Effects of Propolis Crude Hydroalcoholic Extract on Chromosomal Aberrations Induced by Doxorubicin in Rats

• Abstract
• Introduction
• Materials and Methods
• Animals
• Chemicals
• PEE preparation
• Analyses of the green propolis sample by HPLC
• Experimental design
• Bone marrow chromosome aberration test
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

Propolis has been reported to display a broad spectrum of biological activities such as anticancer, antioxidant, anti-inflammatory, antibiotic and antifungal properties, among others. There is great interest not only in the determination of the chemical composition of propolis but also in the understanding of the mechanisms related to its therapeutic actions. In this respect, the aim of the present investigation was to study the influence of both simultaneous (6, 12 and 24 mg/kg b. w.) and subacute (12 mg/kg b. w.) treatment with a crude hydroalcoholic extract of propolis on the frequency of chromosome aberrations induced by the chemotherapeutic agent doxorubicin (DXR) in Wistar rat bone marrow cells. HPLC analysis of the crude extract allowed the quantification of the phenolic compounds: caffeic acid, p-coumaric acid, aromadendrin 4′-methyl ether, 3-prenyl-p-coumaric acid (drupanin), isosakuranetin, kaempferide, 3,5-diprenyl-p-coumaric acid (artepellin C), baccharin and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran. A total of 100 metaphase cells/animal were analyzed for chromosome aberration frequency and 1000 cells/animal were counted to obtain the mitotic index. The results showed that the dose of 12 mg propolis/kg b. w., administered either as a single dose or as subacute treatment, caused a statistically significant decrease in the frequency of chromosome damage induced by DXR compared to the group treated only with DXR. This reduction might be, in part, due to the presence of phenolic compounds in the studied propolis, which are able to capture free radicals produced by chemotherapeutic agents such as DXR.

Introduction

Disease prevention is the most effective strategy for the improvement of human health. There is currently increasing interest in the identification of chemopreventive agents that may block, reverse or prevent the development of cancer induced by environmental mutagens and/or carcinogens. Many classes of compounds, such as antioxidants, have shown a promising chemopreventive activity [1].

Propolis is a natural resinous material produced by honeybees from the gum of various plants. It possesses a highly complex and variable chemical composition, which is intimately related to the ecology of the flora of each region visited by the bees. The main botanical origin of Brazilian propolis is Baccharis dracunculifolia DC (Asteraceae), a shrub widely distributed in the States of São Paulo and Minas Gerais, Brazil [2]. At present, about 300 components, mainly phenolic compounds, have been identified. Most of these isolated compounds belong to three main groups, i. e., flavonoids, phenolic acids and its esters. Propolis and its components quercetin, luteolin, artepillin C, caffeic acid and caffeic acid phenethyl ester, among other compounds, are among the most promising antitumor agents that have been identified [3].

Propolis is widely recommended by herbalists due to its anti-inflammatory [4], hepatoprotective [5], antimicrobial [6], and antioxidant properties [7]. Therefore, propolis has been extensively used in food and beverages to improve health and to prevent several diseases.

The therapeutic effect of propolis on some human diseases claimed by folk medicine has raised the interest in understanding the role of its chemical compounds in the reported biological properties. Several studies have confirmed that the pharmacological properties of propolis are attributed mainly to the presence of flavonoids as a result of their action against free radicals [8], [9], [10]. These compounds interfere not only with the propagation but also with the formation of free radicals both by chelating transition metals and by inhibiting enzymes involved in the initiation reaction. Since the genotoxic activity of the chemotherapeutic agent DXR has been attributed to its ability to produce free radicals [11], the aim of the present investigation was to evaluate the influence of propolis ethanol extract (PEE) on doxorubicin (DXR)-induced genotoxicity in Wistar rat bone marrow cells.

Materials and Methods

Animals

Experiments were carried out on Wistar rats (Rattus norvegicus) obtained from the Animal House of the School of Pharmacy, University of São Paulo, Ribeirão Preto, SP, Brazil. The animals were kept in plastic boxes in an experimental room under controlled conditions of temperature (22 ± 2 °C) and humidity (50 ± 10 %), and under a 12 h light-dark cycle, with standard rat chow and water ad libitum. This study was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the US guidelines (NIH #85 - 23, revised in 1985). The study protocols were approved by the Ethics Committee for Animal Care of the University of Franca, Brazil.

Chemicals

DXR in ampoules containing 50 mg at 98 % of purity was purchased from Pharmacia Brasil Ltda. (São Paulo, Brazil). This compound dissolved in distilled water was used as an inducer of chromosome aberrations in rat bone marrow cells (90 mg/kg body weight, b. w.) [12]. Dimethyl sulfoxide (DMSO) and colchicine were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All other reagents and solvents used were of analytical grade.

PEE preparation

Green propolis extract was prepared from propolis in natura produced in the region of Oliveira (State of Minas Gerais, Brazil), a region rich in native Baccharis dracunculifolia. For this, 20 g of crude propolis were frozen and ground in a blender until a fine powder was obtained. The powder obtained (20 g) was then macerated in 100 mL of aqueous ethanol (7 : 3) solution for 48 h at room temperature. The extract was filtered and kept in a refrigerator for 24 h to allow the precipitation and separation of waxes. The solvent was removed under vacuum to provide the crude extract (12 g). For the assays, the crude extract was dissolved in a mixture of DMSO and sterile distilled water.

Analyses of the green propolis sample by HPLC

The quantitative chromatographic analysis of the green propolis extract was performed using a Shimadzu high performance liquid chromatograph (HPLC) equipped with controller SCL-10Avp, three pumps LC-10AD, detector diode-array model SPD-M10Avp and software controller Shimadzu Class-VP version 5.02. A Shim-Pack CLC-ODS (M), a Shimadzu column (4.6 mm × 250 mm, particle diameter of 5 µm, pore diameter of 100 Å) was used. The mobile phase consisted of a buffer solution in pump A (93.9 % water, 0.8 % acetic acid, 0.3 % ammonium acetate, 5 % methanol) and acetonitrile in pump B. The elution was undertaken using a linear gradient of 25 - 100 % B in 60 min at a flow-rate of 1.0 mL/min. Detection was performed at 280 nm, and veratraldehyde was used as internal standard [13].

The phenolic compounds were identified by comparison with the authentic chromatographic standards available at the compounds library of the Pharmacognosy Laboratory of the School of Pharmacy of Ribeirão Preto. The purity of each standard was estimated by both HPLC and ¹³C-NMR to be higher than 96 %.

Experimental design

Experiment 1: Simultaneous treatment with PEE and DXR: To investigate the influence of simultaneous treatment with PEE on the DXR-induced clastogenicity, the animals, weighing approximately 100 g, were divided into 10 treatment groups of six animals each (three males and three females) as shown in Table [1]. Preliminary studies were undertaken to select the doses of PEE (6, 12 and 24 mg/kg b. w.). The different PEE doses were administered to the animals by gavage in a volume of 1 mL/100 g b. w., followed by intraperitoneal (i. p.) injection of DXR (0.5 mL/100 g b. w.). The different doses of PEE were obtained from a stock solution of 2.4 mg/mL in 25 % DMSO in water. The solvent control group was treated by gavage (1 mL/100 g b. w.) with the same DMSO dose as the animals that received 24 mg PEE/kg b. w. Bone marrow samples were collected 24 h after the treatments.

Experiment 2: Subacute treatment with PEE prior to DXR treatment: The animals were divided into five groups of six animals each (three males and three females) as shown in Table [1]. The PEE dose (12 mg/kg b. w.) was selected on the basis of the results of Experiment 1. For subacute treatment with PEE, rats received the extract in drinking water ad libitum for a period of 10 days, and the PEE solution was replaced daily throughout the undertaken experiment. The dose of 12 mg PEE/kg b. w. was obtained from a stock solution of 0.05 mg/mL in media in 1 % DMSO in water. The solvent control group was treated with the same DMSO dose as the animals that received PEE. Animals of the negative control and DXR groups were given only tap water during the experiment. All animals had free access to standard rat chow, along with the PEE solution, DMSO solution or tap water. DXR (0.5 mL/100 g b. w., i. p.) was administered 24 h before sacrificing the animals (groups 4, 5 and 6) on day 11 of the experiment for the evaluation of chromosome aberrations in bone marrow cells. Body weight and PEE consumption were measured daily throughout the experimental period.

Bone marrow chromosome aberration test

All animals were injected i. p. with 0.5 mL of 0.16 % colchicine per 100 g b. w. 90 min before sacrificing. Bone marrow preparations for the analysis of chromosome aberrations in metaphase cells were obtained using the technique of Ford and Hamerton [14]. One hundred metaphases were analyzed per animal, for a total of 600 cells each for both treated and control groups, to determine the number of chromosome aberrations. The mitotic index (MI) corresponds to the number of metaphase cells in 1000 cells analyzed per animal. The MI is expressed as the mean of six animals.

Statistical analysis

The differences in the numbers of chromosome aberrations, abnormal metaphases, and MI between treatments were analyzed statistically by analysis of variance (ANOVA) for completely randomized experiments, with the calculation of the F statistics and its respective P values. In cases where P < 0.05, the values mean of each treatment were compared by the Tukey test, in which the calculation of the minimum significant difference for P is 0.05. Gaps were recorded but were not included in the statistical analysis since their cytogenetic significance has not been well established.

Results

The quantitative HPLC analysis [13] of the green propolis extract allowed the identification of the following phenolic compounds: (1) caffeic acid; (2) p-coumaric acid; (3) aromadendrin 4′-methyl ether; (4) 3-prenyl-p-coumaric acid (drupanin); (5) isosakuranetin; (6) kaempferide; (7) 3,5-diprenyl-p-coumaric acid (artepellin C); (8) baccharin and (9) 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran (Fig. [1]). It gave the following amounts of the identified compounds (g/100 g) in the crude propolis: 1 (1.50); 2 (0.44); 3 (0.78); 4 (0.85); 5 (0.65); 6 (1.1); 7 (12.70); 8 (0.38) and 9 (0.66). It should be taken into consideration that the hydroalcoholic crude extract yielded 60 %. Therefore, to calculate the amount of compounds in the crude extract, a factor of 1.66 should be used.

Table [2] shows the mean initial and final body weight, body weight gain and water intake during the experimental period. No significant differences in the parameters evaluated were observed between groups.

The results of the chromosome aberration analysis in bone marrow cells of Wistar rats simultaneously treated with different concentrations of PEE combined with DXR, as well as of rats submitted to subacute treatment with PEE before the administration of DXR, are shown in Tables [3] and [4], respectively. The groups treated with PEE showed a frequency of chromosome aberrations similar to that obtained for the DMSO group. The frequencies were slightly higher than those observed for untreated animals. However, these differences were not statistically significant (P > 0.05).

The dose of 12 mg PEE/kg b. w. combined with DXR, administered either as a single dose or as subacute treatment, led to similar frequencies of chromosome aberrations. These treatments resulted in a significant reduction of 34 % and 37 %, respectively, in DXR-induced chromosome aberrations compared to the group that only received DXR (Tables [3] and [4]). The data obtained for the groups simultaneously treated with 6 or 24 mg PEE/kg b. w. plus DXR did not show statistically significant differences when compared with the DXR group (Table [3]).

No significant differences in MI were observed between animals treated with different PEE concentrations and their respective negative controls, indicating that the different doses of PEE were not cytotoxic. The animals treated with DXR and those treated with combinations of PEE plus DXR presented a significantly lower MI compared to controls (Tables [3] and [4]).

Discussion

Since no antimutagenic effect was observed for the groups treated simultaneously with 6 or 24 mg PEE/kg b. w. plus DXR, which was only observed at the medium dose tested, experiments using subacute treatment with PEE prior to DXR treatment was performed in order to better characterize its protective effects on the investigated end-point. Moreover, propolis consumers usually take more than a single dose of propolis, which ranges from 0.3 to 1.5 g daily in tablet, extract or powder form [15]. The dose selected for subacute treatment (12 mg PPE/kg b. w.) is equivalent to a dose of 0.8 g for human consumption.

The present study demonstrated the dose of 12 mg PEE/kg b. w., administered either as a single dose or as subacute treatment, caused a statistically significant decrease in the frequency of chromosome damage induced by DXR. This antimutagenic effect might be due to the ability of PEE to scavenge free radicals, acting as an antioxidant agent, since the genotoxic activity of the chemotherapeutic agent DXR has been attributed to its ability to produce free radicals [11]. The chemical structure of DXR favors the generation of free radicals which can bind to iron and form complexes with DNA, inducing double-strand breaks [16].

The antioxidant activity of propolis and its ability to scavenge reactive oxygen species have been investigated by Simões et al. [17], who studied the biological effects of different extracts and fractions of green propolis. A correlation was observed between the antioxidant activity and chemical composition of its different fractions, with special emphasis on the presence of flavonoids and p-coumaric acid derivatives. The authors concluded that the components of propolis act through different mechanisms sequestering reactive oxygen species. Artepillin C (3,4-diprenyl-p-coumaric acid), a major constituent of green propolis, is also an excellent scavenger of free radicals similar to catechins [18].

Another possible mechanism of PEE in the reduction of chromosome damage caused by DXR might be its action on detoxifying enzymes. Siess et al. [19] reported that propolis and flavonoids enhanced the activities of cytochrome P450 and phase II enzymes. Nevertheless, the induction of P450 enzymes, which metabolize the mutagenic/carcinogenic compounds, usually inhibits the carcinogenic process in experimental animals, probably due to the detoxification mechanism, but has been reported to sometimes enhance carcinogenesis [20]. Therefore, the induction of P450 enzymes by PEE might not only contribute to its antimutagenic effect, but also to the increase in the frequency of chromosome aberrations observed for the groups treated simultaneously with either 6 or 24 mg PEE/kg b. w. plus DXR. Moreover, the increase of enzymes induced by PEE may have played an important role in free radical production induced by DXR in the present study. Thus, it is suggested that the lack of a dose-response relationship observed in simultaneously treatment could be attributed to the activation of different mechanisms depending on the PEE dose levels.

In this regard, in a study on Chinese hamster ovary cells, the results revealed that PEE shows the characteristics of a ”Janus” compound, i. e., PEE is genotoxic at higher concentrations, while at lower concentrations it exerts a chemopreventive effect on DXR-induced mutagenicity. Flavonoids have been suggested to be the components responsible for both the mutagenic and antimutagenic effects of PEE, since these compounds can act either as pro-oxidants or as free radical scavengers depending on their concentration [21].

The present study showed that the dose of 12 mg PEE/kg b. w. combined with DXR led to a similar reduction in DXR-induced clastogenicity when administered either as a single dose or as subacute treatment. The absence of a cumulative effect might be related to the bioavailability of flavonoids in propolis. Maximum concentrations of plasma flavonoids are normally reached within 1 - 2 h after ingestion [22]. For most flavonoids absorbed in the small intestine, the plasma concentration rapidly decreases (elimination half-life of 1 - 2 h). This fast excretion is facilitated by the conjugation of aglycone to sulfate and glucuronide groups [23]. The intestinal absorption of intact artepillin C in Wistar rats was found to be rapid, reaching a peak within 5 min after administration. Thus, maintenance of a high plasma concentration requires repeated intake of the polyphenols over time [24].

In conclusion, the current study showed that PEE significantly reduced DXR-induced chromosome aberrations in rats submitted to simultaneous or subacute treatment with 12 mg PEE/kg b. w., which might be, in part, related to the antioxidant capacity of its active compounds. It can be emphasized that the dose and duration of propolis treatment may be conditional determinants of its antimutagenic effect. Further investigations exploiting different target organs and protocols should be conducted in order to better characterize under what conditions propolis has protective activity, as well as to fully elucidate the mechanisms of action of its potential chemopreventive properties to permit the clinical application of propolis.

Acknowledgements

The authors are grateful to S. A. Neves for valuable technical assistance and J. P. B. Sousa for running the HPLC analysis. We are also thankful to Apis Flora Industrial and Commercial Co for providing propolis samples. This research was supported by FAPESP (Grant 01/12 825-7).

References

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Prof. Dr. Denise Crispim Tavares

Universidade de Franca

Avenida Dr. Armando Salles de Oliveira 201

Parque Universitário

14404-600 Franca

São Paulo

Brazil

Phone: +55-16-3711-8871

Fax: +55-16-3711-8878

Email: denisecrispim2001@yahoo.com

References

• 1 De Flora S, Izzotti A, D'Agostini F, Balansky R M, Noonan D, Albini A. Multiple points of intervention in the prevention of cancer and other mutation-related diseases.  Mutat Res. 2001;  480 - 481 9-22.
  PubMedSearch in Google Scholar
• 2 Park Y K, Paredes-Guzman J F, Aguiar C L, Alencar S M, Fujiwara F Y. Chemical constituents in Baccharis dracunculifolia as main botanical origin of southeastern Brazilian propolis.  J Agric Food Chem. 2004;  52 1100-3.
  CrossrefPubMedSearch in Google Scholar
• 3 Chowdhury S A, Kishino K, Satoh R, Hashimoto K, Kikuchi H, Nishikawa H. et al . Tumor-specificity and apoptosis-inducing activity of stilbenes and flavonoids.  Anticancer Res. 2006;  25 2055-63.
  PubMedSearch in Google Scholar
• 4 Reis C MF, Carvalho J CT, Caputo L RG, Patrício K CM, Barbosa M VJ, Chieff A L. et al . Atividade antiinflamatória, antiúlcera gástrica e toxicidade subcrônica do extrato etanólico de própolis.  Braz J Pharmacog. 2000;  10 43-52.
  PubMedSearch in Google Scholar
• 5 Liu C F, Lin C H, Lin C C, Lin Y H, Chen C F, Lin C K. et al . Antioxidative natural product protect against econazole-induced liver injuries.  Toxicology. 2004;  196 87-93.
  CrossrefPubMedSearch in Google Scholar
• 6 De Vecchi E, Drago L. Propolis antimicrobial activity: what's new?.  Infez Med. 2007;  17 7-15.
  PubMedSearch in Google Scholar
• 7 Nakajima Y, Shimazawa M, Mishima S, Hara H. Water extract of propolis and its main constituents, caffeoylquinic acid derivates, exert neuroprotective effects via antioxidant actions.  Life Sci. 2007;  80 370-7.
  CrossrefPubMedSearch in Google Scholar
• 8 Arvouet-Grand A, Vennat B, Pourrat A, Legret P. Standardization of propolis extract and identification of principal constituents.  J Pharm Belg. 1994;  49 462-8.
  PubMedSearch in Google Scholar
• 9 Mirzoeva O K, Calder P C. The effect of propolis and its components on eicosanoid production during the inflammatory response.  Prostaglandins Leukot Essent Fatty Acids. 1996;  55 441-9.
  PubMedSearch in Google Scholar
• 10 Vynograd N, Vynograd I, Sosnowski Z. A comparative multi-centre study of the efficacy of propolis, acyclovir and placebo in the treatment of genital herpes.  Phytomedicine. 2000;  7 1-6.
  CrossrefPubMedSearch in Google Scholar
• 11 Keizer H G, Pinedo H M, Schuurhuis G J, Joenje H. Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of citotoxicity.  Pharmacol Ther. 1990;  47 219-31.
  CrossrefPubMedSearch in Google Scholar
• 12 Tavares D C, Cecchi A O, Antunes L MG, Takahashi C S. Protective effects of the amino acid glutamine and of ascorbic acid against chromosomal damage induced by doxorubicin in mammalian cells.  Teratog Carcinog Mutagen. 1998;  18 153-61.
  CrossrefPubMedSearch in Google Scholar
• 13 Sousa J PB, Bueno P CP, Gregório L E, da Silva Filho A A, Furtado N AJC, Sousa M L. et al .A reliable quantitative method for the analysis of phenolic compounds in Brazilian propolis by reverse phase high performance liquid chromatography. J Sep Sci 2007, in press.
  Search in Google Scholar
• 14 Ford C E, Hamerton J L. A colchicine, hipotonic citrate, squash sequence for mammalian chromosomes.  Stain Technol. 1956;  31 247-51.
  PubMedSearch in Google Scholar
• 15 Shimizu K, Ashidashida H, Matsuura Y, Kanazawa K. Antioxidative biovailabilitity of artepillin C in Brazilian propolis.  Arch Biochem Biophys. 2004;  424 181-8.
  CrossrefPubMedSearch in Google Scholar
• 16 Eliot H, Gianni L, Myers C. Oxidative destruction of DNA by the adriamycin-iron complex.  Biochemistry. 1984;  23 928-36.
  CrossrefPubMedSearch in Google Scholar
• 17 Simões L MC, Gregório L E, Silva Filho A A, Souza M L, Azzolini A ECS, Bastos J K. et al . Effect of Brazilian green propolis on the production of reactive oxygen species by stimulated neutrophils.  J Ethnopharmacol. 2004;  94 59-65.
  CrossrefPubMedSearch in Google Scholar
• 18 Nakanishi I, Uto Y, Ohkubo K, Miyazaki K, Yakumaru H, Urano S. et al . Efficient radical scavenging of artepillin C, a major component of Brazilian propolis, and the mechanism.  Org Biomol Chem. 2003;  1 1452-4.
  CrossrefPubMedSearch in Google Scholar
• 19 Siess M H, Le Bon A M, Lavier M CC, Amiot M J, Sabatier S, Aubert S Y. et al . Flavonoids of honey and propolis: characterization and effects on hepatic drug-metabolizing enzymes and benzo[a]pyrene-DNA binding in rats.  J Agric Food Chem. 1996;  44 2297-301.
  CrossrefPubMedSearch in Google Scholar
• 20 Wislocki P G, Miller E C, McCoy J A, Rosenkranz H S. Carcinogenic and mutagenic activities of safrole, 1′-hydroxysafrole, and some know or possible metabolic.  Cancer Res. 1977;  37 1883-91.
  PubMedSearch in Google Scholar
• 21 Tavares D C, Barcelos G RM, Silva L F, Tonin C CC, Bastos J K. Propolis-induced genotoxicity and antigenotoxicity in Chinese hamster ovary cells.  Toxicol In Vitro. 2006;  20 1154-8.
  CrossrefPubMedSearch in Google Scholar
• 22 Aziz A A, Edwards C A, Lean M EJ, Crozier A. Absorption and excretion of conjugated flavonols, including quercetin 4′-O-β-glucoside and isorhammetin 4′-O-β-glucoside by human volunteers after the consumption of onions.  Free Radic Res. 1998;  29 257-69.
  CrossrefPubMedSearch in Google Scholar
• 23 Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols.  J Nutr. 2000;  130 2073S-85S.
  PubMedSearch in Google Scholar
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Prof. Dr. Denise Crispim Tavares

Universidade de Franca

Avenida Dr. Armando Salles de Oliveira 201

Parque Universitário

14404-600 Franca

São Paulo

Brazil

Phone: +55-16-3711-8871

Fax: +55-16-3711-8878

Email: denisecrispim2001@yahoo.com