Biological activities of polyphenols-enriched propolis from Argentina arid regions.
Background: Propolis is a bioactive natural product collected by honeybees (Apis mellifera) from plant sources. Purpose: This study was undertaken to determine the effect of propolis extracts from arid region of Argentina, on the activity/expression of pro-inflammatory enzymes, and as potential free radical scavenger, antifungal and anthelmintic agent as well as to get a first insight into the polyphenolic profile of the active fractions. Study design/methods: Two propolis samples were collected in different time from hives located in Tucuman, Argentina. They are representative of the collection time of the raw material for phytotherapeutical purposes. Ethanolic extracts from both propolis were obtained. The PEEs were analyzed for total polyphenol (TP), non-flavonoid phenols (NFP) and flavonoid (FP) content followed by HPLC-DAD analysis and identification of components by HPLC-MS/M[S.sup.n]. The potentiality as anti-inflammatory (LOX, COX, iNOS enzymes), antioxidant, antifungal and nematicidal was determined.
Results: PEEs contain high levels of TP, NFP and FP, including cinnamic acid, caffeic acid prenyl ester, caffeoyl dihydrocaffeate and caffeic acid 3,4-dihydroxyphenethyl ester, liquiritigenin, 2',4'-dihydroxychalcone and 2',4'-dihydroxy-3'-methoxychalcone. The PEEs in vitro reduced the activity of LOX and COX-2. Pretreatment of RAW 264.7 cells with PEEs before the induction of inflammatory state, inhibited NO overproduction and the iNOS protein expression was significantly decreased. The PEEs exhibited antioxidant, antifungal (Candida sp.) and nematicidal effect (C. elegans).
Conclusion: These findings show the potential use of characterized PEEs from arid regions of Argentina as phytomedicine.
The propolis are natural products produced by honeybees (Apis mellifera LJ, they collect resins from bark and leaf bud from plants that grow around of hives. The chemical composition of propolis depend on the vegetation that grow around of hives as well as the time of collection (Bankova et al. 2006; Isla et al. 2005, 2009, 2013; Vera et al. 2011; Solorzano et al. 2012). Several phytogeographical regions with different plant species were found in Argentina. Isla et al. (2013) described at least four different types of propolis in Argentina, based on the chemical composition and botanical origin. Some of them show biological properties, including antibacterial effect against antibiotic-resistant human pathogenic bacteria (Nieva Moreno et al. 1999; 2005; Isla et al. 2009; Vera et al. 2011; Solorzano et al. 2012) and canine pathogenic bacteria (Salas et al. 2014), antifungal activity against dermatophytes and yeasts (Agliero et al. 2010; Isla et al. 2013), free radical scavenging and antimutagenic activities (Isla et al. 2005; Nieva Moreno et al. 2005; Vera et al. 2011; Danert et al. 2014). However, little is known about anti-inflammatory and nematicidal activity of Argentinean propolis. The aim of the present study was to assess the activity of propolis from the arid region of Tucuman, Argentina, as inhibitor of pro-inflammatory enzymes, antioxidant, antifungal and nematicidal effect as well as to get a first insight into the polyphenolic profile of the samples.
Material and methods
Two propolis samples were provided by INTA-PROAPI, Argentina, from hives located in the Agrotechnical School, Monte Region, Tucuman (26[degrees]35'S, 65[degrees]55'W). The samples were collected in December 2012 and March 2013 and are representative of the collection time of the raw material for phytotherapeutical purposes. Samples were weighed and frozen at -20 [degrees]C until processing.
Preparation of propolis extracts
20 g of each crude propolis was extracted with 250 ml ethanohwater 80:20 (v/v) at room temperature during 7 days. Each extract was taken to dryness under reduced pressure and lyophilized to afford the propolis hydroethanolic extracts (PEEs). The extracts were labeled as PEE-1 (December sample) and PEE-2 (March sample). Extracts were stored at -20 [degrees]C in the dark until analysis.
Phenolics content and distribution
TP concentration in the samples was determined spectrophotometrically according to the Folin-Ciocalteu method (Salas et al. 2014). The NFP and FP content were determined according to Isla et al. (2014) and expressed as mg GAE/g PEE. Total flavone/ flavonol (expressed as mg QE/g PEE) and flavanones/ dihydroflavonol (mg NE/g PEE) content was estimated according to Danert et al. (2014).
Biological activities: Assays using cell systems
Cell viability was assessed by the MTT assay as described by Torres Carro et al. (2015) using a density of 2.0 x 105 RAW 264.7 cells (ECACC, Salisbury, UK) per well with or without PEE (12-100 [micro]g/ml). The cells were cultured in the presence or absence of LPS (1.0 p-g/ml) and with or without PEEs (12-50 [micro]g/ml) or dexamethasone (10 |xM, 3.92 [micro]g/ml). The iNOS and COX-2 expression and NO production were determined according to Torres Carro et al. (2015).
Inhibition of pro-inflammatory mediators in a cell free system
The inhibitory activity of the PEE (up to 100 [micro]g/ml) on COX-2 was measured using a COX inhibitor screening assay kit following the manufacturer's instructions, based on measuring PG by ELISA (Cayman Chemical). The effect on LOX was determined according to Torres Carro et al. (2015). In all assays, reference compounds were used.
The antioxidant capacity assay was carried out by using the ABTS cation radial and the system [beta]-carotene-linoleic acid (Zampini et al. 2010; Danert et al. 2014). In both antioxidant assays, BHT was used as positive control.
To assess the antifungal effect, the following microorganisms were used: Candida albicans (A, B), C. tropicalis (C, D), C. krusei (E), C. parapsilosis (F), C. glabrata (G), C. guilliermondii (H), C. albicans ATCC 10231 and C. parapsilosis ATCC 22013. The bioautographic assay for Candida was done according to Svetaz et al. (2007). MIC and MFC values were determined by the broth microdilution methods (M27-A3, 2008) in presence of PEEs (50-1000 [micro]g/ml).
The nematicidal assay was determined according to D'Almeida et al. (2015) using the wild-type strain N2 (Bristol) from Caenorhabditis elegans (Genetics Center of University of Minnesota, Minneapolis, MN, USA).
Fractionation of the PEE for polyphenols identification
The aim of the PEE fractionation was to identify the most active fractions to allow a better chemical identification of the compounds. A sample of the PEE-2 (1.12 g) was permeated on Sephadex LH-20 using MeOH as mobile phase. Ten fractions were obtained with the following yields: I (78 mg), II (142 mg), III (185 mg), IV (222 mg), V (60 mg), VI (44 mg), VII (14 mg), VIII (9 mg), IX (10 mg) and X (4 mg). The fractions III to VIII showed antioxidant and antifungal activities. Then, fraction III (20 [micro]l of a 50 mg/ml solution) was fractionated by HPLC system coupled to a photodiode array detector (Waters Corporation, Milford, MA, USA) in a semi-preparative C18 column (Phenomenex) using a linear gradient solvent system consisting of 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol as follows: 35% A to 20% A over 10 min, 20% A to 0% A from 10 to 20 min, 0% A during 10 min. The yields of the more abundant sub-fraction were as follow: SF-I (13 mg), SF-II (14 mg), SF-III (15 mg) and SF-IV (8 mg). Fraction F-III, the bioactive sub-fractions SF-I to SF-IV as well as fractions F-IV and F-V were analyzed by HPLC using XBridge[TM] C18 column (Waters) according to Costamagna et al. (2015). The fractions and sub-fractions were monitored at 254 nm and UV spectra were recorded from 220 to 500 nm for peak characterization.
Data were recorded on a HPLC-ESI-MS/MS system which consisted of the HPLC HP1100 (Agilent Technologies Inc., Santa Clara, CA-USA) connected through a split to the mass spectrometer Esquire 4000 Ion Trap LC/MS(n) system (Bruker Daltonik GmbH, Bremen, Germany) according to Costamagna et al. (2015).
Results and discussion
In Argentina, the collection of propolis for phytotherapeutical preparations is from December to March (late spring to summer time for the southern hemisphere). Two samples of propolis (December and March) from the same production place were collected and compared to characterize the raw material for phytotherapeutic use.
About 50% of the crude propolis constituents are soluble in ethanohwater (80:20). Both PEEs showed high content of TP (593 [+ or -] 15 and 587 [+ or -] 20 mg GAE/g PEE for PEE-2 and PEE-1, respectively) and NFP (357 [+ or -] 13 and 368 [+ or -] 10 mg GAE/g PEE followed by FP (236 [+ or -] 10 and 219 [+ or -] 18 mg GAE/g PEE). The content of flavone/flavonol (276 [+ or -] 5 and 343 [+ or -] 6 mg GAE/g PEE) was higher than flavanone/ dihydroflavonol content (165 [+ or -] 12 and 185 [+ or -] 15 mg QE/g PEE). No significant differences were found in the polyphenol content between the December and March propolis collection (Supplementary material Table 1).
Effects on pro-inflammatory mediators
PEE-1 was 33% more effective as LOX inhibitor than PEE-2 ([IC.sub.50] values of 94.9 [+ or -] 4.7 and 63.9 [+ or -] 3.2 [micro]g/ml). Both samples were active on COX-2 with similar [IC.sub.50] values, 100 [+ or -] 4 and 106 [+ or -] 5 [micro]g/ml for PEE-2 and PEE-1, respectively (Supplementary material Table 1).
Both PEEs were not toxic against RAW 264.7 cells. Cell stimulation with bacterial LPS induces the production of pro-inflammatory cytokines and the expression of iNOS in macrophages and consequently caused significant increase in NO production. Pretreatment of cells with both PEEs inhibited NO overproduction in a concentration-dependent manner ([IC.sub.50] = 9.4 [+ or -] 0.5 and 8.4 [+ or -] 0.5 [micro]g/ml for PEE-2 and PEE-1, respectively) with a similar potency to dexamethasone, a positive control. The iNOS protein expression was significantly decreased when the cells were treated with PEEs with [IC.sub.50] values of 27.03 and 30.96 [micro]g/ml for PEE-1 and PEE-2, respectively. No changes were observed in the amount of [beta]-actin protein, an internal control, indicating the specific inhibition of iNOS protein expression by PEEs. COX-2 expression was not affected (Supplementary material Fig. 1-I and Fig. 1-II).
The ABT[S.sup.*+] radical scavenging activity for the PEEs showed a [SC.sub.50] value of 14 [micro]g/ml for both extracts, close to that of the commercial synthetic antioxidant BHT ([SC.sub.50] 13 [micro]g/ml). In the [beta]-carotene assay, the PEEs exhibited antioxidant activity with [IC.sub.50] values of 26 and 29 [micro]g/ml while the [IC.sub.50] value for BHT was 2.3 [micro]g/ml.
Antifungal and nematicidal activities
The antifungal activity of both PEEs was evaluated against ten Candida strains. The MIC (125-500 [micro]g/ml) and MFC (375-750 [micro]g/ml) values for the PEE-1 did not differ from the MIC (100-400 [micro]g/ml) and MFC (200-800 [micro]g/ml) values of PEE-2 (Supplementary material Table 2). The antifungal effect of other propolis from Argentina against the yeasts C. albicans, C. tropicalis, Saccharomyces cerevisiae and C. neoformans was reported. Aguero et al. (2010) reported lower (62.5 [micro]g/ml) or similar MIC values (125 [micro]g/ml).
In our assay conditions, both PEEs showed nematicidal effect on adult C. elegans. The [LC.sub.50] of PEEs, defined as the concentration required for killing half of the nematode population within 24 h, was 70 [micro]g/ml. For levamisole, a known anthelmintic drug the [LC.sub.50] was 4.7 [micro]g/ml.
Identification of polyphenolic compounds
Propolis from the arid region of Tucuman, collected in the same place in December and March, showed similar biological activities and fingerprint in HPLC-DAD (Fig. 1A). The PEE from March was selected for the fractionation and identification of polyphenolic compounds. The F-1II, F-IV and F-V were the most abundant bioactive fractions. From fraction F-1II, the following compounds were identified based on the UV spectrum, mass spectra and fragmentation patterns: (1) cinnamic acid in SF-I; (2) caffeic acid prenyl ester in SF-II; (3) liquiritigenin in SF-III; (6) caffeoyl dihydrocaffeate and (7) teucrol in SF-IV (Supplementary material Figs. 2 and 3). The fraction F-IV afforded (5) 2',4,-dihydroxy-3'-methoxychalcone (DHMC) and the fraction F-V yielded (4) 2',4'-dihydroxychalcone (DHC) (Table 1, Fig. 1B-D, Supplementary material Fig. 2). The compounds 4 and 5 were identified by co-injection with standards isolated from Z. punctata.
Cinnamic acid, caffeic acid and its derivatives show antimicrobial activity (Chen et al. 2011) and anti-inflammatory effect (D'Almeida et al. 2013). The dihydroxyphenethyl ester of caffeic acid (teucrol) has strong antioxidant activity and in vivo anticancer effect (Guo et al. 2013). Lee et al. (2009) reported that liquiritigenin protects mice against disseminated candidiasis by stimulation of Thl immune response (induction of interferon-[gamma] and interleukin-2). DHC (4), DHMC (5) and 2',4',4-trihydroxy-6'-methoxychalcone were reported in propolis from arid environments (Solorzano et al. 2012; Vera et al. 2011; Aguero et al. 2010; Nieva Moreno et al. 2005). One of the botanical sources of propolis in arid regions of Argentina is the native shrub Zuccagnia punctata. Propolis from Z. punctata is characterized by the occurrence of compounds 4 and 5, as reported by Aguero et al. (2010) for a propolis sample from Amaicha del Valle, Provincia de Tucuman. The antioxidant, anti-inflammatory, cytotoxic, antitumoral and antimicrobial properties of chalcones were reported (Vera et al. 2011; Solorzano et al. 2012; Nieva Moreno et al. 2005; Aguero et al. 2010).
Our findings add support to the potential use of hydroalcoholic extracts of propolis from the arid region of Tucuman to develop phytotherapeutical products with anti-inflammatory, antioxidant, antifungal and nematicidal properties. More research work is needed to establish the variation in the single bioactive constituents in different samples, covering the whole production area and associating the phenolics with the selected activities using multivariate analysis.
Received 14 June 2015
Revised 10 November 2015
Accepted 19 November 2015
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication.
The authors acknowledge the financial support from SCAIT-UNT, CONICET and ANPCyT, Argentina; and PIEI, Universidad de Talca, Chile.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.n.007.
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Ana Lilia Salas (a), Maria Rosa Alberto (a), Iris Catiana Zampini (a), Ana Soledad Cuello (a), Luis Maldonado (b), Jose Luis Rios (c), Guillermo Schmeda-Hirschmann (d,1), Maria Ines Isla (a,1), *
(a) Laboratorio de Investigation de Productos Naturales (LIPRON), INQUINOA.CONICET. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Universidad National de Tucuman, 4000 San Miguel de Tucuman, Argentina
(b) INTA, Famailla, Tucuman, Argentina
(c) Departamento de Farmacologia, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Espana
(d) Laboratorio de Quimica de Productos Naturales, Instituto de Quimica de Recursos Naturales, Universidad de Talca, Casilla 747, 3460000 Talca, Chile
Abbreviations: PEE, propolis ethanolic extract; TP, total phenolic; NFP, non-flavonoids phenolics; FP, flavonoid phenolics; QE, quercetin equivalents; NE, naringenin equivalents; GAE, gallic acid equivalent; LPS, lipopolysaccharide; PVDF, polyvinylidene fluoride; iNOS, inducible nitric oxide synthase; COX-2, cyclooxigenase-2; PG. prostaglandin; LOX, soy lipooxygenase-1; MTT, thiazolyl blue tetrazolium bromide; DNPH, 2,4-dinitrophenylhydrazine; ABTS. 2,2'-azino-bis3-ethylbenzothiazolilne-6-sulfonic acid; DMSO, dimethyl sulfoxide; BHT, butylated hydroxytoluene; TLC, thin-layer chromatography; MIC, minimal inhibitory concentration; MFC, minimal fungicidal concentration; NGM, nematode growth medium.
* Corresponding author. INQUINOA- CONICET, Universidad Nacional de Tucumdn, San Lorenzo 1469,4000 San Miguel de Tucumdn, Argentina. Tel.: +54 3814203062.
E-mail address: firstname.lastname@example.org, email@example.com (M.I. Isla).
(1) Both authors has the same participation
Table 1 Identification of phenolic compounds in the propolis active fractions by HPLC-MS/MS analysis. Peak UV Rt(min) [[M-H].sup.-] Fragmentation 1 277 40.9 147 103 2 286, 237, 217 46.9 247 178,135 3 305, 270 53.9 255 135,107 4 267, 320, 344 55.5 239 197,135 5 230, 340 55.8 269 254,150,106 6 320, 300 sh 59.5 343 179,135 7 328, 300 sh, 250sh 60.1 315 179,135 Peak Compounds 1 Cinnamic acid 2 Caffeic acid prenyl ester 3 Liquiritigenin/isomer 4 2', 4'-dihydroxychalcone 5 2',4'-dihydroxy-3-methoxychalcone 6 Caffeoyl dihydrocaffeate 7 Teucrol
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|Title Annotation:||Short Communication|
|Author:||Salas, Ana Lilia; Alberto, Maria Rosa; Zampini, Iris Catiana; Cuello, Ana Soledad; Maldonado, Luis;|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jan 15, 2016|
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