Flavonoids and trypanocidal activity of Bulgarian propolis

Journal of Ethnopharmacology 88 (2003) 189–193

Flavonoids and trypanocidal activity of Bulgarian propolis Eliane Prytzyk a , Andreia P. Dantas b , Kelly Salomão b , Alberto S. Pereira a , Vassya S. Bankova c , Solange L. De Castro b,∗ , Francisco R. Aquino Neto a a

c

Ladetec, Instituto de Qu´ımica, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Cidade Universitária, CT Bl. A, 21949-900 Rio de Janeiro, RJ, Brazil b Laboratório de Biologia Celular, DUBC, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria Received 1 July 2002; received in revised form 1 May 2003; accepted 6 June 2003

Abstract Acetone and ethanol extracts of two Bulgarian propolis samples (Bur and Lov) were investigated by high temperature high resolution gas chromatography coupled to mass spectrometry (HT-HRGC-MS), and their activity against Trypanosoma cruzi was evaluated. The ethanol extracts—Et-Bur and Et-Lov—showed similar composition, with a high content of flavonoids, and strong inhibitory activity against T. cruzi proliferative epimastigotes, which were more susceptible than trypomastigotes. In the presence of blood, the activity of Et-Bur or Et-Lov against trypomastigotes was similar to that of the standard drug, crystal violet. Both extracts also showed similar and significant activity against Staphylococcus aureus and Candida albicans, while being inactive against Escherichia coli. The acetone extract, Ket-Bur, was more active than Et-Bur against both forms of T. cruzi. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Propolis; Flavonoids; High temperature gas chromatography; Trypanosoma cruzi; Antibacterial activity; Antifungal activity

1. Introduction Propolis is a resinous substance that honeybees collect from different plant exudates and use to fill gaps and to seal parts of the hive. At least 200 compounds were identified in different propolis samples, including fatty and phenolic acids and esters, substituted phenolic esters, flavonoids, terpenes, ␤-steroids, aromatic aldehydes and alcohols, sesquiterpenes, naphtalene and stilbene derivatives (Walker and Crane, 1987; Greenaway et al., 1991; Bankova et al., 2000). This resinous substance possesses many biological activities, such as antibacterial, antiviral, fungicidal, anti-inflammatory, and antitumor. In most reports, the biological or pharmacological activity was associated with phenolic compounds, mainly to flavonoids and aromatic acids and esters (Burdock, 1998; De Castro, 2001). Flavonoids are potent antioxidants, free radical scavengers and metal chelators: they inhibit lipid peroxidation and exhibit various physiological activities, including antihypertensive and anti-arthritic activities (Wang, 2000; Harborne and Williams, 2000). Methods for identification ∗ Corresponding

author. Tel.: +55-21-2603967; fax: +55-21-5906020. E-mail address: [email protected] (S.L.D. Castro).

of flavonoids are of interest because of the widespread occurrence of these compounds in different natural products. In this context, high temperature high resolution gas chromatography (HT-HRGC) and HT-HRGC-mass spectrometry (HT-HRGC-MS) are established techniques for separation of complex mixtures and identification of high molecular weight compounds, many of which do not elute when analyzed using ordinary HRGC columns. Both techniques are excellent alternatives to classical analytical phytochemistry and a potent tool for the rapid evaluation of the composition of crude natural products, including propolis (Pereira and Aquino Neto, 1999; Pereira et al., 1998, 1999). In the present study, we present the results of our analysis of different extracts from Bulgarian propolis by HT-HRGC-MS and bioassayed their activity against Trypanosoma cruzi, Staphylococcus aureus, Escherichia coli, and Candida albicans. 2. Materials and methods 2.1. Propolis samples Two samples of propolis were collected in 1999 at Burgas (Southeast Bulgaria) (Bur) and at Lovetech (West of

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Bulgaria) (Lov). Each sample (10 g) was cut in small pieces (after cooling at temperatures lower than −10 ◦ C) and extracted with 70% ethanol (1 g propolis in 10 ml of 70% ethanol), at room temperature. Within 24 h, the extract was filtered, and the exact concentration was calculated. An amount of 5 ml of the extract was evaporated to dryness using rotatory evaporator and weighted. This procedure leads to a minimum of waxes and a maximum of active substances (Bankova et al., 1999). The corresponding ethanol extracts were named Et-Bur and Et-Lov. A second methodology was used (Pereira et al., 1999) in which the 4 g powdered propolis from Burgas (Bur) was initially extracted with hexane (1:25, w/v) (Hex-Bur) and the extraction residue was further extracted with acetone at room temperature (Ket-Bur); and the second residue was extracted with methanol (Met-Bur), resulting in 2.1 g, 0.3 g, and 56 mg of crude extracts. This sequential fractionation was performed at room temperature, using an ultrasonic bath, Bransonic 72 (Branson, CA). 2.2. Derivatization and HRGC-MS analysis The trimethylsilyl (TMS) derivatives of the components of Et-Bur and Et-Lov were prepared using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Aldrich, St. Louis, MO). About 3 mg of the extracts were derivatized in 300 ␮l of BSTFA by heating for 60 min at 60 ◦ C. The samples were analyzed in a HP 5890-II gas chromatograph with a HP 5972 MSD detector (Hewlett-Packard, Palo Alto, CA) under electron impact ionization (70 eV). The interface was at 320 ◦ C and the MS scan range was 40–700 Da. The analysis was performed on a borosilicate capillary column (20 m × 0.30 mm i.d.) coated with 0.1 ␮m of PS-086 (15% phenyl–85% methylpolysiloxane) connected to a 2 m piece of 0.25 mm i.d. High Temperature Fused Silica (HTFS) (J and W, CA, USA) served as an interface. The HTFS was purged with hydrogen at 180 ◦ C for 15 min and deactivated by flushing with hexamethyldisilazane (HMDS)/1,3-diphenyl-1,1,3,3-tetramethyldisilazane (DPTMDS) 1:1 v/v (Sigma Chem. Co., MO), sealing the capillary and heating at 400 ◦ C for 12 h. The tubing was then rinsed with hexane, methanol, and diethyl ether. The samples were introduced by a cold on-column injector, with He as the carrier gas, linear velocity 38 m/s and sample volumes 0.2 ␮l. Oven temperatures were, for ethanol underivatized extract, 40 ◦ C (initial temperature), 40 ◦ C/min to 250 ◦ C, 12 ◦ C/min to 390 ◦ C, and a 20 min hold at 390 ◦ C; for acetone underivatized and ethanol derivatized extracts, 40 ◦ C (initial temperature), 15 ◦ C/min to 390 ◦ C and a 20 min hold at 390 ◦ C. 2.3. Characterization of compounds The compounds were characterized by mass spectral interpretation and by comparison with library databases, when

available. The interpretations about chemical fragmentations of flavonoids were based on literature data (Porter and Baldas, 1985; Creaser et al., 1991). Additionally, identification of several compounds was confirmed by analyses of standard compounds (e.g. chrysin, pinocembrin, etc.) by HT-HRGC-MS. Library searches were of relatively limited help in the case of the high molecular weight compounds, because their MS had not previously been recorded. 2.4. Antitrypanosomal activity Stock solutions of the different extracts were prepared in dimethylsulfoxide (DMSO). Experiments showed that in concentrations of up to 4%, DMSO had no deleterious effect on the parasites. The Y strain of T. cruzi was used (Silva and Nussenszweig, 1953). Trypomastigote forms were obtained at the peak of parasitemia from infected albino mice, isolated by differential centrifugation and resuspended with Dulbecco’s modified Eagle medium (DME) to a parasite concentration of 10 × 106 cells/ml in the absence or presence of 10% blood. This suspension (100 ␮l) was added to the same volume of the propolis extracts, previously prepared at twice the desired concentrations also in DME (0.025–4 mg/ml) in 96-well plates and then incubated at 4 ◦ C. Trypomastigote concentration in the wells was 5 × 106 cells/ml containing 0 or 5% blood. Untreated and crystal violet-treated parasites were used as controls (De Castro et al., 1994). We analyzed also the effect of a commercial North American propolis extract (Sigma, code #P1010) that we prepared in 70% ethanol (Et-Sg) and a Brazilian sample (Paraná State) extract with methanol (Met-Pr) (Marcucci et al., 2001). For the assays with epimastigote forms, the parasites were collected at exponential phase of culture in LIT medium (Camargo, 1964), resuspended in the same medium and plated in 24-well plates at the final concentration of 5 × 106 cells/ml, in the absence or presence of the extracts (0.025–4 mg/ml) at 28 ◦ C. Cell counts were performed after 24 h of incubation, and the activity of the extracts was expressed as ED50 values, corresponding to the concentration that causes 50% parasite lysis. 2.5. Antibacterial activity A modification of the bioauthography developed by Kujumgiev et al. (1993) was used to assay the effect of Bulgarian ethanol extracts against Staphylococcus aureus 209 and Escherichia coli WF+. The activity was measured as the diameter of the inhibitory zones in the soft agar layer stained after 72 h of incubation at 37 ◦ C with methylene blue (Doetsch, 1981). The inhibitory zone of 0.4 mg of each extract was measured. Diameter of less than 5 mm was considered as lack of activity, based on previous standardization procedures (Marcucci et al., 2001).

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acterized through their ions typically related to the fragmentation of TMS derivatives of carbohydrates, such as base peak ions at m/z 204 for hexoses and m/z 217 for pentoses. In the methanol extract, no flavonoids or other phenolic compounds were detected, because they were extracted previously with CH2 Cl2 and acetone. The apolar Hex-Bur extract showed the presence of n-alkanes, n-alcohols, and two series of wax acid esters. The results of the tests with the extracts against T. cruzi are presented in Table 2. Ket-Bur was the most active extract against epimastigotes, while Et-Bur and Et-Lov showed similar activities against each form of the parasite, being epimastigotes more susceptible than trypomastigotes. Differences in drug susceptibility have been already described between both forms of the parasite (Schlemper et al., 1977; De Castro et al., 1992, 1993). The differences found in the chemical composition between Et-Bur and Ket-Bur could not be consistently correlated with differences in trypanocidal activity, indicating that different flavonoids may have similar activity towards T. cruzi. The activity of each Bulgarian extract was differently affected by the presence of blood: for Burgas ethanol extract, no substantial interference was observed, while for Lovetech ethanol extract and Burgas acetone extracts a

2.6. Antifungal activity The agar cup method was used (Spooner and Sykes, 1972) to assay the effect of Bulgarian ethanol extracts against Candida albicans 562. The activity was measured as the diameter of the inhibitory zones, using 0.5 mg of each extract. Diameters of less than 10 mm were considered as lack of activity.

3. Results and discussion In Et-Bur, 27 compounds including several flavonoids were characterized and their relative concentration was estimated by using the mass spectrometry detector as if the response factor was equal to one. The ethanol extract obtained from the sample Lov presented a chemical composition similar to that of Et-Bur. Although the ethanol and acetone extracts share several constituents, Et-Bur is richer in fatty acids and flavonoids, while Ket-Bur shows higher levels of monossacharides, glycerol and caffeic acid (Table 1). Chromatograms and fragmentograms are available from the authors upon request. In Met-Bur, the main constituents were monosacchrides and disaccharides, char-

Table 1 Chemical characterization of the ethanol (Et-Bur) and acetone (Ket-Bur) extracts of the Bulgarian propolis sample collected in Burgas No.

Compound

tR a (min)

TMSb groups

Et-Burc area (%)

Ket-Burd area (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Benzoic acid Glycerol Fructose Myristic acid Glucose p-Coumaric acid Palmitoleic acid 3,4-Dimethoxy-cinnamic acid Palmitic acid Ferulic acid Isoferulic acid Caffeic acid Oleic acid Stearic acid Pentenyl ferrulate (isomer) Pentenyl ferrulate (isomer) Pentenyl caffeate (isomer) Pentenyl caffeate (isomer) Pinostrobin Pinocembrin Pinobanksin 3-etanoate Benzyl caffeate Chrysin Pinobanksin 3-butanoate Phenethyl caffeate Squalene Pinobanksin 3-pentanoate

7.11 7.43 12.17 12.26 12.84 12.97 13.48 13.56 13.64 13.90 14.0 14.30 14.74 14.89 15.65 15.72 15.89 15.96 16.46 16.77 17.46 17.65 17.73 17.85 18.07 18.12 18.38

1 3 5 1 5 1 1 1 1 1 1 3 1 1 2 2 2 2 2 2 2 2 1 2 2 0 2

0.14 0.92 0.36 0.65 0.62 0.60 0.87 0.37 2.46 0.40 0.30 2.61 2.33 0.66 0.97 0.79 0.74 1.41 0.40 9.44 11.23 1.64 2.22 9.85 2.10 4.41 2.75

Trace 5.00 10.03 0.95 1.22 0.41 0.24 –e 0.61 –e –e 6.87 0.24 Trace Trace Trace Trace Trace –e 3.60 3.85 0.72 3.85 1.36 0.43 –e 0.58

a

Retention time. Number of trimethylsilyl groups. c Burgas ethanol extract. d Burgas acetone extract. e Not detected. b

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Table 2 Trypanocidal activity of propolis extracts and reference substance expressed as ED50 /24 h in ␮g/ml Samples

Epimastigotes

Et-Bur Ket-Bur Met-Bur Et-Lov Et-Sg Met-Pr Crystal violet

80.6 ± 48.6 ± 6.1 ndb 84.8 ± 8.3 ndb ndb ndb

Trypomastigotes Without blood

a b

6.7a

143.0 108.8 1065.8 160.5 ndb ndb ndb

± ± ± ±

15.0 13.3 144.0 16.8

5% blood 187.4 254.8 1943.7 309.0 2649.0 1098.0 187.0

± ± ± ± ± ± ±

10.4 33.3 464.2 45.6 164.0 91.0 21.0

Mean ± standard deviation of at least four independent experiments. Not determined.

and allows the visualization, in the total ion chromatogram of the Burgas ethanol and acetone extracts, of unknown compounds with retention times up to 20 min at 340 ◦ C. Experiments are under way to compare the trypanocidal activity between Bulgarian and Brazilian samples. In temperate zones, flavonoids make up the main constituents of propolis; in Brazilian samples other classes of bioactive components have been described, such as prenylated acetophenones and specific terpenoids with bactericidal and cytotoxic activities (De Castro, 2001). Natural products rich in flavonoids need to be more thoroughly investigated to explore correlations between compounds or classes of compounds and their biological activity. This could lead to alternative drugs for the chemotherapy of Chagas disease (Coura and De Castro, 2002) and also of other tropical diseases.

Table 3 Antimicrobial activity of ethanol extracts of Bulgarian propolis samples Sample

Staphylococcus aureus

Escherichia coli

Candida albicans

Et-Bur Et-Lov

20.0 ± 1.0a 20.0 ± 1.0a

–b –b

13.7 ± 0.6 13.0 ± 0.6

a b

The inhibitory zone was expressed in mm. No inhibition.

decrease of about two times was observed in the lytic effect against trypomastigotes, when the assays were performed in the presence of blood. The activity of Burgas ethanol extract was similar to that of crystal violet, the standard drug, recommended to prevent transfusional transmission of Chagas disease in endemic areas (WHO, 1984). Inactivation of trypanocidal activity by blood components has been previously described in studies with naphthoquinones and gossypol (Lopes et al., 1978; Rovai et al., 1990; Pinto et al., 1997), preventing their use for blood chemoprophylaxis. Burgas methanol extract showed a very low activity against T. cruzi being 7–10 times less active than the corresponding ethanol and acetone extracts. We can tentatively correlate the lower activity of the methanol extract with the abundance of monosacchrides and disaccharides, therefore implying a lower concentration of possible active compounds. Burgas and Lovetech ethanol extracts showed significant activity against S. aureus and C. albicans (Table 3). On the other hand, they were inactive against the Gram-negative bacteria, E. coli, in accordance with literature data (reviewed by Marcucci, 1995).

4. Conclusions Differences in chemical composition are associated with variations in biological and pharmacological properties of propolis, exacerbating the need of simple, fast and powerful analytical tools, such as HT-HRGC-MS for their characterization. This technique was able to characterize sugars up to tetrasaccharides in Brazilian propolis (Pereira et al., 2000)

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