol was less than or equal to 0.1%. Ethanol (0.1%) was used in the control group. No difference between water as control and 0.1% of ethanol in water was observed in preliminary experiments. 2.3. Mice

2. Materials and methods 2.1. Water-soluble derivative of propolis (WSDP) treatment A water-soluble propolis formulation (WSDP) was prepared by the method described elsewhere (Nikolov et al., 1987). Briefly, Croatian (Cr) propolis from beehives kept at the outskirts of Zagreb, Croatia or Brazilian (Br) propolis (CONAP, Belo Horizonte, Minas Gerais, Brazil) was extracted with 96% ethanol, filtered and evaporated

Animal studies were carried out according to the guidelines in force in the Republic of Croatia (Law on the Welfare of Animals, N.N. #19, 1999) and in compliance to the Guide for the Care and Use of Laboratory Animals, DHHS Publ. #(NIH) 86–123. Male albino mice of the Swiss strain, weighing 20–25 g from our conventional mouse colony were used. In any experiment, mice were of the same sex and were approximately 2 months old at the initiation of each study. The animals were kept not more than five to a cage and were maintained on a pellet diet and water ad libitum. Experimental groups comprised 13–15 mice each.

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45

2.4. Tumor cells Cells of Ehrlich ascites tumor were used and maintained in male Swiss albino mice through serial intraperitoneal inoculation at 7- or 9-day intervals in an ascitis form. After harvesting and preparation of cells, their total number and viability were determined by counting in B¨urker–T¨urk chamber using trypan blue dye. The desired concentration of tumor cells (2 × 106 cells per 0.5 ml) was obtained by dilution with saline (0.9% sodium chloride solution). Viability of tumor cells was always higher than 90%. Below this percentage, the cells were discarded and the entire procedure was repeated. 2.5. Experimental procedure Seven groups of 15 mice were formed for po treatment. The test components (Croatian WSDP, Brazilian WSDP, caffeic acid, chrysin, naringenin, quercetin) were given daily for 7 days starting 2 h prior EAT inoculation (2 × 106 tumor cells/mouse), and the daily dose was 50 mg kg−1 body weight. Seven mice of each group were sacrificed in an ether chamber on the 14th day after tumor cell inoculation. After desinfection of the external abdominal region, each animal was inoculated with 3 ml of saline and after gentle massage of the abdominal wall, the solution containing peritoneal cells was removed for cellular evaluation. The following variables were analyzed: total cells number, differential cell count in the peritoneal cavity, and determination of functional activity of macrophages. The remaining animals of each group were used for the survival analysis.

39

lular suspension obtained in the peritoneal cavity was placed over glass cover slips at room temperature for 15 min. The non-adherent cells were removed by washing with phosphate buffered saline (PBS), and the adherent cells were incubated in culture medium 199 containing 10 nM Hepes at 37 ◦ C for 1 h. Following this, the culture medium was removed and the cells were fixed with 2.5% glutaraldehyde. Then, the cells were stained with a 5% solution of Giemsa and examined under microscope where the percentage of spread cells were determined under ×400 magnification. Spread cells were those that presented cytoplasmic elongation, while the nonspread cells were rounded (Bergman et al., 1995; Fecchio et al., 1990; Rabinovitch et al., 1977; Rabinovitch and DeStefano, 1973), see Fig. 4. Wet adherent cells were photographed at ×400 magnification with a phase-contrast microscope. 2.9. Survival analysis For the survival analysis Swiss mice were given test components po at doses of 50 mg kg−1 for 7 days starting 2 h prior tumor inoculation. Endpoint of experiments was determined by spontaneous death of animals. 2.10. Statistics Results are expressed as means ± S.D. obtained from two experiments. Statistical significance was evaluated using the Student’s t-test. Treatment-dose specific survival curves were calculated according to Kaplan–Meier method, and log–rank test was used to compare survival curves using statistical software STATA 7.0 (Stata Press, College station, TX, USA).

2.6. Total number of cells present in the peritoneal cavity 3. Results The total number of cells present in the peritoneal cavity was determined by counting in B¨urker–T¨urk chamber. 2.7. Differential count of the cells present in the peritoneal cavity The cells in the peritoneal cavity of mice were harvested and stained with May Gr¨unwald and water solution of Giemsa (1 part Giemsa:2 part water) and later differentiated into MN, PMN and tumor cells. To differentiate MN from PMN cells, classic morphological patterns (Bergman et al., 1995) were used. To identify tumor cells, the paper by Fecchio et al. (1990) was used as basic reference. Differential cell counts were determined by counting at least 800 cells in each sample. Wet cell monolayer was photographed at ×400 magnification with a phase-contrast microscope. 2.8. Determination of functional activity of macrophages Functional activity of macrophages in the peritoneal cavity was determined by the spreading technique adapted from Rabinovitch et al. (1977). Thus, 103 cells in 0.1 ml of the cel-

3.1. Determination of total number of cells in the peritoneal cavity The effect of Croatian and Brazilian WSDP and some of its reported polyphenolic compounds on tumor volume, total number of cells and cell viability in mice-bearing Ehrlich ascites tumor were examined by counting total cells in peritoneal cavity in B¨urker–T¨urk chamber using trypan blue. The volumes and total number of EAT cells were significantly reduced in mice-bearing AET protected with WSDP or its polyphenolic compounds (Table 1). Among six individual test components, Croatian WSDP was the most potent, inhibiting proliferation of EAT cell by 79%. Brazilian WSDP and naringenin were intermediate in their inhibitory ability. The effect of test components on the survival rate of micebearing EAT cells is shown in Fig. 1.The results demonstrate that Croatian WSDP, Brazilian WSDP, CA and Naringenin significantly prolonged life span of mice (p = 0.0002, 0.0028, 0.0068 and 0.0007, log–rank test) as compared to control. Chrysin and QU, however, were ineffective in this respect (p = 0.0576 and 0.384, log–rank test).

40

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45

Fig. 1. Kaplan–Meier survival curves for Swiss albino mice treated with Croatian WSDP (a), Brazilian WSDP (b) caffeic acid (c), naringenin (d), chrysin (e) and quercetin (f).

3.2. Percent determination of mononuclear, polymorfonuclear and tumor cells in the peritoneal cavity Figs. 2 and 3 shows the effects of two preparations of WSDP or its polyphenolic compounds on the percentage of MN, PMN and tumor cells. All tumors-bearing groups treated with test components revealed significantly higher percent-

age of PMN and lower percentage of MN cells compared to control. 3.3. Percent determination of macrophages harvested in the peritoneal cavity The results obtained for the variable percentage of spreading of macrophages harvested in peritoneal cavity are shown

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45

41

Table 1 Effect of oral treatment with WSDP and its polyphenolic compounds on viability and tumor volume in mice-bearing Ehrlich ascites tumor Experimental groups

Volume of ascitic fluid (ml)

Total cell number (N × 106 ) X ± S.D.

Control Croatian WSDP WSDP Brazilian WSDP Caffeic acid Naringenin Chrysin Quercetin

7.1 2.65 4.75 5.5 4.05 7.15 5.8

882.49 186.49 285.82 436.62 233.625 773.00 472.47

± ± ± ± ± ± ±

63.70 55.9* 87.13* 86.5* 56.9* 48.75 86.0*

Cell viability (%)

Cell mortality (%)

99.07 94.43 97.56 98.34 95.7 95.64 96.21

0.93 5.57 2.43 1.66 4.3 4.36 3.79

The value was significantly different (* p < 0.001) from the untreated animals analyzed by the Student’s t-test.

Fig. 2. Percentage of the cells present in the peritoneal cavity of the animals on the 14th day of Ehrlich ascites tumor growth after peroral treatment with Croatian and Brazilian WSDP and its polyphenolic compounds. The value was significantly different (* p < 0.01) from the corresponding value of untreated animals analyzed by the Student’s t-test.

in Table 2 and Fig. 4. The treatment with test components produced an increase in the functional activity of macrophages in mice-bearing EAT compared with control group. It should also be pointed out that for the tumor-bearing group treated with test components, there was an increase in the median values of spreading percentage accompanying with an increase in the test components cytotoxicity to EAT cells and in their effect on induction of apoptosis in tumor cells (Fig. 3).

Table 2 Percentage of macrophages spreading in the peritoneal cavity of the animals on the 14th day of Ehrlich ascites tumor growth after oral treatment with WSDP and its polyphenolic compounds Experimental goups

Macrophage spreading (%) X ± S.D.

Range (%)

Control Croatian WSDP WSDP Brazilian WSDP Caffeic acid Naringenin Quercetin Chrysin

18.32 ± 2.55 53.02 ± 15.41*** 51.64 ± 12.98*** 28.72 ± 6.33** 25.04 ± 4.64* 23.52 ± 3.74* 21.78 ± 1.86

15–19 39.67–78.4 31.04–65.98 20.35–39.07 20.50–33.70 20.38–28.40 20.38–24.5

The value was significantly different (* p < 0.05; ** <0.01; ** <0.001) from the untreated animals analyzed by Student’s t-test.

4. Discussion This research was performed to evaluate the effect of two water-soluble propolis preparations (WSDP, Croatian and Brazilian), caffeic acid, quercetin, chrysin, naringenin (components present in WSDP) on the tumoricidal activity of macrophages against Ehrlich ascites tumor (EAT). We have previously described immunomodulatory and antimetastatic effect of WSDP and related polyphenolic compounds (Orˇsoli´c and Baˇsi´c, 2003; Orˇsoli´c et al., 2004a,b). In this study the analysis of the total number of cells present in the peritoneal cavity revealed that all the experimental groups inoculated with tumor cells in the presence of test components, exhibited a significant reduction of tumor size as well as the total number of cells in peritoneal cavity (Table 1). Moreover, the survival rates of EAT-bearing mice were increased after treatment with test components (Fig. 1). It was shown that animals treated with the immunostimulants resist, in various degrees, subsequent inoculation of tumor cells as evidenced by the reduced “tumor take”, slowed growth of the tumors, and prolonged survival of recipients (Scheller et al., 1989; Matsuno, 1995; Kimoto et al., 1998; Hayashi et al., 2000). Scheller et al. (1989) reported that the ethanolic extract of propolis is capable of increasing survival of mice-bearing Ehrlich carcinoma and suggested

42

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45

Fig. 3. Effect of WSDP and its polyphenolic compounds on the functional activity of macrophages to tumor cells and apoptotic process. Cells obtained from ascites fluid on the 14th day of Ehrlich ascites tumor growth after po treatment with WSDP (a, c and f), CA (b and h), naringenin (d), chrysin (e) and QU (g).

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45

43

Fig. 4. Macrophage spreading obtained from ascites fluid on the 14th day of Ehrlich ascites tumor growth. Control group (a and b) and after po treatment with WSDP (c–f).

that immunostimulant activity of propolis may be associated with macrophage activation and enhancement of macrophage phagocytic capacity. Matsuno (1995) reported that various components of propolis have potent anti-inflammatory and antitumor activity. In addition Hayashi et al. (2000) showed that quercetin chalcone and modified citrus pectin reduced the growth of solid colon-25 primary tumor when given to mice. Kimoto et al. (1998) reported that artepilin C (component of propolis) has cytostatic and cytotoxic effect to various malignant tumor cells in vitro and in vivo and that it activates the immune system; especially increased the num-

ber of macrophages and their phagocytic activity as well as the number of lymphocytes, and possessed direct antitumor activity. This paper presents data showing that macrophages activation by propolis and related polyphenolic compounds is likely to be the most important effectors mechanism of antitumor activity of tested compounds in vivo. These data suggest that test components might interfere with the growth of Ehrlich ascites tumor cells directly during early phase of treatment leading to a considerable elimination of these cells. Treatment with test components elicited an increase in the percentage of PMN cells when compared

44

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45

with control group. According to Freire (1994), PMN cells can lead to the elimination of cells that are strange to the host by oxidative and non-oxidative mechanisms. However, in spite of noticing a quantitative increase of PMN cells in test component treated groups, a marked decrease in the percentage of tumor cells was not seen. This suggests that PMN cells alone are unable to inhibit tumor growth. The number of MN cells was decreased due to the presence of test components. Most of the MN cells in the peritoneal cavity were macrophages that are considered the major cells involved in tumor rejection (Sezzi et al., 1972; Fecchio et al., 1990; Orˇsoli´c and Baˇsi´c, 2003). 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 peritoneal macrophages in test component-treated groups. It should also be pointed out that for the tumor-bearing group treated with test components, there was an increase in the median values of spreading percentage accompanying the increase in the test components cytotoxicity on EAT cells and in their effect on induction of apoptosis in tumor cells (Fig. 3). The increase of macrophage activity, possibly due to the test components, might have been responsible for the slower growth of tumor cells. It is well known that MN cells, mainly macrophages, are the major cells involved in tumor rejection. The variable macrophage spreading revealed that treatment with test components affects the functional state of macrophages. Fig. 4 shows macrophage spreading with an increase in size and content large cytoplasmic vacuoles. The best results were achieved with preparations of WSDP (Croatian and Brazilian). It is likely that the antitumor activity of WSDP is the result of synergistic activities of its polyphenolic compounds. Thus, the antitumor effect observed might be due to an increase of peritoneal macrophage activity and not to an increase in macrophage number. Reason of decrease in the percentage of macrophages in test component-treated groups might be due to an increase of phagocytic activity of macrophages. The other possible mechanisms of antitumor influence of test components, as we described previously, include immunomodulatory activity of these products (Orˇsoli´c and Baˇsi´c, 2003; Orˇsoli´c et al., 2003, 2004b) their cytotoxic activity to tumor cells, their capability of inducing changes in the cellular level of glutathione, and their capability to induce apoptosis and/or necrosis (Orˇsoli´c et al., 2004a,b). Thus, test components may have direct and/or indirect action on tumor cells by stimulating the host cells, mainly macrophages. Such stimulation might induce production and release of several cytokines such as IL-1, IL-6, IL-8, TNF-␣ and NO (Dimov et al., 1991, 1992; Orˇsoli´c and Baˇsi´c, 2003; Orˇsoli´c et al., 2004b). Some of these cytokines have direct cytotoxic effect on tumor cells while others act on other cells and activate them: natural killer cells and cytotoxic T lymphocytes. In addition, these cytokines might stimulate production of C-reactive protein and complement factor C3 that would act as opsonins on tumor cells (Bellelli and Sezzi, 1976). The combination of these effects might impede tumor growth and lead to elimination of tumor cells.

In conclusion, we have shown that WSDP and/or its polyphenolic compounds are effective in reducing tumor size as the total number of cells in peritoneal cavity in micebearing EAT. The increase in the animals’ survival time and the important macrophage stimulation after treatment with test components are significant results, which certainly deserve further studies. Since mankind has used propolis from early times, a better understanding of its action as well as their polyphenolic compounds in the immune response will provide a scientific basis for the better therapeutic application in human or veterinary medicine whether it is associated or not with conventional treatments.

References Ames, B.N., Gold, L.S., 1997. Environmental pollution, pesticides, and the prevention of cancer: misconceptions. FASEB Journal 11, 1041–1052 (review). Amoros, M., Lurton, E., Boustie, J., Girre, L., Sauvager, F., Cormier, M., 1994. Comparison of the anti-herpex simplex virus activities of propolis and 3-methylbut-2-enyl caffeate. Journal of Natural Products 57, 644–647. Amoros, M., Sauvager, F., Girre, L., Cormier, M., 1992. In vitro anti viral activity of propolis. Apidologie 23, 231–245. ˇ Maysinger, D., 1983. Cytostatic effect of propolis Ban, J., Popovi´c, S., in vitro. Acta Pharmaceutica Jugoslavica 33, 245–255. Bankova, V., Christov, R., Popov, S., 1994. Volatile constituents of propolis Zeitschrift f¨ur Naturforschung Section C. Biosciences 49, 6–10. Bankova, V., Christov, R., Stoev, G., Popov, S., 1992. Determination of phenolics from propolis by capillary gas chromatography. Journal of Chromatography 607, 150–153. Baˇsi´c, I., Orˇsoli´c, N., Tadi´c, Z., Macedo Fereire Alcici, N., Brbot ˇ Saranovi´ c, A., Bendelja, K., Krsnik, B., Rabati´c, S., 1998. Antimetastatic effect of propolis, caffeic acid phenethyl ester and caffeic acid on mammary carcinoma of CBA mouse. In: Moraes, M., Brentani, R., Bevilaqua, R. (Eds.), 17th International Cancer Congress. Rio de Janeiro, Brazil, pp. 1, 63–75. Bellelli, L., Sezzi, M.L., 1976. C 3 participation in the rejection of some experimental tumors. Oncology 33, 215–218. Bergman, R.A., Afifi, A.K., Heidger, P.M., 1995. Histology. Saunders Publishing, Philadelphia, PA. Brumfitt, W., Hamillton – Miller, J.T.M., Franklin, I., 1990. Antibiotic activity of natural products: 1. Propolis Microbios 62, 19–22. Chiao, C., Carothers, A.M., Grunberger, D., Solomon, G., Preston, A.G., Barrett, C.J., 1995. Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. Cancer Research 55, 3576–3583. Debiaggi, M., Tateo, F., Pagani, L., Luini, M., Romero, E., 1990. Effects of propolis flavonoids on virus infectivity and replication. Microbiologica (Italy) 13, 207–213. Dimov, V., Ivanovska, N., Bankova, V., Popov, S., 1992. Immunodulatory action of propoolis IV. Prophylactic activity against gram-negative infections and adjuvant effect of the water-soluble derivate. Vaccine 10, 817–823. Dimov, V., Ivanovska, N., Manolova, N., Bankova, V., Nikolov, N., Popov, S., 1991. Immunodulatory action of propolis Influence on anti-infectious protection and macrophage function. Apidologie 22, 155–162. Doll, R., 1992. The lessons of life: keynote address to the nutrition and cancer conference. Cancer Research 52, 2024s–2029s (review). Fecchio, D., Sirois, P., Russo, M., Jancar, S., 1990. Studies on inflammatory response induced by Ehrlich tumor in mice peritoneal cavity. Inflammation 14, 125–132.

N. Orˇsoli´c, I. Baˇsi´c / Journal of Ethnopharmacology 102 (2005) 37–45 Focht, J., Hansen, S.H., Nielsen, J.V., 1993. Bactericidal effect of propolis in vitro against agents causing upper respiratory tract infections. Arzneimittel-Forschung/Drug Research 43, 921–924. Freire, B.F.A., 1994. O neutr´ofilo: morfologia, cin´etica e func¸a˜ o. Universidade Estadual Paulista, Faculdade de Medicina, Botucatu, 46 pp. (Monografia). Ghisalberti, E., 1979. Propolis: a review. Bee World 60, 59–84. Grange, J.M., Davey, R.W., 1990. Antibacterial properties of propolis (bee glue). Journal of the Royal Society of Medicine 83, 159–160. Havsteen, B., 1983. Flavonoids, a class of natural products of high pharmacological potency. Biochemical Pharmacology 32, 1141–1148. Hayashi, A., Gillen, A.C., Lott, J.R., 2000. Effects of daily oral administration of quercetin chalcone and modified citrus pectin on implanted colon-25 tumor growth in Balb-c mice. Alternative Medicin Review 5, 546–552. Kimoto, T., Arai, S., Kohguchi, M., Aga, M., Nomura, Y., Micallef, M.J., Kurimoto, M., Mito, K., 1998. Apoptosis and suppression of tumor growth by artepillin C extracted from Brazilian propolis. Cancer Detection and Prevention 22, 506–515. La Torre, A., Guccione, M., Imbroglini, G., 1990. Indagine preliminary sull’azione di preparati a base di propoli nei confronti di Botrytis cinerea Pers della fragola. Apicoltura 6, 169–177. Lori, G.A., 1990. Accion fungicida del propoleos en la dermatomicosis bovina. Indian Apiculture 1, 38–43. Manolova, N., Maximova, V., Manolova, Z., Stoilova, I., Korczak, E., Denis, A., 1987. Immunobiological effect of propolis. I. Effect on Cellular immunity. Acta Microbiologica Bulgarica 21, 76–81 (in Bulgarian). Marcucci, M.C., 1995. Propolis: chemical composition, biological properties and therapeutic activity. Apidologie 26, 83–99. Matsuno, T., 1995. A new clerodane diterpenoid isolated from propolis. Zeitschrift f¨ur Naturforschung Section C 50, 93–97. Meltzer, M.S., Oppenheim, J.J., 1977. Bidirectional amplification of macrophage-lymphocyte activation factor production by activated adherent mouse peritoneal cells. Journal of Immunology 118, 77–82. Meltzer, M.S., Tucker, R.W., Sanford, K.K., Leonard, E.J., 1975. Interaction of Bcg-activated macrophages with neoplastic and non-neoplastic cell line in vitro: quantitation of the cytotoxic reaction by release of tritiated thymidine from prelabeled target cells. Journal of the National Cancer Institute 45, 1177–1184. Monacada, S., Palmer, R.M.J., Higgs, E.A., 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews 43, 109–142. Moreno, M., Isla, M., Sampietro, A., Vattuone, M., 2000. Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. Journal of Ethnopharmacology 71, 109–114. Natan, C.F., Hibbs Jr., J.B., 1991. Role of nitric oxide synthesis in macrophage antimicrobial activity. Current Opinion in Immunology 3, 65–69. Neychev, H., Dimov, V., Vuleva, V., Shirova, L., Slacheva, E., Gegova, G., Manolova, N., Bankova, V., 1988. Immunomodulatory action of propols II. Effect of water-soluble fraction on influenza infection in mice. Acta Microbiologica Bulgarica 23, 58–62. Nikolov, N., Marekov, N., Bankova, V., Popov, S., Ignatova, R., Vladimirova, I., 1987. Method for the preparation of water-soluble

45

derivative of propolis. Bulgarian Journal of Pathology Applied 79903/28, 5. Orˇsoli´c, N., Baˇsi´c, I., 2003. Immunomodulation by water-soluble derivative of propolis (WSDP) a factor of antitumor reactivity. Journal of Ethnopharmacology 84, 265–273. ˇ Orˇsoli´c, N., Bendelja, K., Brbot–Saranovi´ c, A., Baˇsi´c, I., 2004a. Effect of caffeic acid phenethyl ester and caffeic acid, an antioxidant from propolis, on inducing apoptosis in HeLa human cervical carcinoma and Chinese hamster lung V79 fibroblast cells. Periodicum Biologorum 106, 367–372. ˇ Orˇsoli´c, N., Horvat Kneˇzevi´c, A., Sver, L., Terzi´c, S., Baˇsi´c, I., 2004b. Immunomodulatory and antimetastatic action of propolis and related poyphenolic compounds. Journal of Ethnopharmacology 94, 307– 315. ˇ Orˇsoli´c, N., Sver, L., Terzi´c, S., Tadi´c, Z., Baˇsi´c, I., 2003. Inhibitory effect of water-soluble derivative of propolis (WSDP) and its polyphenolic compounds on tumor growth and metastasing ability: a possible mode of antitumor action. Nutrition and Cancer 47, 156–163. Park, Y.K., Alencar, S.M., Agular, C.L., 2002. botanical origin and chemical composition of Brazilian propolis. Journal of Agicultural and Food Chemistry 50, 2502–2506. Rabinovitch, M., DeStefano, M.J., 1973. Macrophage spreading in vitro I. Inducers of spreading. Experimental Cell Research 77, 323–334. Rabinovitch, M., Manejias, R.E., Russo, M., Abbey, E.E., 1977. Increased spreading of macrophages from mice treated with interferon inducers. Cell Immunology 29, 86–95. Rao, C.V., Desai, D., Rivenson, A., Simi, B., Amin, S., Reddy, S.B., 1995. Chemoprevention of colon carcinogenesis by phenylethyl-3methylcaffeate. Cancer Research 55, 2310–2315. Rogers, A.E., Zeisel, S.H., Groopman, J., 1993. Diet and carcinogenesis. Carcinogenesis 14, 2205–2217 (review). Scheller, S., Gazda, G., Pietsz, G., Gabrys, J., Szumlas, J., Eckert, L., Shani, J., 1988. The ability of ethanol extract of propolis to stimulate plaque formation in immunized mouse spleen cells. Pharmacological Research Communications 20, 323–328. Scheller, S., Krol, W., Swiacik, J., Owezarek, S., Gabrys, J., Shani, J., 1989. Antitumoral property of ethanolic extract of propolis in micebearing Ehrlich carcinoma, as compared to bleomycin. Zeitschrift f¨ur Naturforschung Section C 44, 1049–1052. Scheller, S., Stojko, A., 1977. Biological properties and clinical application of propolis VI. Arzneimittel-Forschung/Drug Research 27, 2138–2140. Scheller, S., Wilczok, T., Imielski, S., Krol, W., Gabrys, J., Shani, J., 1990. Free radical scavenging by ethanol extract of propolis. International Journal of Radiation Biology 57, 461–465. Sezzi, M.L., Bellelli, L., Nista, A., 1972. Peritoneal macrophages and neoplastic cells I. Macrophages activity induced by heavily-irradiated or heat-killed cancer cells. Oncology 26, 529–539. Stojko, A., Scheller, S., 1987. Biological properties and clinical application of propolis VIII. Arzneimittel-Forschung/Drug Research 28, 35–37. Walker, P., Crane, E., 1987. Constituents of propolis. Apidologie 18, 327–334. Wleklik, M., Zahorska, R., Luczak, V., 1997. Interferon-inducing activity of flavonoids. Acta Microbiolica Poland 32, 1141–1148.