Original Papers
Authors
Samuel Alday-Provencio 1, Gabriela Diaz 2, Lucila Rascon 1, Jael Quintero 1, Efrain Alday 1, Ramón Robles-Zepeda 1, Adriana Garibay-Escobar 1, Humberto Astiazaran 3, Javier Hernandez 4, Carlos Velazquez 1
Affiliations
The affiliations are listed at the end of the article
Key words " Giardia lamblia l " propolis l " seasonal effect l " chemical compounds l
Abstract
received revised accepted
Dec. 13, 2014 March 28, 2015 April 1, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545982 Published online Planta Med © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Carlos Velazquez University of Sonora Department of Chemistry-Biology Blvd. Luis Encinas y Rosales s/n. Hermosillo Sonora 83000 Mexico Phone: + 52 66 22 59 21 63 Fax: + 52 66 22 59 21 63 [email protected]
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Propolis is a cereus resin with a complex chemical composition that possesses a wide range of biological activities. The aim of this study was to evaluate the in vitro anti-Giardia lamblia activity of Sonoran propolis collected from three different areas of Sonoran Desert in northwestern Mexico (Caborca, Pueblo de Alamos, and Ures) and some of its chemical constituents. Additionally, we also analyzed the seasonal effect on the anti-G. lamblia activity of propolis. G. lamblia trophozoite cultures were treated with different concentrations of Sonoran propolis or chemical compounds during 48 h cell proliferation and cell viability were determined. Ures propolis showed the highest inhibitory activity against G. lamblia (IC50 63.8 ± 7.1 µg/mL) in a dose-dependent manner (Ures > Pueblo de Alamos > Caborca). Season had a significant effect on the in vitro anti-G. lamblia activity of Ures propolis. Summer propolis showed the highest inhibitory effect on the G. lamblia trophozoite growth (IC50 23.8 ± 2.3 µg/mL), followed by propolis collected during winter (IC50 59.2 ± 34.7 µg/mL), spring (IC50 102.5 ± 15.3 µg/mL), and autumn (IC50 125.0 ± 3.1 µg/mL). Caffeic acid phenethyl ester, an Ures propolis exclusive constituent, had the highest growth-inhibitory activity towards G. lamblia [IC50 63.1 ± 0.9 µg/mL (222.1 ±
Introduction !
Giardia lamblia (Lambl, 1859) (Kofoid and Christiansen, 1915) (syn. Giardia intestinalis, Giardia duodenalis) is a flagellated protozoan that inhabits the upper small intestine of humans and other mammals and is the causal agent of giardiasis [1]. It is one of the most frequent causes of intestinal disease worldwide [2]. This parasitosis has a clinical manifestation that varies from asymptomatic infection to acute or chronic infection, and is asso-
3.2 µM)]. To our knowledge, this is the first study showing that caffeic acid phenethyl ester possesses antiparasitic activity against G. lamblia. Naringenin [IC50 125.7 ± 20.7 µg/mL (461.8 ± 76.3 µM)], hesperetin [IC50 149.6 ± 24.8 µg/mL (494.9 ± 82.2 µM)], and pinocembrin [IC50 174.4 ± 26.0 µg/mL (680.6 ± 101.7 µM)] showed weak anti-G. lamblia activity. On the other hand, chrysin and rutin did not show significant antiparasitic activity. In conclusion, our results suggest that Sonoran propolis and some of its chemical constituents had inhibitory effects on the in vitro growth of G. lamblia trophozoites.
Abbreviations !
AP: CAPE: CP: PAP: SP: SuP: UP: WP:
autumn propolis caffeic acid phenethyl ester Caborca propolis Pueblo de Alamos propolis spring propolis summer propolis Ures propolis winter propolis
Supporting information available online at http://www.thieme-connect.de/products
ciated with diarrhea, flatulence, and abdominal pain, among other symptoms. G. lamblia infection is transmitted by contaminated water or food ingestion with G. lamblia cysts (infective form). Among drugs used to control this parasitic infection are the members of the imidazole family such as albendazole, metronidazole, and imidazole; 5nitrofuranes, acrydines, quinolones, and aminoglycosides are also used [3]. The high prevalence of giardia infection as well as the inadequate drug administration could favor the emergence of re-
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Sonoran Propolis and Some of its Chemical Constituents Inhibit in vitro Growth of Giardia lamblia Trophozoites
Original Papers
sistant Giardia strains [4, 5]. For this reason, the identification of potential new drug candidates for anti-G. lamblia is of paramount importance [3, 6]. Propolis is a resinous material collected by bees (Apis mellifera) from different plant exudates [7]. Over 300 chemical compounds have been identified in propolis and among them are aglycans flavonoids, phenolic acids and esters, p-coumaric prenylated acids, and ketophenones. Other identified compounds in propolis are lignans, di- and triterpenic acids, caffeoylquinic acids, monoterpens, sesquiterpens, isoflavonoids, chalcones, and pterocarpans [8–10]. Propolis has a wide spectrum of biological activities such as anti-inflammatory, antiproliferative, antimicrobial, antioxidant, and antiparasitic, among others [9–16]. Previously, we have shown that Sonoran propolis possesses a strong antiproliferative activity against different cancer cell lines [12, 17]. It has been demonstrated that the chemical composition of propolis can vary during the different seasons of the year, which could modify its biological activities [17–19]. There are limited data available about the antiparasitic activities of propolis. It has been reported that Brazilian propolis inhibited the growth of Trypanosoma cruzi [7, 13], Trichomonas vaginalis [20], and Leishmania amazonensis [20–22]. Furthermore, propolis has immunoregulatory properties on peripheral blood mononuclear cells from leishmaniasis patients [23]. The enhancing effect of propolis on immune response in an experimental mouse model of giardiasis, also resulting in the reduction of the parasite load, has been demonstrated [24]. Additionally, it has been reported that Brazilian propolis inhibited the in vitro proliferation of G. lamblia trophozoites [14]. The aim of the present study was to evaluate the antiparasitic activity of Sonoran propolis collected in three different geographic
Alday-Provencio S et al. Sonoran Propolis and …
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areas of Sonora, Mexico, and commercial samples of some of its chemical constituents as well as to analyze the seasonal effect on the anti-G. lamblia activity of Sonoran propolis collected at Ures.
Results !
In order to evaluate the antiparasitic activity of Sonoran propolis on G. lamblia trophozoites, we used several concentrations of propolis extracts (0, 25, 50, 100, and 200 µg/mL). UP showed the highest inhibition of G. lamblia trophozoite growth (IC50 63.8 ± 7.1 µg/mL), followed by PAP (IC50 222 ± 68.4 µg/mL). The CP extract did not show any significant effect on G. lamblia proliferation. The antiparasitic drug albendazole (10 µM) was used as a growth inhibition control, which inhibited more than 90% of the trophozoite proliferation. DMSO did not show any significant effect on proliferation or viability of G. lamblia trophozoites " Fig. 1). (l Microscopic analysis showed that UP induced evident morphological changes on G. lamblia trophozoites. Control G. lamblia cultures showed trophozoite monolayers attached to the surface " Fig. 2 A, B). tubes, showing slightly elongated trophozoites (l " Fig. 2 C) and UP After incubation (48 h) with albendazole (l " Fig. 2 D to G), the trophozoites were detached from the surface (l of the culture tubes, and some of them acquired a round and elongated form. Additionally, in cultures with detached trophozoites (60–80 %), we observed abundant cellular debris as well as swollen trophozoites with slow movements, mainly at the highest propolis concentration evaluated (200 µg/mL). PAP and CP extracts did not show evident morphological changes on trophozoites at the propolis concentrations tested (data not shown). Since UP showed the highest growth-inhibitory activity on G. lamblia trophozoites, we decided to investigate the seasonal effect on the antiparasitic activity of UP. We collected propolis during the different seasons of the year in the region of Ures, Sonora (see Materials and Methods). The collected seasonal samples of propolis (SP, SuP, AP, and WP) were evaluated at different concentrations (0, 25, 50, 100, and 200 µg/mL). All propolis extracts showed antiparasitic activity. The SuP extract showed the highest anti-G. lamblia activity (IC50 23.8 ± 2.3 µg/mL), followed by the WP extract (IC50 59.2 ± 34.7 µg/mL), SP (IC50 102.5 ± 15.3 µg/mL), and AP (IC50 125.0 ± 3.1 µg/mL). DMSO did not affect G. lamblia " Fig. 3). trophozoite growth (l In order to understand more about the antiparasitic activity of UP, we evaluated the growth-inhibitory effect of some of the main chemical constituents of Sonoran propolis on G. lamblia trophozoites. Previously, we identified phenolic compounds, mostly flavonoids, as the main constituents of Sonoran propolis " Table 1) [12]. Therefore, the antiparasitic (UP, PAP, and CP) (l effect of six chemical constituents of Sonoran propolis (CAPE, naringenin, hesperetin, pinocembrin, chrysin, and rutin) was evaluated on G. lamblia trophozoites. CAPE was the chemical constituent that showed the highest anti-G. lamblia activity [IC50 63.1 ± 0.9 µg/mL (222.1 ± 3.2 µM)], followed by naringenin [IC50 125.7 ± 20.7 µg/mL (461.8 ± 76.3 µM)]. Hesperetin [IC50 149.6 ± 24.8 µg/mL (494.9 ± 82.2 µM)] and pinocembrin [IC50 174.4 ± 26.0 µg/mL (680.6 ± 101.7 µM)] had weak inhibitory activity. In contrast, chrysin and rutin did not show antiparasitic ac" Fig. 4). tivity at the evaluated concentrations (l
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Fig. 1 Antiparasitic activity of Sonoran propolis. The antiparasitic activity of the Sonoran propolis was evaluated on the in vitro growth of G. lamblia trophozoites at different concentrations (0, 25, 50, 100, and 200 µg/mL). Propolis tested: A UP, B PAP, C CP. Albendazole was used as a growth inhibition control, which inhibited more than 90 % (IC90 10 µM) of the trophozoite proliferation. The tests were performed in triplicate (± SD) and at least two experiments in each condition were done. The antiparasitic activity was evaluated at 48 h after the addition of propolis extract. The asterisks on the bars show significant differences (p < 0.05) with respect to the control culture.
Original Papers
Discussion
Fig. 2 Morphological changes induced by UP on G. lamblia trophozoites. In order to observe the morphological changes induced by UP on G. lamblia trophozoites, photographs were taken in an inverted optical microscope (100X, total magnification). A Cells in monolayer, B control DMSO (0.06 %), C albendazole (IC90 10 µM), D UP (200 µg/mL), E UP (100 µg/ mL), F UP (50 µg/mL), and G UP (25 µg/mL).
In this study, we evaluated the antiparasitic activity of Sonoran propolis collected from three different areas of Sonora, Mexico, and some of its chemical constituents. Additionally, we analyzed the seasonal effect on the growth-inhibitory activity of Sonoran propolis on G. lamblia trophozoites. Our data showed that UP had the highest growth-inhibitory activity on G. lamblia trophozoites, followed by PAP with moderate activity, and finally CP, which did not show any anti-G. lamblia activity at the tested concentrations. The difference in the antiparasitic activity of Sonoran propolis with a dissimilar geographical origin can be explained by qualitative and quantitative differences in the chemical composition of propolis [25–30]. The antiparasitic activity of propolis has been determined in different parasites, including T. cruzi, L. amazonensis, T. vaginalis, and G. lamblia [7, 13, 14, 20–22]. Specifically on G. lamblia (BTU10 strain recovered from an asymptomatic host), Brazilian propolis showed antiparasitic activity at 125 µg/mL (IC50) [14]. Interestingly, our data showed that UP had a higher anti-G. lamblia activity (GS/M‑83-H7 strain, ATCC 50 581) than Brazilian propolis [twice effective (IC50 63.8 ± 7.1 µg/mL)]. These differences could be due to the propolis chemical composition. Brazilian propolis is mainly constituted by phenolic compounds (flavonoids, aromatic acids, benzopyranes), di- and triterpenes, and essential oils, among others, where artepillin C is its major constituent [14, 31], while UP constitution is mainly comprised by flavonoids, phenolic acids and its esters, with pinocembrin being its major constituent [12, 17]. In addition, it is important to notice that different G. lamblia strains were evaluated against Brazilian and UP propolis; thus, the differences found in the anti-Giardia activity of propolis could be due to the susceptibility of the G. lamblia strains used in both studies. We observed that season had an important effect on the anti-G. lamblia activity of UP. The propolis collected in summer (SuP) showed the highest parasite growth-inhibitory activity, followed by WP > SP > AP. Despite the observed differences on the biological activity, season did not have a significant effect on the chemical profile of UP (Table 1S, Supporting Information) [17]. The dif-
Fig. 3 Seasonal effect on the growth-inhibitory activity of propolis on G. lamblia trophozoites. The seasonal effect was evaluated by using extracts of propolis collected during different seasons of the year. Propolis was evaluated at different concentrations (0, 25, 50, 100, and 200 µg/mL). The propolis collected in A SuP, B WP, C SP, and D AP inhibited the growth of G. lamblia trophozoites. Albendazole was used as a growth inhibition control, which inhibited more than 90 % (IC90 10 µM) of the trophozoite proliferation. The tests were performed in triplicate (± SD) and at least two experiments in each condition were done. The antiparasitic activity was evaluated at 48 h after the addition of propolis extract. The asterisks on the bars show significant differences (p < 0.05) with respect to the control culture.
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Original Papers
Table 1 Identification and quantification of the major constituents of Sonoran propolis by HPLC‑DADa. Compound
R. T. (min)
λmax
Rutin Naringenin Hesperetin Pinocembrin CAPE Chrysin
22.1 30.9 33.1 46.7 52.3 54.0
256, 355 292 243, 289 243, 294 242, 328 270, 313
Propolisb UP
PAP
CP
N. d. N. d. N. d. 218.4 ± 1.5 11.4 ± 0.5 58.9 ± 0.6
4.1 ± 0.0 21.9 ± 0.1 3.7 ± 0.0 62.7 ± 1.3 N. d. 6.3 ± 0.1
N. d. N. d. N. d. 60.6 ± 1.2 N. d. 11.4 ± 0.8
Fig. 4 Antiparasitic activity of Sonoran propolis chemical constituents. The anti-G. lamblia activity of several chemical constituents of Sonoran propolis were evaluated at different concentrations (0, 25, 50, 100, and 200 µg/mL or its equivalent in µM). A CAPE (0, 88, 176, 352, and 703 µM), B naringenin (0, 92, 184, 367, and 734 µM), C hesperetin (0, 83, 165, 331, and 662 µM), D pinocembrin (0, 97, 195, 390, and 780 µM), E chrysin (0, 89, 197, 393, and 787 µM), and F rutin (0, 41, 82, 164, and 328 µM). Albendazole was used
ferences found on anti-G. lamblia activity of seasonal UP could be due to small quantitative variations in its constituents, which are secondary metabolites generated by the botanical source under the influence of climatic conditions of the Ures region. Another possible explanation for those observations could be quantitative variations of unidentified compounds with potent anti-G. lamblia activity in the seasonal propolis tested. Further experiments are needed to know the chemical basis of the seasonal effect on antiG. lamblia activity of the Sonoran propolis. In this study, we evaluated some chemical constituents present in the Sonoran propolis. Pinocembrin and chrysin are compounds present in the three propolis samples evaluated. CAPE is a UP exclusive constituent, while rutin, naringenin, and hespere" Table 1). CAPE was the tin are exclusive constituents of PAP (l most effective chemical constituent tested of Sonoran propolis and showed the highest growth-inhibitory activity on G. lamblia trophozoites [IC50 63.1 µg/mL (222.1 µM)]. These results could Alday-Provencio S et al. Sonoran Propolis and …
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as a growth inhibition control, which inhibited more than 90% (IC90 10 µM) of the trophozoite proliferation. The tests were performed in triplicate (± SD) and at least two experiments in each condition were done. The antiparasitic activity was evaluated at 48 h after the addition of propolis extract. The asterisks on the bars show significant differences (p < 0.05) with respect to the control culture.
suggest that CAPE is one of the chemical constituents involved in the anti-G. lamblia activity of UP. However, the antiparasitic activity of the control drug albendazole (IC90 10 µM) was more effective than CAPE. It is important to consider that propolis has a complex chemical composition, where CAPE represents only 1.14 % of the UP total weight (Table 1S, Supporting Information). When we compared the antiparasitic activity (IC50) of CAPE (63.1 ± 0.9 µg/mL) and UP (63.8 ± 7.1 µg/mL), it was evident that UP is more efficient than CAPE, suggesting that there are other compounds in UP that could be involved in anti-G. lamblia activity. Further experiments need to be conducted in order to establish the chemical basis of anti-G. lamblia activity of this natural product and CAPE. To our knowledge, this is the first work that reports the antiparasitic activity of CAPE. Pinocembrin is widely distributed in nature, and it is one of the main components of the propolis of various geographic areas, which presents a broad spectrum of biological activities [9, 32,
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All values represent the mean of triplicate determinations ± standard deviation; a Data obtained from Hernandez et al. [16]; b Quantification (mg/g) propolis; N. d.: Not detectable
Original Papers
Antiparasitic activity of Sonoran propolis and chemical constituents In order to evaluate the growth-inhibitory capacity of propolis or chemical compounds against G. lamblia trophozoites, we used propolis obtained from three different Sonoran regions (PAP, CP, and UP) and some of its main chemical constituents (CAPE, naringenin, hesperetin, pinocembrin, chrysin, and rutin). G. lamblia trophozoites cultures (1 × 105/tube) were incubated at 37 °C for a period of 12 h in 13 × 100 glass tubes with a screw cap. Then, trophozoite cultures were treated with different concentrations of propolis or chemical compounds (0, 25, 50, 100, and 200 µg/ mL) during 48 h. Each experiment was performed in triplicate, and at least two experiments were done in each condition evaluated. Dimethyl sulfoxide (DMSO; Sigma-Aldrich) was used as a solvent control, and albendazole (10 µM) as a positive control for parasite inhibition. Cell proliferation and cell viability were determined by using a Neubauer chamber and by trypan blue (Sigma-Aldrich) dye exclusion assay, respectively. The results were analyzed using GraphPad Prism v5.01 software.
Microscopic analysis Materials and Methods !
Giardia lamblia culture G. lamblia trophozoites were obtained from the American Type Culture Collection (GS/M‑83-H7, ATCC 50 581), and were axenically maintained in TYI‑S‑33 culture medium supplemented with 10 % newborn calf serum (NBCS; Gibco) with antibiotics (ceftriaxone 100 µg/mL) at 37 °C [34].
Propolis and methanolic extracts from propolis Sonoran propolis was collected in different arid and semiarid regions in northwest Mexico (Sonora), Pueblo de Alamos, Caborca, and Ures regions [11, 12]. PAP was collected between June 2001 and February 2003 (N29°15′, W 109°59′), CP (N30°46′, W 112° 05′) was collected between March and June 2006, and UP (N29° 27′, W 110°22′398) was collected between March 2008 and March 2009. Previously, we observed that there were not qualitative variations in the chemical composition of UP during the year (Fig. 1S, Supporting Information) [17]. Therefore, to perform the antiparasitic activity of Sonoran propolis on G. lamblia, we decided to use UP collected in summer (2008) to compare with PAP and CP. To evaluate the seasonal effect on the antiparasitic activity of Sonoran propolis, UP was collected in different seasons of the year (SuP: June 22, 2008 to September 22, 2008; SP: March 21, 2008 to June 21, 2008; AP: September 23, 2008 to December 21, 2008; and WP: December 22, 2008 to March 20, 2009). Seasonal propolis was collected from several hives in the Ures area, as reported by Valencia et al. 2012. The propolis extractions were performed according to Hernandez et al. 2007 and Valencia et al. 2012 and, subsequently, the propolis methanolic extracts were stored at − 80 °C [12, 17].
Morphological changes induced by Sonoran propolis on G. lamblia trophozoites were observed and photographed at 100× on a Nikon inverted optical microscope (Inverses Mikroskop Eclipse TS 100) and analyzed with Lumera Corporation Infinity Analyze software (Current revision 6.0.0).
Statistical analysis Data were processed and analyzed by the nonparametric method of Kruskal-Wallis. The statistical analyses were made in SPSS software. Statistical significance was accepted when p < 0.05.
Supporting information HPLC‑DAD/UV chromatograms of UP, and the chemical structures and relative abundance of its constituents are available as Supporting Information.
Acknowledgements !
We are grateful to the professional beekeeper Gilberto Valenzuela for all the facilities provided during the collection of propolis. We appreciate the support provided for the partial realization of this work by the project CONACYT (CB-2010-01/155224).
Conflict of Interest !
The authors declare that they have no financial or nonfinancial competing interests.
Affiliations 1
Sonoran propolis chemical compounds The following authentic flavonoids standards were commercially acquired from INDOFINE: naringenin, hesperetin, chrysin (≥ 98 % purity), and rutin (≥ 95 % purity). Albendazole (≥ 98 % purity) was acquired from Sigma-Aldrich. Pinocembrin (≥ 95 % purity) was purified from UP in our lab (HPLC‑DAD‑MS). CAPE (≥ 97% purity) was synthetized by our group [35].
2
3
4
Department of Chemistry-Biology, University of Sonora, Hermosillo, Sonora, Mexico Department of Chemistry-Biology and Agropecuary Sciences, University of Sonora, Navojoa, Sonora, Mexico Department of Nutrition and Metabolism, Centro de Investigación en Alimentación y Desarrollo A. C. Hermosillo, Sonora, México Unidad de Servicios de Apoyo en Resolución Analítica, Universidad Veracruzana, Xalapa, Veracruz, México
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33]. Pinocembrin is the most abundant constituent in UP (218.4 mg/g) and has weak antiparasitic activity (IC50 174.4 ± 26.0 µg/mL or 680.6 ± 101.7 µM), suggesting that pinocembrin is not properly related to the antiparasitic activity of UP. The exclusive constituents of PAP, naringenin (IC50 125.7 ± 20.7 µg/mL or 461.8 ± 76.3 µM), and hesperetin (IC50 149.6 ± 24.8 µg/mL or 494.9 ± 82.2 µM) showed significant anti-G. lamblia activity. Our data suggested that naringenin and hesperetin are involved in the anti-G. lamblia activity of PAP. Additional studies will be needed to determine the structural-activity relationship of these chemical compounds of Sonoran propolis. In conclusion, our results suggest that Sonoran propolis and some of its chemical constituents had an effect on the in vitro growth of G. lamblia trophozoites. Additionally, season had a significant effect on the anti-G. lamblia activity of Sonoran propolis. Although the antiparasitic activity of propolis from other sources is known, to our knowledge, this is the first study reporting the antiparasitic activity of propolis collected from arid and semidesert areas of Sonora, Mexico.
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18 Sforcin JM, Fernandes A jr., Lopes CAM, Bankova V, Funari SRC. Seasonal effect on brazilian propolis antibacterial activity. J Ethnopharmacol 2000; 73: 243–249 19 Teixeira EW, Message D, Negri G, Salatino A, Stringheta PC. Seasonal variation, chemical composition and antioxidant activity of Brazilian propolis samples. Evid Based Complement Alternat Med 2010; 7: 307–315 20 Monzote FL, Sariego RI, García PM, Cuesta-Rubio O, Márquez HI, Campo FM, Piccinelli AL, Rastrelli L. Activity of Cuban propolis extracts on Leishmania amazonensis and Trichomonas vaginalis. Nat Prod Commun 2011; 6: 973–976 21 Ferreira FM, Castro RA, Batista MA, Rossi FM, Silveira-Lemos D, Frézard F, Moura SA, Rezende SA. Association of water extract of green propolis and liposomal meglumine antimoniate in the treatment of experimental visceral leishmaniasis. Parasitol Res 2014; 113: 533–543 22 Santana LC, Carneiro SM, Caland-Neto LB, Arcanjo DD, Moita-Neto JM, Citó AM, Carvalho FA. Brazilian brown propolis elicits antileishmanial effect against promastigote and amastigote forms of Leishmania amazonensis. Nat Prod Res 2014; 28: 340–343 23 Amarante MK, Watanabe MA, Conchon-Costa, Fiori LL, Oda JM, Búfalo MC, Sforcin JM. The effect of propolis on CCL5 and IFN-γ expression by peripheral blood mononuclear cells from leishmaniasis patients. J Pharm Pharmacol 2012; 64: 154–160 24 Abdel-Fattah NS, Nada OH. Effect of propolis versus metronidazole and their combined use in treatment of acute experimental giardiasis. J Egypt Soc Parasitol 2007; 37: 691–710 25 Bankova V. Recent trends and important developments in propolis research. Evid Based Complement Alternat Med 2005; 2: 29–32 26 Falcão SI, Vilas-Boas M, Estevinho LM, Barros C, Domingues MR, Cardoso SM. Phenolic characterization of Northeast Portuguese propolis: usual and unusual compounds. Anal Bioanal Chem 2009; 396: 887–897 27 Ghedira K, Goetz P, Le Jeune R. Propolis. Phytothérapie 2009; 7: 100– 105 28 Lotti C, Campo Fernandez M, Piccinelli AL, Cuesta-Rubio O, Marquez Hernandez I, Rastrelli L. Chemical constituents of red mexican propolis. J Agric Food Chem 2010; 58: 2209–2213 29 Salatino A, Teixeira EW, Negri G, Message D. Origin and chemical variation of Brazilian propolis. Evid Based Complement Alternat Med 2005; 2: 33–38 30 Weinstein E. Seasonal variation, chemical composition and antioxidant activity of Brazilian propolis samples. Evid Based Complement Alternat Med 2008; 10: 1093–1102 31 Bankova V, Boudourova-Krasteva G, Sforcin JM, Frete X, Kujumgiev A, Maimoni-Rodella R, Popov S. Phytochemical evidence for the plant origin of Brazilian propolis from São Paulo state. Z Naturforsch C 1999; 54: 401–405 32 Diaz NG, Carpinella MC, Palacios SM. Antifeedant activity of ethanolic extract from Flourensia oolepis and isolation of pinocembrin as its active principle compound. Bioresour Technol 2009; 100: 3669–3673 33 Rasul A, Millimouno FM, Eltayb WA, Muhammad A, Li J, Li X. Pinocembrin: a novel natural compound with versatile pharmacological and biological activities. Biomed Res Int 2013; 10: 5 34 Keister DB. Axenic culture of Giardia lamblia in TYI‑S‑33 medium supplemented with bile. Trans R Soc Trop Med Hyg 1983; 77: 487–488 35 Grunberger D, Banerjee R, Eisinger K, Oltz EM, Efros L, Caldwell M. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 1988; 44: 230–232
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Original Papers
Authors
Samuel Alday-Provencio 1, Gabriela Diaz 2, Lucila Rascon 1, Jael Quintero 1, Efrain Alday 1, Ramón Robles-Zepeda 1, Adriana Garibay-Escobar 1, Humberto Astiazaran 3, Javier Hernandez 4, Carlos Velazquez 1
Affiliations
The affiliations are listed at the end of the article
Key words " Giardia lamblia l " propolis l " seasonal effect l " chemical compounds l
Abstract
received revised accepted
Dec. 13, 2014 March 28, 2015 April 1, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545982 Published online Planta Med © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Carlos Velazquez University of Sonora Department of Chemistry-Biology Blvd. Luis Encinas y Rosales s/n. Hermosillo Sonora 83000 Mexico Phone: + 52 66 22 59 21 63 Fax: + 52 66 22 59 21 63 [email protected]
!
Propolis is a cereus resin with a complex chemical composition that possesses a wide range of biological activities. The aim of this study was to evaluate the in vitro anti-Giardia lamblia activity of Sonoran propolis collected from three different areas of Sonoran Desert in northwestern Mexico (Caborca, Pueblo de Alamos, and Ures) and some of its chemical constituents. Additionally, we also analyzed the seasonal effect on the anti-G. lamblia activity of propolis. G. lamblia trophozoite cultures were treated with different concentrations of Sonoran propolis or chemical compounds during 48 h cell proliferation and cell viability were determined. Ures propolis showed the highest inhibitory activity against G. lamblia (IC50 63.8 ± 7.1 µg/mL) in a dose-dependent manner (Ures > Pueblo de Alamos > Caborca). Season had a significant effect on the in vitro anti-G. lamblia activity of Ures propolis. Summer propolis showed the highest inhibitory effect on the G. lamblia trophozoite growth (IC50 23.8 ± 2.3 µg/mL), followed by propolis collected during winter (IC50 59.2 ± 34.7 µg/mL), spring (IC50 102.5 ± 15.3 µg/mL), and autumn (IC50 125.0 ± 3.1 µg/mL). Caffeic acid phenethyl ester, an Ures propolis exclusive constituent, had the highest growth-inhibitory activity towards G. lamblia [IC50 63.1 ± 0.9 µg/mL (222.1 ±
Introduction !
Giardia lamblia (Lambl, 1859) (Kofoid and Christiansen, 1915) (syn. Giardia intestinalis, Giardia duodenalis) is a flagellated protozoan that inhabits the upper small intestine of humans and other mammals and is the causal agent of giardiasis [1]. It is one of the most frequent causes of intestinal disease worldwide [2]. This parasitosis has a clinical manifestation that varies from asymptomatic infection to acute or chronic infection, and is asso-
3.2 µM)]. To our knowledge, this is the first study showing that caffeic acid phenethyl ester possesses antiparasitic activity against G. lamblia. Naringenin [IC50 125.7 ± 20.7 µg/mL (461.8 ± 76.3 µM)], hesperetin [IC50 149.6 ± 24.8 µg/mL (494.9 ± 82.2 µM)], and pinocembrin [IC50 174.4 ± 26.0 µg/mL (680.6 ± 101.7 µM)] showed weak anti-G. lamblia activity. On the other hand, chrysin and rutin did not show significant antiparasitic activity. In conclusion, our results suggest that Sonoran propolis and some of its chemical constituents had inhibitory effects on the in vitro growth of G. lamblia trophozoites.
Abbreviations !
AP: CAPE: CP: PAP: SP: SuP: UP: WP:
autumn propolis caffeic acid phenethyl ester Caborca propolis Pueblo de Alamos propolis spring propolis summer propolis Ures propolis winter propolis
Supporting information available online at http://www.thieme-connect.de/products
ciated with diarrhea, flatulence, and abdominal pain, among other symptoms. G. lamblia infection is transmitted by contaminated water or food ingestion with G. lamblia cysts (infective form). Among drugs used to control this parasitic infection are the members of the imidazole family such as albendazole, metronidazole, and imidazole; 5nitrofuranes, acrydines, quinolones, and aminoglycosides are also used [3]. The high prevalence of giardia infection as well as the inadequate drug administration could favor the emergence of re-
Alday-Provencio S et al. Sonoran Propolis and …
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Sonoran Propolis and Some of its Chemical Constituents Inhibit in vitro Growth of Giardia lamblia Trophozoites
Original Papers
sistant Giardia strains [4, 5]. For this reason, the identification of potential new drug candidates for anti-G. lamblia is of paramount importance [3, 6]. Propolis is a resinous material collected by bees (Apis mellifera) from different plant exudates [7]. Over 300 chemical compounds have been identified in propolis and among them are aglycans flavonoids, phenolic acids and esters, p-coumaric prenylated acids, and ketophenones. Other identified compounds in propolis are lignans, di- and triterpenic acids, caffeoylquinic acids, monoterpens, sesquiterpens, isoflavonoids, chalcones, and pterocarpans [8–10]. Propolis has a wide spectrum of biological activities such as anti-inflammatory, antiproliferative, antimicrobial, antioxidant, and antiparasitic, among others [9–16]. Previously, we have shown that Sonoran propolis possesses a strong antiproliferative activity against different cancer cell lines [12, 17]. It has been demonstrated that the chemical composition of propolis can vary during the different seasons of the year, which could modify its biological activities [17–19]. There are limited data available about the antiparasitic activities of propolis. It has been reported that Brazilian propolis inhibited the growth of Trypanosoma cruzi [7, 13], Trichomonas vaginalis [20], and Leishmania amazonensis [20–22]. Furthermore, propolis has immunoregulatory properties on peripheral blood mononuclear cells from leishmaniasis patients [23]. The enhancing effect of propolis on immune response in an experimental mouse model of giardiasis, also resulting in the reduction of the parasite load, has been demonstrated [24]. Additionally, it has been reported that Brazilian propolis inhibited the in vitro proliferation of G. lamblia trophozoites [14]. The aim of the present study was to evaluate the antiparasitic activity of Sonoran propolis collected in three different geographic
Alday-Provencio S et al. Sonoran Propolis and …
Planta Med
areas of Sonora, Mexico, and commercial samples of some of its chemical constituents as well as to analyze the seasonal effect on the anti-G. lamblia activity of Sonoran propolis collected at Ures.
Results !
In order to evaluate the antiparasitic activity of Sonoran propolis on G. lamblia trophozoites, we used several concentrations of propolis extracts (0, 25, 50, 100, and 200 µg/mL). UP showed the highest inhibition of G. lamblia trophozoite growth (IC50 63.8 ± 7.1 µg/mL), followed by PAP (IC50 222 ± 68.4 µg/mL). The CP extract did not show any significant effect on G. lamblia proliferation. The antiparasitic drug albendazole (10 µM) was used as a growth inhibition control, which inhibited more than 90% of the trophozoite proliferation. DMSO did not show any significant effect on proliferation or viability of G. lamblia trophozoites " Fig. 1). (l Microscopic analysis showed that UP induced evident morphological changes on G. lamblia trophozoites. Control G. lamblia cultures showed trophozoite monolayers attached to the surface " Fig. 2 A, B). tubes, showing slightly elongated trophozoites (l " Fig. 2 C) and UP After incubation (48 h) with albendazole (l " Fig. 2 D to G), the trophozoites were detached from the surface (l of the culture tubes, and some of them acquired a round and elongated form. Additionally, in cultures with detached trophozoites (60–80 %), we observed abundant cellular debris as well as swollen trophozoites with slow movements, mainly at the highest propolis concentration evaluated (200 µg/mL). PAP and CP extracts did not show evident morphological changes on trophozoites at the propolis concentrations tested (data not shown). Since UP showed the highest growth-inhibitory activity on G. lamblia trophozoites, we decided to investigate the seasonal effect on the antiparasitic activity of UP. We collected propolis during the different seasons of the year in the region of Ures, Sonora (see Materials and Methods). The collected seasonal samples of propolis (SP, SuP, AP, and WP) were evaluated at different concentrations (0, 25, 50, 100, and 200 µg/mL). All propolis extracts showed antiparasitic activity. The SuP extract showed the highest anti-G. lamblia activity (IC50 23.8 ± 2.3 µg/mL), followed by the WP extract (IC50 59.2 ± 34.7 µg/mL), SP (IC50 102.5 ± 15.3 µg/mL), and AP (IC50 125.0 ± 3.1 µg/mL). DMSO did not affect G. lamblia " Fig. 3). trophozoite growth (l In order to understand more about the antiparasitic activity of UP, we evaluated the growth-inhibitory effect of some of the main chemical constituents of Sonoran propolis on G. lamblia trophozoites. Previously, we identified phenolic compounds, mostly flavonoids, as the main constituents of Sonoran propolis " Table 1) [12]. Therefore, the antiparasitic (UP, PAP, and CP) (l effect of six chemical constituents of Sonoran propolis (CAPE, naringenin, hesperetin, pinocembrin, chrysin, and rutin) was evaluated on G. lamblia trophozoites. CAPE was the chemical constituent that showed the highest anti-G. lamblia activity [IC50 63.1 ± 0.9 µg/mL (222.1 ± 3.2 µM)], followed by naringenin [IC50 125.7 ± 20.7 µg/mL (461.8 ± 76.3 µM)]. Hesperetin [IC50 149.6 ± 24.8 µg/mL (494.9 ± 82.2 µM)] and pinocembrin [IC50 174.4 ± 26.0 µg/mL (680.6 ± 101.7 µM)] had weak inhibitory activity. In contrast, chrysin and rutin did not show antiparasitic ac" Fig. 4). tivity at the evaluated concentrations (l
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Fig. 1 Antiparasitic activity of Sonoran propolis. The antiparasitic activity of the Sonoran propolis was evaluated on the in vitro growth of G. lamblia trophozoites at different concentrations (0, 25, 50, 100, and 200 µg/mL). Propolis tested: A UP, B PAP, C CP. Albendazole was used as a growth inhibition control, which inhibited more than 90 % (IC90 10 µM) of the trophozoite proliferation. The tests were performed in triplicate (± SD) and at least two experiments in each condition were done. The antiparasitic activity was evaluated at 48 h after the addition of propolis extract. The asterisks on the bars show significant differences (p < 0.05) with respect to the control culture.
Original Papers
Discussion
Fig. 2 Morphological changes induced by UP on G. lamblia trophozoites. In order to observe the morphological changes induced by UP on G. lamblia trophozoites, photographs were taken in an inverted optical microscope (100X, total magnification). A Cells in monolayer, B control DMSO (0.06 %), C albendazole (IC90 10 µM), D UP (200 µg/mL), E UP (100 µg/ mL), F UP (50 µg/mL), and G UP (25 µg/mL).
In this study, we evaluated the antiparasitic activity of Sonoran propolis collected from three different areas of Sonora, Mexico, and some of its chemical constituents. Additionally, we analyzed the seasonal effect on the growth-inhibitory activity of Sonoran propolis on G. lamblia trophozoites. Our data showed that UP had the highest growth-inhibitory activity on G. lamblia trophozoites, followed by PAP with moderate activity, and finally CP, which did not show any anti-G. lamblia activity at the tested concentrations. The difference in the antiparasitic activity of Sonoran propolis with a dissimilar geographical origin can be explained by qualitative and quantitative differences in the chemical composition of propolis [25–30]. The antiparasitic activity of propolis has been determined in different parasites, including T. cruzi, L. amazonensis, T. vaginalis, and G. lamblia [7, 13, 14, 20–22]. Specifically on G. lamblia (BTU10 strain recovered from an asymptomatic host), Brazilian propolis showed antiparasitic activity at 125 µg/mL (IC50) [14]. Interestingly, our data showed that UP had a higher anti-G. lamblia activity (GS/M‑83-H7 strain, ATCC 50 581) than Brazilian propolis [twice effective (IC50 63.8 ± 7.1 µg/mL)]. These differences could be due to the propolis chemical composition. Brazilian propolis is mainly constituted by phenolic compounds (flavonoids, aromatic acids, benzopyranes), di- and triterpenes, and essential oils, among others, where artepillin C is its major constituent [14, 31], while UP constitution is mainly comprised by flavonoids, phenolic acids and its esters, with pinocembrin being its major constituent [12, 17]. In addition, it is important to notice that different G. lamblia strains were evaluated against Brazilian and UP propolis; thus, the differences found in the anti-Giardia activity of propolis could be due to the susceptibility of the G. lamblia strains used in both studies. We observed that season had an important effect on the anti-G. lamblia activity of UP. The propolis collected in summer (SuP) showed the highest parasite growth-inhibitory activity, followed by WP > SP > AP. Despite the observed differences on the biological activity, season did not have a significant effect on the chemical profile of UP (Table 1S, Supporting Information) [17]. The dif-
Fig. 3 Seasonal effect on the growth-inhibitory activity of propolis on G. lamblia trophozoites. The seasonal effect was evaluated by using extracts of propolis collected during different seasons of the year. Propolis was evaluated at different concentrations (0, 25, 50, 100, and 200 µg/mL). The propolis collected in A SuP, B WP, C SP, and D AP inhibited the growth of G. lamblia trophozoites. Albendazole was used as a growth inhibition control, which inhibited more than 90 % (IC90 10 µM) of the trophozoite proliferation. The tests were performed in triplicate (± SD) and at least two experiments in each condition were done. The antiparasitic activity was evaluated at 48 h after the addition of propolis extract. The asterisks on the bars show significant differences (p < 0.05) with respect to the control culture.
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!
Original Papers
Table 1 Identification and quantification of the major constituents of Sonoran propolis by HPLC‑DADa. Compound
R. T. (min)
λmax
Rutin Naringenin Hesperetin Pinocembrin CAPE Chrysin
22.1 30.9 33.1 46.7 52.3 54.0
256, 355 292 243, 289 243, 294 242, 328 270, 313
Propolisb UP
PAP
CP
N. d. N. d. N. d. 218.4 ± 1.5 11.4 ± 0.5 58.9 ± 0.6
4.1 ± 0.0 21.9 ± 0.1 3.7 ± 0.0 62.7 ± 1.3 N. d. 6.3 ± 0.1
N. d. N. d. N. d. 60.6 ± 1.2 N. d. 11.4 ± 0.8
Fig. 4 Antiparasitic activity of Sonoran propolis chemical constituents. The anti-G. lamblia activity of several chemical constituents of Sonoran propolis were evaluated at different concentrations (0, 25, 50, 100, and 200 µg/mL or its equivalent in µM). A CAPE (0, 88, 176, 352, and 703 µM), B naringenin (0, 92, 184, 367, and 734 µM), C hesperetin (0, 83, 165, 331, and 662 µM), D pinocembrin (0, 97, 195, 390, and 780 µM), E chrysin (0, 89, 197, 393, and 787 µM), and F rutin (0, 41, 82, 164, and 328 µM). Albendazole was used
ferences found on anti-G. lamblia activity of seasonal UP could be due to small quantitative variations in its constituents, which are secondary metabolites generated by the botanical source under the influence of climatic conditions of the Ures region. Another possible explanation for those observations could be quantitative variations of unidentified compounds with potent anti-G. lamblia activity in the seasonal propolis tested. Further experiments are needed to know the chemical basis of the seasonal effect on antiG. lamblia activity of the Sonoran propolis. In this study, we evaluated some chemical constituents present in the Sonoran propolis. Pinocembrin and chrysin are compounds present in the three propolis samples evaluated. CAPE is a UP exclusive constituent, while rutin, naringenin, and hespere" Table 1). CAPE was the tin are exclusive constituents of PAP (l most effective chemical constituent tested of Sonoran propolis and showed the highest growth-inhibitory activity on G. lamblia trophozoites [IC50 63.1 µg/mL (222.1 µM)]. These results could Alday-Provencio S et al. Sonoran Propolis and …
Planta Med
as a growth inhibition control, which inhibited more than 90% (IC90 10 µM) of the trophozoite proliferation. The tests were performed in triplicate (± SD) and at least two experiments in each condition were done. The antiparasitic activity was evaluated at 48 h after the addition of propolis extract. The asterisks on the bars show significant differences (p < 0.05) with respect to the control culture.
suggest that CAPE is one of the chemical constituents involved in the anti-G. lamblia activity of UP. However, the antiparasitic activity of the control drug albendazole (IC90 10 µM) was more effective than CAPE. It is important to consider that propolis has a complex chemical composition, where CAPE represents only 1.14 % of the UP total weight (Table 1S, Supporting Information). When we compared the antiparasitic activity (IC50) of CAPE (63.1 ± 0.9 µg/mL) and UP (63.8 ± 7.1 µg/mL), it was evident that UP is more efficient than CAPE, suggesting that there are other compounds in UP that could be involved in anti-G. lamblia activity. Further experiments need to be conducted in order to establish the chemical basis of anti-G. lamblia activity of this natural product and CAPE. To our knowledge, this is the first work that reports the antiparasitic activity of CAPE. Pinocembrin is widely distributed in nature, and it is one of the main components of the propolis of various geographic areas, which presents a broad spectrum of biological activities [9, 32,
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All values represent the mean of triplicate determinations ± standard deviation; a Data obtained from Hernandez et al. [16]; b Quantification (mg/g) propolis; N. d.: Not detectable
Original Papers
Antiparasitic activity of Sonoran propolis and chemical constituents In order to evaluate the growth-inhibitory capacity of propolis or chemical compounds against G. lamblia trophozoites, we used propolis obtained from three different Sonoran regions (PAP, CP, and UP) and some of its main chemical constituents (CAPE, naringenin, hesperetin, pinocembrin, chrysin, and rutin). G. lamblia trophozoites cultures (1 × 105/tube) were incubated at 37 °C for a period of 12 h in 13 × 100 glass tubes with a screw cap. Then, trophozoite cultures were treated with different concentrations of propolis or chemical compounds (0, 25, 50, 100, and 200 µg/ mL) during 48 h. Each experiment was performed in triplicate, and at least two experiments were done in each condition evaluated. Dimethyl sulfoxide (DMSO; Sigma-Aldrich) was used as a solvent control, and albendazole (10 µM) as a positive control for parasite inhibition. Cell proliferation and cell viability were determined by using a Neubauer chamber and by trypan blue (Sigma-Aldrich) dye exclusion assay, respectively. The results were analyzed using GraphPad Prism v5.01 software.
Microscopic analysis Materials and Methods !
Giardia lamblia culture G. lamblia trophozoites were obtained from the American Type Culture Collection (GS/M‑83-H7, ATCC 50 581), and were axenically maintained in TYI‑S‑33 culture medium supplemented with 10 % newborn calf serum (NBCS; Gibco) with antibiotics (ceftriaxone 100 µg/mL) at 37 °C [34].
Propolis and methanolic extracts from propolis Sonoran propolis was collected in different arid and semiarid regions in northwest Mexico (Sonora), Pueblo de Alamos, Caborca, and Ures regions [11, 12]. PAP was collected between June 2001 and February 2003 (N29°15′, W 109°59′), CP (N30°46′, W 112° 05′) was collected between March and June 2006, and UP (N29° 27′, W 110°22′398) was collected between March 2008 and March 2009. Previously, we observed that there were not qualitative variations in the chemical composition of UP during the year (Fig. 1S, Supporting Information) [17]. Therefore, to perform the antiparasitic activity of Sonoran propolis on G. lamblia, we decided to use UP collected in summer (2008) to compare with PAP and CP. To evaluate the seasonal effect on the antiparasitic activity of Sonoran propolis, UP was collected in different seasons of the year (SuP: June 22, 2008 to September 22, 2008; SP: March 21, 2008 to June 21, 2008; AP: September 23, 2008 to December 21, 2008; and WP: December 22, 2008 to March 20, 2009). Seasonal propolis was collected from several hives in the Ures area, as reported by Valencia et al. 2012. The propolis extractions were performed according to Hernandez et al. 2007 and Valencia et al. 2012 and, subsequently, the propolis methanolic extracts were stored at − 80 °C [12, 17].
Morphological changes induced by Sonoran propolis on G. lamblia trophozoites were observed and photographed at 100× on a Nikon inverted optical microscope (Inverses Mikroskop Eclipse TS 100) and analyzed with Lumera Corporation Infinity Analyze software (Current revision 6.0.0).
Statistical analysis Data were processed and analyzed by the nonparametric method of Kruskal-Wallis. The statistical analyses were made in SPSS software. Statistical significance was accepted when p < 0.05.
Supporting information HPLC‑DAD/UV chromatograms of UP, and the chemical structures and relative abundance of its constituents are available as Supporting Information.
Acknowledgements !
We are grateful to the professional beekeeper Gilberto Valenzuela for all the facilities provided during the collection of propolis. We appreciate the support provided for the partial realization of this work by the project CONACYT (CB-2010-01/155224).
Conflict of Interest !
The authors declare that they have no financial or nonfinancial competing interests.
Affiliations 1
Sonoran propolis chemical compounds The following authentic flavonoids standards were commercially acquired from INDOFINE: naringenin, hesperetin, chrysin (≥ 98 % purity), and rutin (≥ 95 % purity). Albendazole (≥ 98 % purity) was acquired from Sigma-Aldrich. Pinocembrin (≥ 95 % purity) was purified from UP in our lab (HPLC‑DAD‑MS). CAPE (≥ 97% purity) was synthetized by our group [35].
2
3
4
Department of Chemistry-Biology, University of Sonora, Hermosillo, Sonora, Mexico Department of Chemistry-Biology and Agropecuary Sciences, University of Sonora, Navojoa, Sonora, Mexico Department of Nutrition and Metabolism, Centro de Investigación en Alimentación y Desarrollo A. C. Hermosillo, Sonora, México Unidad de Servicios de Apoyo en Resolución Analítica, Universidad Veracruzana, Xalapa, Veracruz, México
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33]. Pinocembrin is the most abundant constituent in UP (218.4 mg/g) and has weak antiparasitic activity (IC50 174.4 ± 26.0 µg/mL or 680.6 ± 101.7 µM), suggesting that pinocembrin is not properly related to the antiparasitic activity of UP. The exclusive constituents of PAP, naringenin (IC50 125.7 ± 20.7 µg/mL or 461.8 ± 76.3 µM), and hesperetin (IC50 149.6 ± 24.8 µg/mL or 494.9 ± 82.2 µM) showed significant anti-G. lamblia activity. Our data suggested that naringenin and hesperetin are involved in the anti-G. lamblia activity of PAP. Additional studies will be needed to determine the structural-activity relationship of these chemical compounds of Sonoran propolis. In conclusion, our results suggest that Sonoran propolis and some of its chemical constituents had an effect on the in vitro growth of G. lamblia trophozoites. Additionally, season had a significant effect on the anti-G. lamblia activity of Sonoran propolis. Although the antiparasitic activity of propolis from other sources is known, to our knowledge, this is the first study reporting the antiparasitic activity of propolis collected from arid and semidesert areas of Sonora, Mexico.
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