Abstract
Propolis is a resinous material produced by bees from the selective collection of plant exudates that are subsequently mixed with beeswax and salivary bee secretions. Propolis has been used in folk medicine, and certainly, several studies have validated its biological properties. The chemical composition and pharmacological activities of propolis collected through North (including Central America and Caribbean) and South America have been studied in the last years, and several papers have reported differences and similarities among the analysed geographical samples. Propolis has been classified according to its aspect and plant source; however, the ecological diversity present along the Americas provides a plethora of botanical resins. Herein, we summarize and discuss most of the studies performed at present on this profitable product for apiculture, attempting to compare the bioactivity, phytochemical diversity and botanical sources of honeybee propolis produced in Americas.
Keywords
- Propolis from Americas
- biological properties
- chemical constitution
- Apis mellifera
- botanical sources
1. Introduction
Bees are the most ecologically important pollinators for flowering plants, a coevolutive activity they have been performing for more than 100 millions of years. In particular, eusocial bees have reached an evolutionary success by living in perennial colonies (approximately 50,000 individuals) and developing sophisticated recruitment communication mechanisms to foraging and profit the chemistry of plants via manufacture and application beehive products for their own benefit [1–5]. According to their significant role as vectors of pollen in agricultural crops and the impact of beehive products for human societies, bees have earned an important position in different civilizations through history and geographies. Indeed, the bee management practice has been described since ancient times, including two types of beekeeping: apiculture (Apinae) and meliponiculture (Meliponinae). The first includes the Asian honeybees (
Inside the hive, the cooperative behaviour of eusocial bees is reflected by the contribution of each individual into colony maintenance, resulting in a suitable community health termed social immunity, which is characterized by hygienic practices, accompanied with the removal of diseased brood to avoid the persistence of pathogens and parasites inside the hive. However, one of the expensive consequences of social living is the disease transmission due to high interaction among individuals, and in order to supply additional immunological benefits, bees collect antimicrobial natural resins to produce a substance called propolis [4, 11–13]. Propolis, such as honey, beeswax and royal jelly, is one of the beehive products that have been valuable for human societies through the ages. In general, honeybees (
Propolis varieties produced by honeybee and stingless bee possess mechanical and biological properties, and these materials are used inside the hive in order to seal cracks, to prevent structural damage and to act as a thermoregulatory resource; in addition, those products are used as chemical weapons to protect the colony from diseases, acting as antimicrobial and as embalming substances that avoid putrefaction of killed intruders [1, 14–18, 23, 24]. Moreover, in several human traditional medicine systems, propolis has been used as a remedy due to its properties. In fact, Ancient Egyptians, Greeks and Romans employed this sticky material mainly as a wound healing and as an antiseptic agent. In addition, in Central and South America, Maya and Inca civilizations used cerumen and geopropolis produced by stingless bees as a folk remedy [15, 20, 25–28]. Nowadays, propolis is used in alternative medicine in Japan, and as a remedy to treat wounds, burns, sore throat and stomach ulcer in the Balkan states, meanwhile geopropolis is employed by the population of some tropical countries in Americas as empirical remedy for wound healing, gastritis, infections among others. In this context, propolis and geopropolis represent a promising source of bioactive compounds for pharmacological research [1, 14–18, 21, 28, 29].
Propolis has been extensively studied, and in the last decades, propolis has aroused scientific attention and many reports have been published concerning its broad spectrum of pharmacological activities and its bioactive components [14, 15, 18, 28]. At present, the biological activities reported for propolis include antibacterial [30–32], antioxidant [31, 33], antiparasitic [34–38], antifungal [39–41], antiviral [42], local anaesthetic [43], anti-inflammatory [44, 45], immunomodulatory [46, 47], antitumor [48–50], and antiproliferative activity on cancer cell growth [50–54], among others. There are remarkable differences in the biological activities of propolis from dissimilar geographical origin, and those mainly depend on the qualitative and quantitative variations of its characteristic chemical constituents, which are provided by botanical sources. Thus, the chemical diversity of propolis is dictated by the phytogeographical conditions and the climatic characteristics, and finally by the honeybee species involved in its production [55–57]. In that sense, the chemical composition of propolis from temperate zones (Europe, North America, Southern South America, and West Asia) differs from those of tropical zones (Central and South America, South and Southern Asia and Africa), as several studies have reported it in recent years.
Exudates from poplar buds (
Propolis must be purified in order to proceed into pharmacological and chemical investigations. The removal of inert material, which is mainly wax, is generally performed by preparing alcoholic or hydro-alcoholic extracts of the macerated raw material, where ethanol, ethanol 70% and methanol are often used. Several analytical techniques have been used to identify and characterize the chemical constituents of propolis samples from different geographical origins, including chromatographic and spectroscopic methods, such as thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC) and their coupled techniques to mass spectrometry (MS) [1, 58]. GC–MS analysis has been used for the identification of volatile and semi-volatile components of propolis; however, many propolis constituents are not sufficiently volatile for GC–MS analysis, even with derivatization procedures. In that sense, electrospray ionization (ESI) has been extensively used to ionize nonvolatile, thermally unstable, heavy and polar molecules; therefore, ESI-MS and its tandem version ESI-MS/MS have been applied for analyse propolis. ESI ionizes more efficiently polar compounds with acid [negative ion mode (−)] or basic sites [positive ion mode (+)], an important characteristic for the chemical study of propolis, since most of the pharmacologically active constituents identified in propolis possess certain polarity, commonly acidic of phenolic moieties [65, 66]. In addition, nuclear magnetic resonance (NMR) techniques have been used to fully characterize the isolated compounds from propolis [1, 58].
The chemical composition and pharmacological activities of propolis samples collected throughout North America (including Central America and Caribbean) and South America have been studied in the last years, and several papers have reported differences and similarities among the analysed geographical samples. At present, propolis from Americas has been classified according to its aspect and plant source, due to the ecological diversity present along the continent that provides different botanical resins. The major classes include the North and Southern South American propolis described as ‘poplar’ type, as well as Cuban, Brazilian and Mexican ‘red’ propolis type, in addition to the Brazilian “green” type, and finally the Cuban and Venezuelan “Clusia” type, reflecting a propolis diversity produced as consequence of adaptive responses of European honeybees to the beehive necessities and vegetation present in Americas, both in tropics and in temperate zones. Moreover, propolis produced by the stingless bees in Americas has gain attention and different samples have been chemically and biologically analysed in recent years, exhibiting in some cases similarities in bioactivity and composition with propolis of honeybees from the tropical areas. However, in this chapter, we were exclusively focused on honeybee propolis.
Some authors have reviewed and presented an extensive number of reports in propolis research with different approaches [1, 14, 15, 18, 21, 28, 55, 58, 62], although the discussion of analyses carried out in propolis from Americas in an attempting to compare the bioactivity, phytochemical diversity and botanical sources has not been approached. Herein, we summarize and discuss for the first time; to the best of our knowledge, most of the studies on honeybee propolis produced in Americas, including recent advances in research for this profitable product for apiculture. Most of the studies performed on propolis from Americas are depicted in Figure 1.
2. North American propolis
North America comprises the geographical region occupied by the countries of Greenland, Canada, United States and Mexico, in addition to the countries included in Central America and the Caribbean Islands. As a continent, North America is a terrestrial portion of the Americas, which is considered rich in biodiversity and a land with a plethora of natural resources that are distributed along its extension. Moreover, this landmass has many topographical variations, from valleys to extensive chains of tall mountains, and is characterized by possessing a wide range of climates and biomes starting from tundra, subarctic forest, followed by temperate forest, plains, deserts and tropical forests [67, 68].
Although the honeybee is not native from North and South American continents, it is wide distributed throughout the Americas. European colonists have brought
2.1. Canada
Chemical analysis on Canadian propolis by chromatographic technics, such as GC–MS and HPLC–MS, has identified the presence of poplar characteristic compounds. To the best of our knowledge, one of the initial studies on chemical composition of Canadian propolis was performed on a sample collected from Sydenham region in Ontario State [73]. Fifteen flavonoids, including pinobanksin-3-
In North America, poplars from section
More studies have been done regarding the chemical composition of commercialized extract formulations of Canadian propolis, such as Herstat® propolis extract ACF® (antiviral complex of flavonoids), which is patented and manufactured with propolis (at 3% concentration) collected from a specific area in Western Canada (probably Manitoba), which is rich in poplar trees. Bankova et al. [75] have found by GC–MS that the chemical composition of the marketed ointment formulation was mainly comprised by benzoic and
Furthermore, another commercial Canadian propolis acquired from TrophicTM products was analysed with the aim to determine the composition and to assess its antioxidant properties. Raw commercial propolis was extracted using a two-step sequential process with ethanol and water, wherein the higher antioxidant activity (by FRAP and DPPH methods) was exhibited by the ethanolic extracts, and a higher polyphenol and flavonoid content. Furthermore, the ESI-MS fingerprints revealed a resemblance with Brazilian brown propolis, in addition to the presence of chrysin and pinocembrin, among other flavonoids [78]. All these studies concerning to Canadian propolis provide enough phytochemical evidence about the botanical origin of those bioactive resins, which are mainly collected from different poplars species along the country, confirming the chemical composition of a propolis characteristic of temperate zones.
2.2. United States
The arrival of
To our knowledge, the subsequent analyses performed in propolis from the United States were focused on the identification of main chemical constituents of samples from Western Ohio, and both North and South Georgia (Athens and Claxton, respectively), where some similarities to the flavonoid profile of European propolis were found in Ohio and North Georgia samples, in addition to the identification of kaempferol, galangin, 3,3′-dimethoxyquercetin and 3-methoxykaempferol in Ohioan propolis [81]. Those flavonoids occur naturally in some species of Populus, Pinus, Betula, Alnus and Aesculus (horse chestnut), plant sources that are visited by honeybees for resins [16] and were proposed by the authors as possible botanical origin of Ohioan propolis. Interestingly, none of the aromatic acids (ferulic, caffeic and cinnamic acid) commonly present in European propolis were found in Ohioan sample, which established a difference among this propolis [81].
Moreover, in a comparative study with different geographical samples around the world, Kumazawa et al. [82] determined by HPLC analyses that a propolis sample from United States (supplied by Api corporation and Tamagawa University, Japan) was mainly composed by aromatic acids, flavonoids, and their ester and methyl ether derivatives. In particular, the high amounts of p-coumaric acid, pinobanksin-5-methyl ether, pinobanksin, chrysin, pinocembrin, galangin, pinobanksin-3-
In another comparative analysis, propolis from Eastern United States (Indiana and New York), Europe (Bulgaria, Finland and England) and Brazil was studied by ESI-MS/MS. Chrysin, pinocembrin and
Recently, a comprehensive analysis by GC–MS of ten geographically distinct propolis samples collected throughout the United States, including the cold North, the wet Southeast and the dry Southwest Regions, has provided a classification system by applying a chemometric approach principal component analysis (PCA) based on the relative amounts of main chemical classes found in the samples. Propolis from New York (NY-2,3,8,10), Pennsylvania (PN-6), Louisiana (LA-1), Minnesota (MN-9), Nebraska (NE-4), Nevada (NV-5) and North Carolina (NC-7) was analysed, and over 60 chemical constituents grouped in main compound types (benzoic and cinnamic acid derivatives, chalcones, flavanones and dihydroflavonols, flavones and flavonols, phenolic glycerides, and terpenes) were identified [87]. As expected, different geographical samples presented distinct chemical profiles. In all samples, poplar-type propolis compounds were found (aromatic acids and their esters, flavonoids and chalcones). Three main groups were obtained: group I) propolis rich in cinnamic acid derivatives (samples NY-2, NY-3, MN-9 and NY-10), such as benzoic, cinnamic, p-coumaric, and ferulic acids and benzyl-
Group II) propolis with high concentrations of flavonoids (NE-4, PA-6 and NY-8), such as pinocembrin, pinobanksin, pinobanksin-3-
In another study, with the aim to characterize the antimicrobial activity of propolis against beehive pathogens, 12 samples were collected from different geographical regions of the United States (Chaska, MN; Baton Rouge, LA; Ithaca, NY; Jamestown, ND; Lincoln, NE; Raleigh, NC; Wakinsville, GA; Tucson, AZ; Aspen, CO; Vacaville, CA; Beaumont, TX; Fallon, NV) and were evaluated against the bee pathogens
In order to track the botanical origin of antimicrobial resins (against
Further chemical studies have been done in propolis collected outside the temperate poplar zone of North America, specifically in the Sonoran Desert (Southwestern United States). Wollenweber and Buchmann [89] have analysed several propolis samples from managed and feral honeybee colonies located in Arizona State with the aim to determine the botanical origin of propolis from desert zones. The widespread vegetation in Sonoran Desert is comprised by xeromorphic shrubs and cacti rather than poplars, which are scarcely found in some watercourse zones. Wollenweber and Buchmann determined by TLC and GC–MS that some propolis samples presented the fingerprint pattern of
In addition to the mainland United States, the Hawaiian Islands chain is included in the political territory of this country, and it represents a totally different ecological scenario with tropical characteristics. In that sense, poplars are not available for propolis production in Hawaii; thus, honeybees must use other botanical sources. In a recent study, Inui et al. [90] have chemically investigated Hawaiian propolis by HPLC–ESI-MS in order to identify its botanical origin. A family of prenylflavonoids was identified in this propolis sample [90], chemical compounds that are additionally present in the Pacific propolis type (Okinawa, Taiwan, Indonesia and Myanmar propolis) [61]. By comparison of the chemical profile
All these studies on United States propolis revealed that
2.3. Mexico
Mexico is included among the five countries of the world with a great richness of endemic species, and this consideration is mainly related to the wide range of topographical diversity and the variety of climatic zones that lie between North American deserts and Mesoamerican forests [91]. Propolis collected from different ecological regions of Mexico has been analysed, including samples from North American deserts (Sonoran Desert), tropical forests, southern semi-arid highlands and temperate sierras.
One of the most investigated Mexican propolis types is Sonoran Desert propolis. Since almost a decade, several studies have been reporting the biological activities and main chemical constitution of samples collected from arid and semi-arid lands in the Sonora State [Ures (UP), Pueblo de Alamos (PAP) and Caborca (CP)]. Hernandez et al. [51] reported that the chemical composition of Sonoran propolis was mostly comprised by phenolic acids, flavonoids and their ester derivatives; moreover, pinocembrin, chrysin and pinobanksin-3-
Additionally, the chemical constitution of propolis from CP was further investigated in another study carried out by Li et al. [54], which resulted in the NMR characterization of three new flavonoids: (2
More studies have been done in order to determine the biological activities of Mexican propolis samples collected in semi-arid and arid zones (UP, PAP and CP). Sonoran propolis showed a strong antiproliferative effect on human and murine cancer cell lines A549 (IC50: 58.6 µg/mL), HeLa (IC50: 31.7–49.8 µg/mL), LS-180 (IC50: 53.3–84.9 µg/mL), RAW 264.7 (Abelson murine leukemia virus transformed macrophages; IC50: 0.8–5.2 µg/mL) and M12.C3.F6 (murine B-cell lymphoma cells; IC50: 3.1–6.8 µg/mL). Moreover, CAPE, galangin, xanthomicrol and chrysin induced a significant antiproliferative effect on most of the cancer cell lines evaluated (IC50: 3.2–95.4 µM) [51]. Since DNA harvested from cancer cells treated with UP exhibited a ladder of internucleosomal DNA cleavage pattern characteristic of apoptosis, in addition to the morphological changes observed in treated cells, a study conducted with the aim to determine biochemical events produced at earlier stages of apoptosis has been done. By annexin V-FITC/Propidium iodide double labelling, it has been demonstrated that Sonoran propolis treatment induced antiproliferative effect on M12.C3.F6 cells through apoptosis induction, and this apoptotic effect resulted to be mediated by modulations in the loss of mitochondrial membrane potential and through activation of caspases signalling pathway (3, 8 and 9). Additionally, some of the constituents of Sonoran propolis that induce apoptosis in cancer cells were characterized by an HPLC–PDA–ESI-MS/MS analysis, followed by isolation procedures and NMR spectroscopy that yield eighteen flavonoids, commonly described in poplar-type propolis, including two esters of pinobanksin, pinobanksin-5-methylether-3-
The antibacterial and FRS activities of Sonoran propolis (UP, PAP and CP) have been tested by broth microdilution method and by DPPH assay, respectively. Sonoran propolis exhibited antibacterial activity against only Gram-positive bacteria, and UP presented the highest inhibition against
Since UP showed to be one of the most biologically active of the Sonoran propolis tested, the evaluation of the seasonal effect on the chemical composition and biological activities (antiproliferative, antiparasitic and antioxidant activities) of UP has been done. The collected seasonal samples [spring (sp), summer (s), fall (f) and winter (w)] were analysed by an HPLC–DAD–UV method, wherein from the qualitative point of view, the chemical profile of the seasonal samples was similar; however, the results for antiproliferative effect on M12.C3.F6 cell line [sp (IC50: 11.6 µg/mL) > w (IC50: 26.6 µg/mL) > s (IC50: 49.7 µg/mL) > f (IC50: 54.5 µg/mL)] and antiparasitic activity on
Recently, the immunomodulatory properties of UP were tested (0.2–20.0 μg/mL) in a comparative study with Brazilian (Botucatu, Sao Paulo) and Cuban (Havana) propolis on pro- and anti-inflammatory cytokine production [tumor necrosis factor (TNF)-α and interleukin (IL)-10, respectively] by human monocytes. Brazilian propolis stimulated both TNF-α and IL-10 production by monocytes; meanwhile, Cuban propolis stimulated TNF-α and inhibited IL-10 production. UP exerted the opposite effect, inhibited TNF-α and stimulated IL-10 production. These results are due to qualitative and quantitative differences in the chemical constitution of the three samples, since different constituents that may exert pro- and anti-inflammatory activity depending on concentration. It is reported that the major compounds found in Brazilian, Cuban and Mexican propolis samples used in this study are artepillin C, isoflavonoids and pinocembrin, respectively, [46].
In a recent chemical comparative study performed with the aim to develop and validate a suitable RP–HPLC method to determine and quantify flavonoid markers in Mexican propolis, 11 samples collected at different ecological regions in six states (Estado de Mexico, Puebla, Chiapas, Zacatecas, Tlaxcala and Guanajuato) were analysed. Acacetin, 4′,7-dimethyl naringenin and 4′,7-dimethyl apigenin were used as marker components in this study, and the method was applied to establish some quantitative variations related to seasonal and geographical conditions of the propolis samples. 4′,7-Dimethyl apigenin was selected as an appropriate marker of Mexican propolis, followed by 4′,7-dimethyl naringenin. Both chemical compounds were considered useful for quality control procedures in the geographical origin validation of Mexican propolis [95].
The chemical constitution of a Mexican red-type propolis collected from Champoton at Southern Mexico (Campeche State) was analysed. Three new compounds 1-(3′,4′-dihydroxy-2′-methoxyphenyl)-3-(phenyl)propane, (
Moreover, in a comparative study about the volatile constituents of propolis from honeybees and stingless bees collected in the Yucatan peninsula, ninety-nine compounds were identified by GC–MS, wherein common compounds were present in both types of propolis. However, styrene, phenylacetaldehyde, trans-sabinene hydrate, nonanal, decanal, 2-undecanone, cyperen, cis-α-bergamotene, massoia lactone, ar-curcumene, cis-calamenene, cardina-1,4-diene, α-cadinene, β-eudesmol, α-bisabolol, neryl linalool, geranyl linalool, manoil oxide, kaur-16-ene, pentacosane and heptacosane were identified only in honeybee propolis [96].
In general, these studies confirm the chemical diversity present in propolis produced by
2.4. Cuba
The isle of Cuba is the largest in the Caribbean Sea, and together with Island of Youth and over 4000 islands comprise the Cuban Archipelago. The moderate tropical climate together with the exposure to different wind currents, topographical variations, diversity in moisture levels and types of soil produce heterogeneous ecological regions, such as the wetlands in the southern coast, tropical desert-like conditions in eastern coast, and pine forest at mountains, which make Cuba an example of almost every ecosystem present throughout the Antilles Islands [97]. Cuban propolis is the most investigated propolis type from North America (including Caribbean and Central America) and several studies have been published in regard to its composition and properties.
The first studies carried out on Cuban propolis were mainly focused on its biological properties, since it is considered as a traditional homemade remedy in this country. However, most of these studies were performed without a certain information on the chemical constitution of Cuban propolis, which makes it difficult to draw a direct correlation between bioactivity reported 20 years ago and the chemical constituents identified at present [58]. The antioxidant effect of ethanolic extracts of Cuban propolis from Baracoa and Pinar del Rio province was analysed by their scavenging action against different species of oxygen radicals (superoxide and alkoxy) using luminol-sensitized chemiluminescence, both propolis preparations showed a high antioxidant activity against superoxide (IC50: 5.0 and 9.5 µg/mL, respectively) and alkoxy (IC50: 0.5 and 0.6 µg/mL, respectively) radicals [98].
Moreover, the hepatoprotective effects of Cuban red propolis (CRP) from Havana region were evaluated in different models of acute liver injury induced by paracetamol (600 mg/kg) [99, 100], and by allyl alcohol (64 mg/kg) [101], both in mice. The intraperitoneal administration of ethanolic extract of propolis (25, 50 and 100 mg/kg) showed to decrease significantly the enzymatic activity of alanine aminotransferase (ALAT), the levels of reduced gluthatione (GSH), and as well showed to reduce liver damage, these protective effects of propolis were produced both, before (30 min.) and after (2 h) paracetamol hepatotoxicity induction [99, 100]. Similar results were obtained with CRP administration before allyl alcohol (30 min.) [101].
Additionally, CRP was evaluated in other two models of acute hepatotoxicity induced by carbon tetrachloride (CCl4) and by galactosamine (1000 mg/kg) in Sprague–Dawley rats [102–104]. The treatment with CRP (5, 10 and 25 mg/kg) in rats with hepatotoxicity induced by CCl4, showed to reduce ALAT and hepatic malondialdehyde (MDA) levels in blood serum, and as well decreased the triglyceride (TG) levels in liver in comparison with control group [102]. Furthermore, histopathological evaluation revealed rats treated with CRP (25, 50 and 100 mg/kg) exhibited a significant reduction in liver injury, according to the low count of affected cells and the limited extension of steatosis area in comparison with those rats with control treatment [102, 103]. Similar biochemical histopathological results were obtained by CRP against the hepatotoxicity induced by galactosamine [104]. After these experiments, it was suggested that CRP probably exerted its hepatoprotective effects by antioxidant properties (scavenging action against oxygen radicals) [100–104].
The antipsoriatic, anti-inflammatory and analgesic effects of CRP were additionally assessed in another study, wherein CRP induced the formation of granular layer in a mouse tail model, reflecting its antipsoriatic activity; meanwhile, its anti-inflammatory effect was observed using three different test models [Cotton-pellet granuloma assay in rats Sprague–Dawley (dose: 50 mg/kg i.g.), croton oil-induced edema (dose: 25%; 2.5 µL) and the peritoneal capillary permeability test in Swiss albino mice (dose: 10 mg/kg)]. CRP exhibited an analgesic activity using the models of acid-acetic-induced writhing (25 mg/kg i.g.) and hot plate test in Swiss albino mice (40 mg/kg) [105]. Moreover, the sensitizing properties of CRP were assessed, and CRP did not induce erythema, edema, and the study also revealed the absence of dermal and ocular toxicity in guinea pigs and New Zealand rabbits at 24 h. However, a moderate contact allergy potential was identified in CRP treatment by slight induction of erythema [105].
Since Cuban propolis presented several biological activities, subsequent studies came as consequence of the renewed interest in exploring the chemical composition of propolis for drug development [61, 106]. An ethanolic extract of Cuban propolis collected from Nuevitas, Cuba has yielded the isolation and structural determination of propolone A, the first polyisoprenylated benzophenone isolated from tropical propolis [106]. Although the presence of polyisoprenylated benzophenones in propolis from the tropical areas has been previously proposed, the full characterization of individual benzophenone molecules has not happened until this study [107]. In addition, propolone A showed significant antimicrobial and fungicidal activities against several Actinomyces, yeasts and Gram-positive and Gram-negative bacteria [106] that encouraged the study and isolation of other bioactive prenylated benzophenone derivatives present in Cuban propolis.
The presence of polyisoprenylated benzophenones in Cuban propolis suggested that resins from genus
By virtue of the differences of endemic plants in tropical and temperate regions and also the ecological diversity of Cuba, further investigations on the chemical composition of propolis from different regions of Cuba were carried out. A scrupulous chemical analysis of a CRP from Pinar del río provided a cutting edge input on composition of propolis in general by reporting for the first time the presence and structural determination of isoflavonoids (isoflavones, isoflavans and pterocarpans) in a propolis sample, in addition to gallic acid, isoliquiritigenin, and (-)-liquiritigenin [57]. The occurrence of isoflavonoids is pretty restricted in nature and is characteristic of Leguminosae family, and these findings yield two different Cuban propolis types, suggesting the participation of at least two diverse botanical sources, one that provides prenylated benzophenones and another the isoflavonoids [57].
With the aim to study the chemical similarities and differences on Cuban propolis and to establish a classification system according to the presence of secondary metabolites, 65 propolis samples from different regions of Cuba were chemically analysed by HPLC–PDA, HPLC–ESI/MS and NMR. Cuban propolis types were grouped in three groups, including CRP rich in isoflavonoids; Cuban brown propolis (CBP), characterized by a high amount of polyisoprenylated benzophenones; and Cuban yellow propolis (CYP) that contains aliphatic compounds, which was sub-classified depending on its content on triterpenic alcohols (type A) and polymethoxylated flavonoids (type B) [58, 111–113]. Once CBP was identified as the major type of propolis produced in Cuba, biological assays were performed, especially on its capacity to inhibit
Other studies showed that BCP induced significant antiproliferative activity on human breast cancer cell lines, preferentially on MCF-7 (estrogen receptor positive; ER α+) rather than MDA-MB-231 (estrogen receptor negative ER α-) in a dose-dependent manner. The antiproliferative effect on MCF-7 was partially related to apoptosis induction after an arrest in G1 phase of cell cycle was detected. Moreover, the co-administration of 17-β-estradiol and an antagonist (ICI 182,780) allowed to hypothesize that BCP possesses an estrogen-like activity, although the effect would not be exclusively considered ER-dependent because the mortality also induced on MDA-MB-231 by BCP [115].
Otherwise, the treatment with nemorosone on MCF-7, MDA-MB-231 and LNCaP cells induced a selective antiproliferative effect on MCF-7 by arresting the cell cycle in G0/G1 phase, in addition to a reduction in the expression of pERK1/2 and PAkt. Nemorosone did not induce antiproliferative effect on MDA-MB-231 nor human prostate cancer cells LNCaP (which express ERβ but not ERα), which suggested that nemorosone is the main responsible for the antiproliferative effect of BCP on ERα+ breast cancer cells, and it could have therapeutic applications in breast cancer treatment since its activity on ERα+ cells [116]. Moreover, nemorosone exhibited cytotoxicity in neuroblastoma cell lines, including their clone selected for resistance to chemotherapeutic compounds. It induced a G0/G1 arrest on cell cycle that yields a reduction in S-phase population, in addition to the detection of an upregulation of p21Cip1, presence of apoptotic DNA laddering, the activation of caspase 3 activity, dephosphorylation of ERK1/2 in LAN-1 and the inhibition of Akt/PKB [114]. Due to the reported correlation of nemorosone cytotoxicity on cancer cell lines to direct action on estrogen receptor (ERs), other studies have been done. By
In addition to the presence of nemorosone in CBP, there have been identified mucronulatol and plukenetione A as cytotoxic and antiproliferative compounds that could contribute to the potent inhibitory activity of CBP on cancer cell proliferation. The occurrence of plukenetione A has previously been identified in
Other different biological properties of nemorosone have been evaluated, including the mutagenic, antimutagenic and estrogenic effects. The mutagenic and antimutagenic activity of nemorosone were assessed by the Ames test on
Red propolis has also been described in other tropical countries, including Brazil, Mexico and Venezuela. Brazilian red propolis (BRP) is a propolis type produced by honeybees from the resinous exudates of
All these studies contribute to understand the chemical diversity present in propolis from tropical zones, wherein
2.5. El Salvador
As observed, honeybees had to find other different plant sources of bioactive resins in the tropics to those commonly visited in the temperate zones; thus, the chemical diversity present in tropical propolis, and the pharmacological properties of its constituents has attracted so much interest. In tropical propolis, it has been reported a variety of compounds, including the presence of polyprenylated benzophenones, isoflavonoids, and triterpenes in Cuban propolis [57, 111, 113], the occurrence of isoflavonoids and polyisoprenylated benzophenones in Brazilian propolis and, and 1,3-diarylpropane derivatives, flavonoids, and isoflavonoids from Mexican propolis [56].
With the aim to continue the studies of propolis from tropical Central America, a sample collected in the vicinities of Usulutan, El Salvador, was studied and two new chalcones were isolated and characterized by NMR (2′,3′-dihydroxy-4,4′-dimethoxychalcone and 2′,3′ 4-trihydroxy-4′-methoxychalcone) by first time in propolis. Both compounds presented a good antibacterial activity on
In another study, two diterpene glycosides (
2.6. Honduras
In Central America, there are a high richness of endemic species, and since chalcones and diterpene glycosides were found in Salvadoran propolis [126, 127], and not in other tropical propolis analysed (Cuban, Brazilian and Mexican), the chemical investigation of other sample from Central America was carried out in order to establish differences and similarities in tropical propolis from Americas. To the best of our knowledge, only one study of Honduran propolis has been reported, wherein a sample collected in Marcala was fractionated and led to the isolation of cinnamic ester derivatives, including a new (E,Z)-cinnamyl cinnamate, in addition to flavanones, triterpenes, aromatic acids and one chalcone. Honduran propolis inhibited the ATPase activity of Pdr5p (70% at 100 μg/mL), a protein responsible for a multidrug resistance phenotype in
The presence of these cinnamic ester derivatives has been described in exudates and volatile fractions of
3. South American propolis
South America is characterized by possessing the highest plant diversity of any other region in the world, which is due to several aspects, including its continental size and location (latitude and longitude), the presence of the largest extension of tropical forest and finally the Andes Mountains that form the biggest mountain system in the world, representing a linkage between tropical and temperate latitudes across South America [128, 129]. Propolis samples from different regions of South America have been studied. Brazilian propolis is by far the most analysed propolis in South America, followed by Argentinean, Chilean, Uruguayan and Venezuelan. Recently, propolis from Colombia and Bolivia has been studied; meanwhile, Peruvian propolis has only been included in biological comparative studies. In this section, most relevant and recent advances in South American propolis research topic are summarized.
3.1. Colombia
Colombian vegetation possesses a great biodiversity with a high number of endemic plant species distributed in a variety of tropical forests, steppe and grasslands, representing a source of great variety in phytochemical substances [130]. Propolis is used in Colombia as a folk remedy, and in cosmetic and food industries, however, few studies in Colombian propolis have been done [131]. In a recent study, propolis collected from Medellin region, and subsequently extracted with n-hexane/methanol and fractioned with dichloromethane, exhibited an inhibitory effect on mycelial growth against
The presence of isocupressic acid and other labdan diterpenes has been described in Brazilian green propolis, a propolis type that has its plant sources in the resins of
3.2. Venezuela
As Colombia, Venezuela is a tropical region in South America that represents an enormous source of endemic plants and a great variety of bioactive compounds. One of the first chemical compositional analysis in tropical propolis was performed with 38 samples collected in different tropical areas of Venezuela, including Bolívar, Nueva Esparta, Cojedes, Yaracuy, Monagas, Mérida, Táchira, Portuguesa, Sucre, Baringas, Aragua and Carabobo, with the aim to identify phytochemical evidence for the botanical origin of Venezuelan propolis. Most of these propolis samples contained similar chromatographic patterns characteristic of phenolic compounds, specifically of prenylated benzophenones, since flavonoids were identified only in few samples, and they were methylated 6-oxygenated flavones. The HPLC profile of resins excreted by the flowers of
Moreover, in a later study on Venezuelan propolis from Trujillo State, the presence of the already reported polyisoprenylated benzophenones scrobiculatones A and B was identified, together with their derivatives 18-ethyloxy-17-hydroxy-17,18-dihydroscrobiculatone A and 18-ethyloxy-17-hydroxy-17,18-dihydroscrobiculatone B (first time described in this study). Additionally, a mixture of scrobioculatones A-B exhibited a significant antibacterial activity against
3.3. Peru
Peruvian propolis has not yet been analysed from the chemical compositional point of view; however, there is a comparative report wherein propolis from Peru, Brazil, Netherlands and China was studied to determine the cytotoxic, hepatoprotective and FRS capacity (DPPH method) of the collected samples. Methanol (MeOH ext.) and water (Wt ext.) extracts were prepared for each propolis samples. Peruvian propolis showed the lowest FRS activity among the other propolis samples (ED50: MeOH ext. 82.3 µg/mL and Wt ext. 94.9 µg/mL), although it exhibited moderate cytotoxicity on murine colon 26-L5 carcinoma (51.1 µg/mL) and HT-1080 fibrosarcoma cells (76.4 µg/mL). The hepatoprotective activity of propolis was tested on primary cultures of mouse hepatocytes induced to cell death with D-galactosamine (D-GalN) and tumor necrosis factor-α (TNF-α), wherein the MeOH extract of Peruvian sample showed toxicity at 200 µg/mL; meanwhile, the water extract exhibited significant hepatoprotective activity at 200 µg/mL (approximately 45%) [136].
3.4. Bolivia
Bolivia is located at the centre of South America, and its territory comprises a transition into humid tropical and dry subtropical climatic zones. The geographical characteristics result in a great biodiversity present along the country; however, scarce phytochemical studies of Bolivian flora have been done. Propolis is used in Bolivia as antimicrobial remedy to treat infections and respiratory illnesses [137]. Bolivian propolis has been recently studied, and according to the chemical profile of ten samples collected from the main centres of beekeeping in Bolivia, two main types of propolis have been identified. The first correspond to propolis from the valley regions (Cochabamba, Chuquisaca and Tarija) which were characterized by the presence of prenylated phenylpropanoids in a high amount, including
Moreover, the antimicrobial properties of those ten Bolivian propolis samples and their main constituents were tested against 11 bacterial pathogenic strains of clinical relevance (
3.5. Brazil
Brazil has an extensive territory that comprises the richest flora in the world, a huge plant biodiversity with over 56,000 species, which represents approximatively 19% of the total flora of the world [140]. Thus, Brazilian propolis possess a particular chemical diversity, involving different plant resins from dissimilar eco-regions, resulting in a variety of propolis types, including those with characteristic chemical composition of both tropical and temperate regions. At present, Brazilian propolis has been by far one of the most investigated propolis types, and a significant number of chemical compounds have been identified in samples collected throughout the country. Due to the large amount of chemical and pharmacological studies of Brazilian propolis, herein, we present the most significant reports in an attempt to summarize hundreds of studies.
One of the first studies, focused on chemical characterization of bioactive constituents of Brazilian propolis, was carried out with a sample collected in Sao Paulo state, which yielded the isolation of three new compounds: 3,5-diprenyl-4-hydroxycinnamic acid, 3-prenyl-4-dihydrocinnamoloxycinnamic acid and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran [141]. These Brazilian propolis constituents presented antimicrobial activity against
In other studies, the occurrence of fifteen more cinnamic acid derivatives, in addition to nine
In order to establish the basis of Brazilian propolis classification, 500 samples were collected from different regions, including southern (Paraná and Rio Grande do Sul), Southeastern (Sao Paulo, Minas Gerais) and Northeastern (Bahía, Piauí, Ceará and Pernambuco) of Brazil. According to their chemical profile, the samples were classified into 12 groups, wherein five groups were identified in the Southern, one from Southeastern and six from Northeastern Brazil, suggesting a greater plant diversity on Northeastern and Southern than Southeastern region [145, 146]. The antimicrobial activity against
The main compounds identified in propolis from group 3 were flavonoids and organic acids, and the chemical profile of that sample was similar to that of poplar exudates, in particular to
In group 12, the presence of prenylated derivatives of
With the aim to establish a more precise correlation among Brazilian propolis types, thirty-eight samples were collected, from Minas Gerais, Sao Paulo, Paraná, Mato Grosso do Sul, Bahia and Algoas, and were analysed by ESI-MS and tandem mass spectrometry (ESI-MS/MS). A principal component analysis (PCA) has been applied to determine similarities and differences in the secondary metabolite fingerprint of propolis samples. Brazilian propolis was divided into groups, according to their chemical profile and geographical origins. BRP from Algoas and Bahia was divided into two main groups (R1 and R2), and as well propolis from South and Southeastern Brazil were divided in one BGP group, which contained the greatest number of samples, and two groups of BBP (B1 and B2) from Paraná state. Seven compounds were used as markers and allowed the classification of Brazilian propolis into those five groups according to PCA analysis, wherein the presence of intersection of constituents among samples was detected. Pinocembrin was in BRP R2 and BBP B1, and chsysin in BBP B1, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran in BBP B2, 3-prenyl-4-hydroxycinnamic acid, 3,5-diprenyl-4-hydroxycinnamic acid and dicaffeoylquinic acid were identified in BGP and BBP B2,
In regard to the chemical composition of BRP, recent studies have reported the presence of prenylated benzophenones, such as guttiferone E/xanthochymol mixture, the isoflavonoids isosativan and medicarpin, the triterpenoid ketone 20(29)-lupen-3-one, and also a naphthoquinone epoxide (isolated for the first time from a natural source) in a sample collected from the state of Algoas [124]. The presence of these compounds suggested a mixed plant origin, since prenylated benzophenones are described in floral resins of
More studies on chemical composition of BRP collected from Algoas have been done, identifying methyl o-orsellinate, methyl abietate, medicarpin, 2,4,6-trimethylphenol, and the isoflavonoids, homopterocarpin, 4′,7-dimethoxy-2′-isoflavonol and 7,4′-dihydroxyisoflavone. Biological tests have been carried out for the BRP extract, including antimicrobial against
The chemical composition of an essential oil extract obtained from Brazilian propolis collected in Rio de Janeiro was characterized, and 26 constituents were identified, wherein β-caryophyllene (12.7%), acetophenone (12.3%) and β-farnasene (9.2%) were the major constituents, in addition to the new compounds found linalool (6.47%), ϒ-elemene (6.25%), methyl hydrocinnamate, ethyl hydrocinnamate, α-ylangene and valencene. This essential oil extract presented antimicrobial activity against
Since Brazilian propolis is continuously produced all over the year, it was important to determine the seasonal effect on propolis composition and evaluate the influence of season on its biological activities. By GC and GC–MS, the seasonal variations in chemical composition of BGP collected from Sao Paulo state were investigated. All the seasonal samples contained phenolic compounds, mainly cinnamic acid derivatives as major constituents; however, the presence of diterpenes appeared in summer sample and reached a predominate percentage in the autumn sample, but being absent during the other seasons, which suggested the participation of at least two plants as botanical source [59]. These chemical variations could be important for the practical application of propolis, and in order to evaluate the effect of these slight changes on Brazilian propolis composition, antibacterial assays on bacterial strains isolated from human infections were carried out. The growth of Gram-positive bacteria,
Propolis possesses immunomodulatory activities, and it has been reported that its immunostimulant effect is produced via macrophage activation, which enhances macrophage phagocytic capacity [28, 151, 152]. The effect of BGP on activation by reactive oxygen (H2O2) and nitrogen (NO) species was determined on peritoneal macrophages obtained from male BALB/c mice. Both
The role of toll-like receptors (TLR) in microbial pattern recognition and as well as the action of pro-inflammatory cytokines are important to trigger the initial events of immune response. Thus, the immunomodulatory action of BGP has been evaluated in regard to those mechanisms of innate immunity, and BGP treatment resulted to upregulate TLR-2 and TLR-4 expression, together with the production of pro-inflammatory cytokines (IL-1β and IL-6) in peritoneal macrophages and spleen cells of Male BALB/c mice treated with BGP of Sao Paulo State (200 mg/kg) for 3 days, suggesting the favourable action of BGP by enhancing immune responses in macrophages and spleen cells of treated mice [154]. Moreover, the action of BGP on antibody production was studied in rats immunized with bovine serum albumin, wherein BGP (10%), independently of the season of the year, stimulated antibody production, which was concluded to be a consequence of synergic effects, since isolated compounds and
Other studies in regard to the effects of BGP on immune response of acutely and chronically challenged stressed mice have been also investigated. After 3 days treatment under acutely stress conditions, BGP (200 mg/kg) restored TLR-2 and TLR-4 expression and increased IL-4 production in mice, in comparison with control [156, 157]. Meanwhile, BGP (200 mg/kg) during 7 days treatment in stressed mice increased the production of H2O2 in macrophages and reduced the alterations found in spleen [158]. Moreover, the BGP treatment (200 mg/kg) for 14 days in mice submitted to chronic stress exerted similar effects, demonstrating these experiments the immunomodulatory activity of BGP under
Once the effect of BGP on the initial steps of the immune response was evaluated in murine models, the immunomodulatory effect of BGP was evaluated on receptors expression, cytokine production and fungicidal activity of human monocytes. The BGP treatment (5, 10, 25, 50 and 100 μg/mL) resulted to upregulate TLR-4 and CD80 expression and decreased the production of TNF-α and IL-10 as concentration treatment increased. The fungicidal activity of human monocytes after incubation with BGP and challenged with
Later, in an attempt to investigate the BGP constituents involved in the immunomodulatory effects on the innate immunity, caffeic and cinnamic acids (5, 10, 25, 50 and 100 μg/mL) have been assessed on human monocytes. Caffeic acid downregulated TLR-2 and HLA-DR expression. Otherwise, cinnamic acid downregulated TLR-2, HLA-DR and CD80; meanwhile, it upregulated TLR-4 expression depending on concentration. Both phenolic acids inhibited TNF-α and IL-10 production, whereas they increased the fungicidal activity of monocytes against
In another study, the antioxidant and anti-inflammatory action of BGP and caffeic acid were determined. By DPPH assay, FRS activity of caffeic acid (EC50: 2.5 μg/mL) resulted to be more effective than that of BGP (EC50: 18.51 μg/mL), and the treatment with BGP and caffeic acid (5, 10, 25, 50 and 100 μg/mL) exerted anti-inflammatory action, by inhibiting nitric oxide (NO) production in RAW 264.7 cells. Moreover, both treatments suppressed LPS-induced signalling pathways, namely p38 MAPK, JNK1/2 and NF-κB, and did not induce hepatotoxicity at the tested concentrations, suggesting that caffeic acid may be involved in the antioxidant and anti-inflammatory effects of BGP [164]. BGP and its main botanical source,
All these results obtained for Brazilian propolis reflect the enormous plant biodiversity of the country, obtaining different phytochemical patterns in samples collected from different geographical origins. Therefore, it is necessary to further investigate the chemical composition of samples produced in different zones in order to understand the plant origin of the bioactive compounds present in propolis from entire Brazil. The broad pharmacological activities tested for Brazilian green, red and brown propolis are a great example of biological interest in this beehive product, which makes it one of the most characterized propolis in the world. It is important to continue the studies on Brazilian propolis to provide a more precisely insight about the possibilities of tropical, temperate and a mixture of both types of propolis from this biodiverse country.
3.6. Uruguay
Uruguay is covered by the temperate sub-humid grasslands, a biome extended through the vast plains of Southern South America, including part of Northeast and Central Argentina and Southern Brazil [169]. Uruguayan propolis has been investigated, and one of the first studies reported the identification of 22 different phenolic acids and flavonoids in six samples collected from different regions of the country. The presence of these constituents was detected by HPLC–PDA–UV, and resins from
In another study, the chemical constitution of propolis collected from Montevideo was further investigated using HPLC–MS and NMR techniques and led to the isolation of eighteen flavonoids, four aromatic carboxylic acids and eleven phenolic acid esters, in addition to 3 new compounds elucidated: pinobanksin-3-
Subsequently, a propolis sample from Montevideo was included in a comparative study with different geographical samples, wherein Uruguayan propolis was qualitatively similar to those from the United States, New Zealand, Hungary, China and other temperate samples included. Additionally, Uruguayan propolis presented a moderate antioxidant activity by a FRS method (DPPH assay: ≥30% at 20.0 µg/mL) and by β-carotene–linoleic acid system (≥45% at 10.0 µg/mL) [82]. The antioxidant properties of ten propolis samples collected in Southern Uruguay were further evaluated by
The volatile compounds of three samples from Central-southern Uruguay were analysed by static headspace technique coupled with a GC–MS, and compared with volatile content of Brazilian, Estonian and Chinese propolis. Monoterpenes (α- and β-pinenes) were predominant, and the volatile profile of Uruguayan and Brazilian propolis was composed mainly by α-pinene and β-pinene (64.6–77.6%). Brazilian propolis was distinguished by a high amount of β-methyl crotonaldehyde (10.1%), and one of Uruguayan samples displayed the presence of limonene (15.6%). By principal component analysis of the volatile profile found in samples showed high differences, falling into separate clusters. In this study, the geographical origin of Uruguayan and Brazilian propolis is not specified; however, by the results obtained, it could be suggested that Brazilian propolis is from the southern region, since the similarities in volatile composition to Uruguayan propolis [173]. The results obtained in these studies for Uruguayan propolis confirm the chemical characteristics of propolis from temperate zones.
3.7. Argentina
Argentina has a vast territory and a great diversity of ecological regions, including subtropical rain forests and temperate sub-humid grasslands or “Pampas.” However, two thirds of Argentinean mainland is comprised by extensive arid and semi-arid region between the Andes and cold subantartic zones. Natural arid and semi-arid plain regions include western Chaco, Monte and Patagonian steppe, and these are ecologically similar to those semi-arid regions of North America, present in Sonora, Sinaloa and Great Basin, respectively [174, 175]. Several studies have differentiated propolis into two main groups, one from temperate zones and the other from tropical regions; however, few studies have been done in regard to propolis from arid and semi-arid areas [89]. Propolis from different regions of Argentina has been investigated, twenty-five samples collected in temperate, arid and semi-arid lands in Northern Argentina (Santiago del Estero, Tucumán, Chaco, Salta, Catamarca, Jujuy and Misiones) were chemically and biologically analysed. By absorption spectra, RP–HPTLC and RP–HPLC analysis, it was found that 16 of the 25 samples presented a similar phenolic profile to that exhibited by samples from Southern Brazil and Uruguay. The main phenolic constituents identified in those 16 samples were pinobanksin, pinocembrin, chrysin, galangin, tectochrysin and 1,1-dimethylallylcaffeic acid [176]. Propolis samples from Catamarca and Tucumán presented the highest phenolic content, exhibiting greater amounts of pinocembrin, 1,1-dimethylallylcaffeic acid, ferulic acid and cinnamic acid in comparison with the other 20 samples analyzed by Isla et al. [176]. Additionally, these two propolis presented the higher antibacterial activity against
Moreover, other biological properties of propolis from Tucumán have been tested, including cytoxicity with the lethality test of
Similar results have been obtained from Tucumán propolis against human opportunistic and pathogenic fungi, specifically on dermatophytes and yeasts (
Other studies focused on the characterization of propolis from Catamarca Province in Northwestern Argentina have been done. Propolis collected from El rincón presented FRS activity by ABTS assay (SC50: 6.9 µg/mL) and by β-carotene–linoleic acid antioxidant assay (SC50: 2.0 µg/mL), additionally exhibited antibacterial effect on methicillin resistant
FRS activity by DPPH assays of Argentinean propolis collected in the regions of Mendoza, Rio Negro, La Pampa, and Entre Rios was evaluated. Almost all of the propolis samples presented FRS activity (40-60% at 20 µg/mL), in exception to the samples from La Pampa and Entre Rios. Greater amounts of caffeic acid, ferulic acid, caffeic acid phenetyl ester were found by HPLC–PDA–UV analysis in propolis samples with the stronger FRS activity. In general, the presence of flavonoids, such pinocembrin, chrysin, pinobanksin, pinobanksin-3-
The chemical composition of propolis collected from the Andean locality of Bauchaceta in San Juan province was characterized by HPLC–ESI-MS/MS and GC–MS techniques and revealed a lignan and volatile organic acid profile that matched with the exudates of
A reverse phase LC–DAD–MS method has been developed to quantify phenolic acids and flavonoids present in propolis and found that the most abundant constituents from Argentinean, European and Chinese propolis were chrysin (2–4%), pinocembrin (2–4%), pinobanksin-acetate (1.6–3%) and galangin (1–2%) [185]. Furthermore, the typical fingerprint of propolis from Argentina, Italy and Spain was determined by on-line HPLC–ESI/MS analysis, and those propolis samples showed the same total ion chromatogram (TIC) profile, in addition to a similar amount of pinocembrin (39-49% of the total identified flavonoids), suggesting a poplar-type origin for Argentinean propolis [186]. Interestingly, the subtropical montane forest of “El Siambón” in Tucumán region is characterized by the presence of native vegetation including members of Lauraceae, Myrtaceae, Fabaceae, Juglandaceae, Salicaceae and Nyctaginaceae families, as well as the introduced poplar, eucalyptus and pinus trees, which are described as the most visited trees by honeybees for resins in the Tucuman region [178].
However, in another study, propolis samples from 30 different regions of Santiago del Estero province were analysed and classified into three groups according to the main arboreal species in the region. Nineteen chemical constituents were identified in these 30 samples, pinocembrin, quercetin, kaempferol, chrysin, salicylic, 4-hydroxybenzoic, benzoic, ferulic and gallic acids were observed in most of the analysed samples. Some quantitative differences of each component were found in the three groups, and interestingly, no
Furthermore, the antibacterial, antiradical and antioxidant activities of those 30 propolis samples from Santiago del Estero province were analysed, and variability in bioactivity was found. All samples presented a substantially similar inhibitory effect on
All these studies carried out to characterize the chemical composition and biological properties of Argentinean propolis have demonstrated that samples collected from different ecological regions throughout the country exhibited different chemical profiles; however, flavonoids are present in the most of the analysed samples, and chemical compounds such as lignans and chalcones could be used as markers to suggest the participation of a particular botanical source; thus, the phytochemical diversity found in Argentinean propolis has its botanical origin on several plants, including
3.8. Chile
Chile extends along the Southwestern coast of South America, between the geographical barriers of the Andes on the East and Pacific Ocean on the West, resulting in a unique flora developed as consequence of the environmental isolation that consists in many endemic plant species. Chile owns the Atacama Desert on the North, Chilean Mediterranean-type region in North-central, temperate forest in South-central region and subpolar forests at south [189, 190].
Some studies have been carried out in Chilean propolis collected from different regions. A propolis sample from Quebrada Yaquil in Santa Cruz (Region VI) in the Mediterranean semi-arid region of central Chile have led to the isolation and structural determination by NMR of five lignans, including 3 novel compounds: 1-(4-hydroxy-3-methoxyphenyl)-1,2-bis{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propan-3-ol acetate, and two different optical isomers ([α]25D + 8.8° and [α]25D–15.6°, respectively) of 1-(4-hydroxy-3-methoxyphenyl)-2-{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propan-1,3-diol 3-acetate, in addition to the already reported 3-acetoxymethyl-5-[(E)-2-formylethen-1-yl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran, and 3-acetoxymethyl-5-[(E)-3-acetoxypropen-1-yl)]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran. The hives were located in a region with the dominant species sclerophyllous shrubs and herbaceous species, such as
In another study, propolis was collected from hives located at Cuncumen (also Mediterranean-type climate), and fractionated to yield viscidone, vanillin, 3′,4′-(methylendioxy)acetophenone, 3-ethoxy-4-methoxybenzaldehyde, cinnamic acid and 3-methoxy-4-hydroxymethyl ester. These chemical compounds were already described; however, this was the first report on propolis composition of an arid, and a Mediterranean-type climate area. Additionally, the frequency of pollen grains observed in this Cuncumen propolis by optical and scanning electronic microscopy (SEM) allowed to suggest that resins of
Other studies have been carried out in propolis from Central Chile. Six samples collected from the region of Santiago (Curacaví, Lo Cañas, Buin, Caleu, Cajón del Maipo and pireque) were analyzed by HPLC–ESI-MS/MS, and 30 chemical phenolic compounds were identified, including pinocembrin, pinobanksin, pinobanksin-3-
In addition, a propolis sample collected from San Vicente de Tagua-Tagua (VI Region) was biologically assessed, and showed FRS activity by DPPH (100% at 80 µg/mL) and scavenger effect on superoxide anion (100% at 0.78 µg/mL), as well this Chilean propolis presented antiproliferative activity (at 80 µg/mL for 72 h incubation) by MTT assays on human tumour cell lines KB, Caco-2 and DU-145 ( 9, 45 and 23% cell viability, respectively). Galangin, caffeic acid, p-coumaric acid, ferulic acid and CAPE were identified by HPLC analysis in San Vicente sample, and since the most abundant plant species in this region were
Moreover, the chemical constitution, the botanical origin and antibacterial activity of twenty samples collected from Central and Southern regions of Chile (Valparaíso, Metropolitana, Libertador Bernardo O’Higgins and La Araucanía Regions) were investigated. Quercetin, myricetin, kaempferol, pinocembrin, coumaric acid, caffeic acid and CAPE were identified in propolis samples by HPLC–MS. All Chilean propolis samples presented a growth inhibitory effect on
With the aim to determine whether the bioactivity against
These reports provide important information about chemical composition of Chilean propolis. At present, two main types of Chilean propolis are described, those with lignans as main compounds, and the others with flavonoids and phenolic acids related to propolis collected from temperate regions; however, the palynological evidence obtained indicated that native plants, such as
4. Conclusions
Propolis produced by honeybees in Americas represents an important source of diverse bioactive compounds, such as nemorosone and other prenylated benzophenones, artepillin C, CAPE, terpenes labdane type, chalcones, flavonoids, lignans, among others. These secondary metabolites are biosynthesized by different plants present in diverse ecological regions throughout the continent. Thus, propolis from Americas possess constitutional particularities, and some samples share both temperate and tropical chemical constitution, and even characteristic compositional mixtures are present in some propolis from North and South America, resulting in a huge propolis diversity. In addition, propolis from temperate zones is not restricted to poplar, birch, pines and horse chestnut exudates as main and exclusive botanical source, since recent findings on propolis from United States and Honduras tracked liquidambar species as botanical source, providing a peculiar and transcontinental alternative source of resinous material.
At present, the chemical constitution of North American and South American propolis is partially characterized; however, more studies are needed to understand and identify the bioactive compounds found in those samples and to determine the mechanism of action through which they exert their pharmacological activities. Additionally, more studies should be done in order to understand the plant resin bee foraging behaviour, the role of propolis inside the hive and the benefits of botanical chemistry available along the ecological regions present in America, which imply interdisciplinary work to draw a relationship between bee health and the ecosystem implied. Finally, in accordance to the broad spectrum of biological activities, the high variability and complexity of North American and South American propolis, it becomes necessary to develop a more precise classification of this propolis diversity that combine the qualitative and quantitative plant origin fingerprint, and as well as the biological properties of this beehive product.
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