Comparison of the Biological Potential and Chemical Composition of Brazilian and Mexican Propolis

Abstract

Propolis is a resinous substance collected by bees from plants and its natural product is available as a safe therapeutic option easily administered orally and readily available as a natural supplement and functional food. In this work, we review the most recent scientific evidence involving propolis from two countries (Brazil and Mexico) located in different hemispheres and with varied biomes. Brazil has a scientifically well documented classification of different types of propolis. Although propolis from Brazil and Mexico present varied compositions, they share compounds with recognized biological activities in different extraction processes. Gram-negative bacteria growth is inhibited with lower concentrations of different types of propolis extracts, regardless of origin. Prominent biological activities against cancer cells and fungi were verified in the different types of extracts evaluated. Antiprotozoal activity needs to be further evaluated for propolis of both origins. Regarding the contamination of propolis (e.g., pesticides, toxic metals), few studies have been carried out. However, there is evidence of chemical contamination in propolis by anthropological action. Studies demonstrate the versatility of using propolis in its different forms (extracts, products, etc.), but several potential applications that might improve the value of Brazilian and Mexican propolis should still be investigated.

1. Introduction

The etymology of the word “propolis” comes from the Greek “pro” (in defense of) and “polis” (city/community), which defines this word as a “natural product in defense of the community” [1]. Examples and evidence of ancient use of propolis (“black wax” or “balsam”) for therapeutic purposes are found in biblical records [2]. Use by ancient civilizations aimed different purposes such as embalming antiputrefactive (Egyptians) and medical (Greek and Roman) and antipyretic agent (Incas) [3]. In the 17th century, propolis became popular in Europe for its antimicrobial activity, so London pharmacopeias listed it on the official drug list [2].

In 1908, the first scientific work [4] appeared on the chemical properties and composition of propolis, indexed in the Chemical Abstracts. In 1968, the summary of the first patent, from Romania, appeared in Chemical Abstracts, using propolis for the production of bath lotions (RO 48101) [5]. During wars (South Africa, Russia), propolis was widely used as a wound healing agent and also for tuberculosis treatment. In the 1980s, with the knowledge of its pharmacological properties, propolis began to be industrially incorporated into food and pharmaceutical products (topical applications) as a disease preventative [6].

Mexican propolis is a product that has been used since ancient times and has maintained its appeal status through the years due to its medicinal properties. Since pre-Hispanic times, beekeeping was as important as the cultivation of corn, considering honey as an essential food product for the Maya culture. In Puebla, Mexico, the Nahua community continues to market products such as sweets, eye drops, soap, shampoo, and other cosmetic products whose main ingredients are honey, pollen, and propolis [7]. However, the first scientific studies on Mexican propolis did not begin until the early 2000s. Scientific research is motivated by the great potential and positioning of propolis. In 2017, to establish the quality standards of Mexican propolis, the Secretary of Agriculture, Livestock, Rural Development, Fishing and Feeding established the Official Mexican Standard for the Production and Specifications of Propolis (NOM-003-SAG/GAN-2017-Propolis, production and specifications for its processing) [8].

In Brazil, the first scientific study on propolis was published in 1981 and is related to antibacterial capabilities. Regarding patents, the first deposit in Brazil was made in 1992 (Japanese patent—JP 92033270), while the first Brazilian patent was deposited in 1994 (BR 94042861). As of 2006, the number of patent deposits has increased; however, there are still few deposits made [9,10,11].

Brazil (8,516,000 km²) and Mexico (1,973,000 km²) are large countries in surface area and present different types of biomes (humid jungles, coastal jungle, deserts, mangroves, savannah, and others). In this diversity of ecosystems, there are different species of bees and, consequently, different types of propolis [12]. Both Mexico and Brazil have carried out researches on biological properties of propolis from different locations (Figure 1); however, contamination in Brazilian propolis by herbicides and heavy metals is also reported.

Due to the different beneficial properties attributed to propolis from different geographical origins, the scientific community has been researching on this matrix in different areas in the last decades. Between 2013 and 2017, over 2000 scientific articles related to the study of propolis were published, with investigations on Brazilian propolis corresponding to 16% among a total of 94 countries responsible for all the studies [13].

In this review, the scientific production on native propolis of two countries (Brazil and Mexico) is assessed considering different geographic locations and ecosystems and collecting relevant information on the antimicrobial and anticancer activities of propolis, as well as chemical composition and potential contaminants.

2. Methods

This work gave priority to relevant scientific articles (published from 2010) referring to research conducted using propolis from Brazil and Mexico. For the preparation of tables and figures, data (antimicrobial activity, cytotoxicity, contaminants) reported in the articles were extracted. Graphs were plotted to summarize propolis’ inhibitory activities against microorganisms and cancer cell lines.

Table 1. Number of occurrences of inhibitory concentrations divided in intervals.

Distribution of Brazilian Propolis Minimal Inhibitory Concentrations
Type of Propolis Extract	Concentration (µg/mL)
	0–31.3	50–250	400–1600	3000–10,000	29,000–300,000
Ethanolic Extract of Green Propolis (EtGP)	4	9	5	3	0
Supercritical Extract of Green Propolis (ScGP)	0	2	8	0	0
Ethanolic Extract of Red Propolis (EtRP)	8	11	3	4	3
Supercritical Extract of Red Propolis (ScRP)	0	9	5	1	0
Ethanolic Extract of Brown Propolis (EtBP)	0	2	9	1	0
Supercritical Extract of Brown Propolis (ScBP)	0	0	9	0	0
Distribution of Mexican Propolis Minimal Inhibitory Concentrations
	0–64	100–512	750–3750	7500–15,000	20,000–30,000
Aqueous Extract (Aq)	0	0	20	37	60
Ethanolic Extract (Et)	12	31	54	19	15
Methanolic Extract (Met)	10	15	0	0	0
Distribution of Brazilian and Mexican Propolis Inhibitory Concentration (IC50)
	1–15	15–60	60–100	100–550
Brazilian Propolis	2	23	7	3
Mexican Propolis	8	9	2	3

was generated to classify the data obtained from the different articles validated in this study. For general antibacterial activity (Table 1), the species inhibited by different extracts of Brazilian and Mexican propolis were counted and displayed in 2 graphs, one for the Brazilian and another for Mexican propolis. Figures to show the distribution of Minimal Inhibitory Concentrations (MICs) of each extract were also made (Table 1). For that purpose, all MICs from 13 papers (for Brazilian and Mexican Propolis) were converted to µg/mL and added to a spreadsheet, and then divided in 5 intervals to facilitate visualization. Papers that did not determinate the MIC were excluded. Following, the frequency of a MIC in each interval was measured by counting every time a MIC was found in each interval. In Table 1, the frequency is shown, for example, as follows: the ethanolic extract of Brazilian green propolis had 4 MIC in the interval of 0–31.3, 9 in the 50–250, 5 in the 400–1600, 3 in the 3000–10,000, and none in the 29,000–300,000 µg/mL interval.

The same methodology was used for the cytotoxic activity figures. Cell line and inhibitory concentration (IC₅₀) frequency graphs were made for both countries (Table 1). The different types of cell lines and the times they appeared in 16 papers of Brazilian propolis and in 5 papers of Mexican propolis were listed. Then, the cells were grouped by the human body system they belong to and 2 graphs were made for each country: one for cells grouped in systems and another for the cell lines. The IC₅₀ frequency graph followed the same methodology as the MIC frequency graph, except that it was not divided by the type of extract.

Classification of propolis (color, botanical sources, and chemical composition), extraction methods, antifungal, antiviral, antiprotozoal activity, veterinary application, and propolis contamination were discussed based on tables assembled or discussed directly in the text, based on information obtained from the articles analyzed.

3. Discussion

3.1. Propolis Composition

Propolis is an apicultural product obtained mainly from Apis mellifera bees that has compounds such as wax, resins, and minor constituents, including pollen and minerals [1]. One cannot fail to mention the contaminants commonly found in propolis, such as toxic metals and herbicides [14,15], among others. Salatino and Salatino [16] point out that the composition of propolis declared in many scientific articles for many years (resins and vegetable balsam 50%, bee wax 30%, pollen 5%, and essential and aromatic oils 10%) are generic (without details of the methodologies used to determine these parameters), considering that the composition varies mainly with the geographic location and ecosystem. Propolis samples from Italy [17], Brazil [18], Guinea-Bissau [19], Ethiopia [20], and Morocco [21] had different percentages in this composition. Propolis with wax content above 25% is rejected for commercial use [22].

3.2. Propolis Production in Mexico and Brazil

There is no official information on crude propolis trade. Mexico is among the primary honey producers worldwide and ranks the eighth in exports, being Germany its leading importer. However, Mexico is not among the exporting countries of propolis globally, and the leading exporters of this product are China, Brazil, Argentina, Cuba, Chile, Uruguay, and Canada [23]. Brazilian propolis is highly valued in the international market, and Brazil is one of the largest exporters of propolis globally. Brazilian propolis can reach a value up to three times higher (between 100 and 150 euros per kg) than Chinese propolis, mainly due to the differential in quality [24].

Minas Gerais (MG), Rio Grande do Sul (RS), Bahia (Ba), Santa Catarina (SC) and São Paulo (SP) are the Brazilian states that stand out for their production of propolis. The main market for Brazilian propolis is Asia, 92% of all fresh propolis consumed in Japan is from Brazil. European countries and the United States are also consumers of Brazilian propolis [9,14,25,26,27].

Unlike Brazil, Mexico does not have reliable statistics indicating propolis production, nor does it have updated patents on this beekeeping product. In all regions of Mexico, propolis production is potential, and the propolis producing regions are classified as follows: the Northern Region, Pacific Coast Region, Gulf Region, Altiplano Region, and Peninsula of Yucatán. Compared to the other regions, the Peninsula of Yucatan is more organized, producing 6 tons per year of propolis [28].

In order to encourage the beekeeping industry for the production and regulation of propolis, the Mexican Official Standard for the Production and Specifications of Propolis, NOM-003-SAG/GAN-2016 [8], was approved by the Federal Commission for Regulatory Improvement (COFEMER) and recently promoted, regulating the product’s quality and its sale at competitive prices in the international market. In Brazil, regulation of propolis production is promoted by a specific Normative Instruction—SDA No. 03, of 19 January 2001 [22]—Technical Regulation of Identity and Quality of Apitoxin, Bee Wax, Royal Jelly, Lyophilized Royal Jelly, Bee Pollen, Propolis and Propolis Extract (https://www.gov.br/agricultura/pt-br (accessed on 23 October 2021). However, this regulation lacks many specifications regarding the production and quality of propolis.

3.3. Propolis: Color, Botanical Sources, and Chemical Composition

The coloration of propolis will depend on the plant’s origin and its ecosystem since bees collect substances from plants. For example, the Brazilian green propolis is derived mainly from the species Baccharis dracunculifolia. On the other hand, in the Gulf Region of Mexico, the majority of propolis found is from the green variety, unlike the northern region, where the ecosystem is more desertic than the other regions, and mesquite (Prosopis laevigata) is a predominant vegetable source [29]. Each plant species has its secondary metabolites responsible for its biological activities, and propolis contains the metabolites of the plant that will define its biological activity. In Mexico, the most remarkable locations with diversity of tropical vegetation are in the Gulf and Pacific Coasts of the country; for this reason, a diversity of propolis colors is seen (chestnut-green, red, yellow-red, dark yellow, dun, or black) [28].

In Brazil, the classification of propolis by color is strongly associated with its botanical source [30]. Park et al. [31] classified propolis samples collected in the country (except the north region) into 12 groups, according to the appearance and color of the extracts. Subsequently, a new propolis variation was found in hives along the coast and mangroves in northeastern Brazil, which was classified as group 13, standing for red propolis [32].

In the southern region of Brazil, five types of propolis were described according to the classification of color (yellow, light brown, dark brown, greenish-brown, and reddish brown). In the northeast region, 6 were described (greenish-brown, dark brown, yellow, dark yellow, and red), and in the southeast region one type of propolis was described as green (or greenish-brown). It is noteworthy that these regions have diverse and varied ecosystems (Figure 1a), so although the name of the color may be similar, the composition of propolis is different mainly due to the different botanical sources. Propolis is a complex mixture, and more than 300 chemical moieties have been identified [33].

According to the verified articles (Tables S1 and S2 of supplementary material) [33], Brazilian and Mexican propolis composition analyses found many shared compounds, even considering different geographic regions, ecosystems, and types (color differences) [34,35,36,37,38,39,40,41,42,43]. Many of these chemical compounds stand out for their biological activity, for example, phenolic acids, phenolic acid esters, flavonoids, terpenoids, luteolin, galantine, trans ferulic acid, caffeic acid, chrysin, pinobanksin 5-methyl ether, quercetin, apigenin, kaempferol, naringenin, rutin, catechin, p-coumaric, pinobanksin, pinocembrin, and pinobanksin 3-acetate, and many others [21,25,26,27,28,29,30,31,32]. On the other hand, some compounds that are considered propolis-type markers, such as in propolis from Brazil, are artepillin C (3,5-diphenyl-4-hydroxycinnamic acid) for green propolis [44,45,46,47] and formononetin (3-hydroxy-8-9-dimethoxypterocarpan) for red propolis [32,48,49,50]. These chemical markers described for each propolis are phytologically associated with the main botanical source. The primary source of green propolis is wild rosemary (alecrim do campo—Baccharis dracunculifolia) and rabo-de-bugio or marmelo-do-mangue (Dalbergia ecastaphyllum (L.) Taub), with 35.68% of artepillin C [51], while the red propolis presents 67.59% formononetin in its chemical composition [49,50].

In the case of brown propolis, its composition varies according to several botanical sources (Eucalyptus, Mimosa caesalpiniaefolia, Mimosa scabrella, Cecropia, Anacardiaceae, Asteraceae, Citrus, Cocos and Poaceae, Populus), without a specific marker described [52,53].

In Mexico, among the botanical sources described for red propolis, in the Gulf, Pacific Coast, and Peninsula of Yucatan regions are Bursera simaruba (L.) Sarg, Dalbergia glabra, Cordia alliodora, Cardiospermum halicacabum, Dombeya wallichii, Antigonon leptopus, Sapindus saponaria, and Dalbergia species [54,55,56]. Valencia et al. [57] refer to brown-green-ocher (chestnut-green) propolis that differs in composition from the Brazilian green propolis, despite the similar color-based nomenclature. Distinctive chemical compounds are present due to botanical sources such as Encelia farinosa, Ambrosia deltoidea, Ambrosia ambrosioides, Bursera laxiflora, Populus fremontii S. Prosopis laevigata, and Acacia greggii. However, this propolis contains compounds also found in other types of propolis, such as gallic acid, cinnamic acid, p-coumaric acid, naringenin, quercetin, luteolin, kaempferol, apigenin, pinocembrin, pinobanksin 3-acetate, CAPE, chrysin, galangin, acacetin, and pinostrobin [29].

3.4. Extraction Methods Commonly Used to Produce Propolis Extracts

Usually extractions are carried out with different solvents [58]. These extracts can be prepared by maceration of crude propolis with solvents. Solvents such as water [59], methanol [60], ethanol (50–80%) [26,34,61], chloroform, and ethyl acetate [60] are commonly used, with ethanol (70–80%) being the most used extraction method, whether in research or industrially [12]. In recent years, the process of extraction with supercritical fluid (SFE) has been used as an alternative to obtaining propolis extracts, usually applying as the solvent CO₂ (which has low cost, is easily available in high purity levels, non-toxic, non-flammable, and non-explosive) [62].

This process has high selectivity and reduced use of organic solvents, allowing the obtention of extracts with high biological value compared to conventional ethanolic extraction [34,35,39,63]. To improve the performance of SFE, many authors use co-solvents (methanol, ethanol) to intensify the extraction of compounds with a lower lipophilic nature in propolis, increasing the yield of the extractive process with CO₂ and obtention of relevant compounds [62,63]. Thus, the data reported on the evaluation of propolis extracts in various biological functions are generally associated with these extraction forms. Ultrasound-assisted extraction (UAE) is considered a green and economically viable alternative to conventional SC-CO₂ techniques for propolis extraction [64]. Promising results have been reported for successfully extracting bioactive constituents from propolis using microwave irradiation [65,66]. Commercially, most propolis extracts are ethanolic.

In most studies, SC-CO₂ extracts have been compared to ones obtained with ethanol using maceration and UAE. The lower polarity of SC-CO₂ expectedly resulted in the low content of bioactive compounds in extracts from different propolis types [34,35,67,68,69]. On the other hand, in experiments with propolis from different origins, some selectivity of SC-CO₂ to flavonoids [70,71], artepillin C, and p-coumaric acid from green Brazilian propolis [34,72] has been detected. SC-CO₂ extraction has been suggested as a pretreatment of propolis before a second classic extraction with ethanol, and this two-stage process resulted in an ethanol extract with high concentrations of active components [73,74].

While several scientific works applied mainly ethanol and supercritical extraction in Brazil, the works in Mexico are based on ethanol extractions, as seen in the evaluation of antimicrobial activity (Figure 2, Figure 3, Figure 4 and Figure 5).

3.5. Antibacterial Activity

From the analysis of articles published in the last ten years, graphs were generated to show the number of bacterial species treated with different concentrations of extracts (Figure 2 and Figure 3).

Figure 2A shows that among the 3 colors of Brazilian propolis represented, the red propolis had a broader antibacterial activity, affecting more species. This type of propolis was the most studied one in the last decade, followed by the green and brown propolis extracts, respectively. Red propolis was characterized in the first decade of the 2000s; thus, more related scientific works emerged. Brown propolis had a very low spectrum of species affected, and the ethanolic extract of green propolis had an intermediate range. All supercritical extracts had a low variability of species affected. Figure 2B shows the MICs grouped in 5 different intervals.

Regarding the distribution of the MICs, it was verified that the supercritical extract (Sc) of brown propolis inhibited microorganisms only in the 400–1600 µg/mL concentration range (Figure 2B). The ethanolic extract (Et) of green propolis had a good inhibitory effect in low concentrations, as more than half of occurrences was on the 3.8–250 µg/mL interval. The red propolis extracts, mainly the ethanol extracts, had a higher MIC spectrum (3.8 to 250 µg/mL), followed by the green and brown propolis extracts, although 2 thirds of occurrences were in the 3.8–250 µg/mL range. The Sc extract was not so effective in all the 3 colors of propolis.

Scientific work related to the Mexican propolis extracts is limited for alcoholic (methanolic and ethanolic) and aqueous extracts. The ethanol extract was the most used one to verify the antibacterial activity of propolis, reaching a total of 16 species (Figure 3A). The ethanol extract showed a higher MIC spectrum when compared to other types of extracts (Figure 3B). The methanolic extract had all its MIC on the 100–512 µg/mL range, and the aqueous extract was only effective in high concentrations (7500–30,000 µg/mL). In comparison with the Brazilian propolis, the ethanolic extract of Mexican propolis was also the most used one, but Mexican propolis was effective in higher concentrations.

Many variables interfere with the antibacterial capacity or potential of a propolis extract, mainly geographic region, flora, and the species of bee. Some studies indicated that the compounds found in propolis extract act on the cell wall of bacteria and the function of the ribosome-inhibiting protein synthesis and consequently bacterial growth (cell division), and disorganize the cytoplasmic membrane and cell wall, causing partial bacteriolysis (permeability alteration). The antibacterial mechanism is associated with chemical compounds of propolis already described in the literature as presenting biological activities, such as phenolic compounds/flavonoids (pinocembrin, galantine, caffeic acid, pinobanksin, artepillin C, ferulic acid, umbellic acid, p-coumaric acid, kaempferol, catechin, epicatechin, formononetin, isoformononetin, luteolin, naringenin, calycosin, quercetin, and others) [12,35,37,44,56,89,90].

In Figure 4 and Figure 5, the antimicrobial effects of Brazilian and Mexican propolis in Gram-positive and -negative bacteria were compared according to the distribution of the minimal inhibitory concentrations (MICs). In scientific articles carried out with Brazilian propolis, it was observed that the Gram-negative bacteria were significantly affected by the action of different extracts applied at concentrations up to 1600 µg/mL. The Gram-positive bacteria show a greater variation in MICs, where supercritical extracts reach the concentration range of 400–1600 µg/mL. EtGP, EtRP, and ScRP extracts have a higher frequency of inhibition concentrations between the range of 50 to 250 µg/mL.

In the case of Mexican propolis extracts, it is verified that ethanol extracts have a greater concentration spectrum for inhibition of both classes of bacteria, and the concentration range from 0 to 3750 µg/mL presents a higher frequency of inhibition. Aqueous extracts are the least efficient in terms of MICs.

As expected, the extracts from both Brazilian and Mexican propolis samples showed higher activity against the Gram-positive strains than against the Gram-negative strains. These results following those from several authors, which can easily be explained by the structural differences of the bacterial cell wall [91,92,93,94,95,96,97]. Compared to the extraction method, EtOH extracts showed the best antimicrobial activities, and as previously shown, these extracts also had the best antioxidant activities and the highest content of total phenolic acids and flavonoids. Although the mechanism of action of these compounds in the antimicrobial function of propolis is poorly understood, some studies suggest that certain isolated constituent compounds have antimicrobial activity [98]. Among the more than 300 compounds already identified in different propolis, the following are examples. (1) Gallic acid and ferulic acid can cause irreversible changes in membrane properties and consequently the occurrence of local rupture or formation of pores in cell membranes, causing leakage of essential intracellular constituent substances. Furthermore, ferulic acid enhances the antibacterial activity of quinolone antibiotics against A. baumannii [99,100]. (2) Artepillin C has bacteriostatic activity with membrane blebbing [101]. (3) Cinnamic acid and its derivatives inhibit bacteria by division of the cell membrane, inhibiting ATPases, cell and biofilm formation [102]. (4) Catechins in vitro studies have demonstrated antimicrobial effects in bacteria (G+ and G−) and have been reported as effective antivirulence agents [103].

3.6. Antifungal Activity

The increase in mycosis and the appearance of resistant fungal strains in patients has been increasingly observed throughout the world. This has generated the search and development of new compounds that meet the requirements of an antifungal and in that sense, Mexico and Brazil have studied the antifungal properties of ethanolic extracts of propolis in different species of yeast [104] and filamentous fungi [105,106].

Table 2. Antifungal properties of propolis of different regions of Brazil and Mexico.

Microorganism	Color	Region of the Country	Extraction	MIC (μg/mL)	Reference
Fungi Mexico
Candida albicans	ND	South	Ethanolic	32	[106]
	ND	Southeast	Ethanolic	1.6–2.30	[107]
	Brown	Southeast	Ethanolic	750
		Center	Ethanolic	750–2400	[85]
Cryptococcus neoformans	ND	South	Ethanolic	32	[106]
Candida albicans (ATCC 14065) Aspergillus flavus	Green, Brow, Yellow	Southeast	Ethanolic	1.6–2.30	[108]
Aspergillus fumigatus	ND	Center	Ethanolic	32	[106]
Fungi Brazil
Candida albicans	Red	Northeast	Ethanolic/Sc-CO2	≥1000	[26]
	Red	Northeast	Ethanolic/Sc-CO2	1.56	[76]
	Brown	Northeast	Ethanolic/Sc-CO2	≥1000	[105]
Malassezia pachydermatis	Red	Northeast	Ethanolic/Sc-CO2	4000–8000	[105]
	Green	Southeast	Ethanolic	4000–8000
	Browns	South	Ethanolic	≥16,000

ND. Not declared.

shows these various studies and as can be seen, most studies have been restricted to the study of ethanolic extracts of propolis against Candida albicans. Likewise, when comparing the antifungal activity of propolis from both countries, it is observed that the extracts from Mexico require a lower concentration to achieve antifungal activity.

3.7. Antiviral Activity

Around the world, many types of research with antiviral activity (human immunodeficiency virus (HIV), adenovirus, HSV-1, HSV-2, Newcastle virus disease, bovine rotavirus, pseudorabies virus, canine adenovirus type 2, feline calicivirus, bovine viral diarrhea virus, influenza virus types A and B, parainfluenza virus, infectious bursal disease virus, and avian reovirus) have already been conducted [109,110,111,112,113,114].

Few scientific articles with the antiviral activity of propolis extracts have been verified. For example, González (102) studied the antiviral effect of Mexican propolis, when it has been evaluated different treatments with propolis, flavonoids individually, and a mixture of the three flavonoids that are normally present in propolis (quercetin, naringenin, and pinocembrin). They demonstrated that the best antiviral activity was the administration of propolis or the mixture of flavonoids than the individual compounds. On the other hand, Brazilian propolis has been verified in MS2 and Av-08 bacteriophages showing antiviral reduction ~3 and ~4.5 Log 10 PFU/mL respectively [115,116]. In addition, in the current COVID-19 pandemic, propolis from Brazil, specifically a standardized green propolis extract (EPP-AF^®), has been evaluated in clinical treatment, using doses of 400–800 mg/day of the preparation, and a reduction in hospitalization time has been observed [117].

3.8. Antiprotozoan Activity

Some scientific articles have identified the effect of different concentrations of extracts (methanolic, ethanolic, and supercritical) of different propolis (brown, green, and red) against protozoa were identified. The extracts showed good inhibition against these organisms (Trypanosome brucei, Trypanosoma cruzi Y, Leishmania (V.) braziliensis, Leishmania amazonenses) particularly when using the green and red propolis extracts from Brazil [26,34,35,37]). Red propolis extract showed a better reduction of L. (V) braziliensis comparing with green propolis extract, corroborating the results that red propolis had more effective antioxidant activity [26,34,35,64,118].

For Mexican propolis, only one scientific report on the effect of propolis on protozoa was found, and a good inhibition of Giardia lamblia was verified.

3.9. Antiproliferative and Cytotoxic Activity

Propolis cytotoxic activity has been extensively studied, particularly in cancer research [119]. The chemical composition of propolis is well known and quite varied. Some compounds found in different types of propolis have been reported and associated with cytotoxic activity [120,121]. This cytotoxic and antiproliferative activity against tumor cells may not be due to isolated compounds nonetheless to the synergism between all the compounds in propolis [98]. Many of these compounds that are associated with these activities have previously been identified with antioxidant capabilities [122].

Figures showing the comparison of the cell lines used in researches carried out with Brazilian propolis are presented (Figure 6A,B). A greater variety of cell types was observed where the cytotoxicity of the extracts was tested, especially the cells of the reproductive system (≈40%). Among the articles verified, more than 80% of the cytotoxicity tests with the different cells were carried out with different types of extracts (ethanolic and supercritical) of red propolis, originating from the northeast region of Brazil (SE, AL, BA, RN). As mentioned before, in the last decade, studies with this type of propolis gained notoriety. The most used cell lines in the tests were HCT-116, OVCAR-8, SF-295, HL-60, PC3 [50,64,123,124,125].

In studies related to the use of Mexican propolis extracts (Figure 7A,B) with cytotoxic activity, it appears that most of the research was carried out on murine cells (40%) and cells of the reproductive system (40%). The M12.C3.F6 murine cell line was the most used on tests.

Altiplano region propolis was its effectiveness proven compared with anti-cancer drugs and show an antiproliferative effect on glioma cells better than temozolomide, despite this proliferation and viability in cervical cancer cells (HeLa, SiHa, and CaSki) is lower than cisplatin [41]. The method used in the study was the MTT test, being this the most frequently used test to analyze the metabolic activity of a cell and evaluate its cytotoxic activity.

In the northern region of Mexico, murine B cell lymphoma (M12.C3.F6 cancer cell line) has been reported to evaluate the antiproliferative activity on cancer cells in Sonora desert propolis due to its high sensitivity to anti-cancer drugs [57,137,138] having excellent results. It has been proven that propolis and its components show proapoptotic activity inducing extrinsic and intrinsic pathways for cell apoptosis [119]. However, desert propolis samples showed a low antiproliferative activity on the murine normal cell line L-929, demonstrating that propolis shows a much lower antiproliferative effect in murine non-cancerous than in murine cancerous cell lines [137]. Another main result is that the antiproliferative effect is affected by quantitative fluctuations in its desert propolis polyphenolic profile due to its collection time [138,139]. Sonora desert propolis has been extensively studied by Li et al. [140,141]. Results showed more cytotoxicity against human lung adenocarcinoma (A549 cells) than with other five cancer cell lines, and PANC-1 human pancreatic cancer cells observed resolute preferential cytotoxic activity with similar results in propolis from Brazil [126]. The compounds with potent and preferential toxicity in PANC-1 is (6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan and (7″R)-8-[1-(4′-hydroxy-3′-methoxyphenyl)prop-2-en-1-yl]galangin in Brazilian and Mexican propolis, respectively.

The antiproliferative effect of propolis extracts generally depends on their chemical composition, as described above. Chemical compounds present in various types of propolis, to which biological activities are related, could act as powerful cytocidal effects and induced levels of apoptosis in all the cellular lines [122,143,144,145].

The antitumor action of propolis (from the northern region, specifically from Sonora, Mexico) has been reported in several studies showing antiproliferative effects in humans (pancreatic cell lines, B-cell lymphoma, human lung carcinoma, human colon adenocarcinoma). Likewise, various studies have shown that propolis from southern Mexico has anti-inflammatory activities, inhibiting TNF alpha and stimulating interleukin 10 system [137,138,142].

In the case of studies carried out with different Brazilian propolis, 35 different inhibitory concentrations were found (in some cases, the same article tests several different cells and propolis) and 22 with Mexican propolis. Figure 8 shows that 63% of the IC50 studies were in the concentration range of 15–60 µg/mL, whereas for Mexican propolis this concentration range represented 41%. It is observed that Mexican propolis in the verified articles presented an inhibition range of 1 to 60 µg/mL for 77.3% of the tested samples, thus demonstrating a better IC50 for this type of studies.

3.10. Veterinary Application

With scientific proof of the positive effects of propolis associated with antimicrobial, anticancer activities, and others, its application is also successful in the veterinary field [146]. The application of propolis extracts is associated with producing a vehicle, usual formulations of topical ointments, and soap, which are mainly used with an antimicrobial function. Examples of the recent application of propolis in the veterinary field can be seen in

Table 3. Veterinarian products with propolis extract.

Veterinary Diseases	Microorganisms	Product with Propolis Extract	Author
Effect canine dermatophytosis effect in dogs	Microsporum gypseum Microsporum canis	Soap	[147]
Treatment of Dermatomycosis in Horses	Trichophyton mentagrophytes Candida albicans	Shampoo	[148,149]
Post-surgical Treatment of Caseous Lymphadenitis in Sheep	Corynebacterium pseudotuberculosis	Ointment	[79]

.

4. Propolis Contamination

4.1. Xenobiotics in Propolis

Honeybees and their products are considered bioindicators of environmental pollution [150,151,152]. Xenobiotic substances, such as inorganic compounds, bactericides, fungicides, insecticides, herbicides are deposited on the soil when sprayed in the air, and can be transported to the hive through bee food or in other necessary compounds (water, nectar, pollen from trees and flowers, etc.) where they can remain for years [153]. Hence, propolis can be polluted directly or indirectly for pesticides and toxic metals of anthropogenic origin [154].

4.1.1. Pesticides in Brazilian and Mexican Propolis

Propolis can be polluted in direct or indirect form principally for pesticides and heavy metals of anthropogenic origin. Pesticides and heavy metals have been reported in many Hispanic and Portuguese countries.

Much of the bee production is found close to regions where conventional crops (soybeans, corn, wheat) that use herbicides regularly are found [155]. These crops can serve as a source of raw material for propolis production by bees, or they can be close to their range of action. According to Moreira et al. [156], a significant part of the agricultural production areas are close to the different biomes of each region. Due to the contamination of these productive areas and the volatilization of pesticides, such as contaminating precipitation.

Pesticides (mainly systemic herbicides) can be deposited on plants and trees. These plants can absorb toxic compounds through their leaves or roots and translocate them to other parts of the plant, such as buds [157,158,159], so bees will come into contact and can transfer these compounds to bee products, including propolis.

Studies of pollutants in Mexican and Brazilian propolis are very scarce. As shown in

Table 4. Pesticides research in Brazilian and Mexican propolis.

Mexican Propolis
Number of Samples	Geographical Origin	Elements Analyzed	Toxic Elements Found	Analytical Methods	References
N/E	South	Dichlorvos, Diazinon, Methyl parathion, Malathion, and Coumaphos	No found	GC/MS-SIM	[164]
4	South	Dichlorvos, Diazinon, Methyl parathion, Malathion, and Coumaphos	Organophosphate pesticides (≤025 μg/mL)	GC-MS	[160]
Brazilian Propolis
19	Northeast (Ba, Se, Al); South (SC, Pr). Southeast (MG) and Centersouth (MT)	AMPA Atrazine Glyphosate Picloram	AMPA (10.2 ± 1.39–11.3 ± 2.65) Atrazine (9.7 ± 0.11–17.4 ± 2.60)	HPLC	[15]
50	South (SP)	Organochlorines, Organophosphates, Pyrethroids, Carbamates, Herbicides, Fungicides, and Acaricides any pesticide	ND	(GC/ECD), (GC/NPD)	[165]

ND. Not detected.

, there are two scientific works in each country regarding the verification of the presence of pesticides. Among the various pesticides investigated, only Organophosphate [160] was found in a propolis sample from southern Mexico. In a study carried out with propolis from Brazil [15], they verified the presence of AMPA and Atrazine in 32% of the samples evaluated.

Pesticides in Chilean raw propolis detected triadimefon on concentrations in mg/kg⁻¹ until to 0.95 and Dicofol 0.54 and in Spanish propolis triadimefon 2.65, dicofol 1.2, dichlofluanid 0.38, procymidone 0.36, Folpet 3.74, and metazachlor 1.3 mg kg⁻¹ [161]. In processed propolis (capsules, tablets, tinctures, candies, and syrups) from Spain, Portugal, and Chile report pesticide in mg/kg of quintozene 1.06, procymidone 0.11, metazachlor 6.09, folpet 11.31, dichlofluanid 0.29, and chlorfenson 1.05 [162]. Other study found coumaphos in an Argentinean propolis candy sample of 0.36 mgkg⁻¹, whereas chlorpyrifos was detected at 0.02 mg/kg in one Uruguayan sample [163].

Generally, if there is a presence of toxic compounds in propolis, other bee products, mainly honey, can be contaminated. Valdominos–Flores et al. [166] present a complete pesticide analysis in honey and wax from Altiplano, Northern, and Peninsula of Yucatan in the highest concentrations were the phenyl phenol and the organochlorine 2,4′-DDT in the south; malathion, chlorpyrifos, phenyl phenol, and thiabendazole in the Northern Mexico; and chlorpyrifos and imidacloprid in Altiplano. These results demonstrate the pesticide exposure in the Mexican beehives. In Brazil in 1% and 44% of honey samples evaluated were found pesticides [167,168], respectively. Oliveira et al. [167] found pesticides in 7% of pollen samples analyzed.

Overall, environmental contamination by agrochemicals is being associated as a significant factor in the decline of insects, including bees and other pollinators [169,170,171]. Researchers relate to evidence of a decrease in the population of bees and even their mortality to exposure to pesticides [172,173,174,175,176].

4.1.2. Toxic Metals in Brazilian and Mexican Propolis

As described above, propolis is composed basically of a mixture of substances that can be contaminated by metals of different sources such as bees, air, water, plants, and soil [26,27,28]. Anthropogenic actions (industrial activities, mining, increased urbanization, fertilizers, and pesticides) or natural process (eruptions, leaching) also contribute to the contamination of the environment and consequently bees and their products [9,28,30].

In the survey of studies referring to toxic metals in samples of Brazilian propolis, six studies were found (

Table 5. Brazilian propolis toxic metals contamination.

Number of Samples	Toxic Elements Found	Analytical Methods	References
19	Pb, Cd, As, Cu	GFAAS and FAAS	[14]
106	Hg, Cd, Pb, Sn	FAAS	[177]
6	Pb, Cd, Cr	FAAS	[178]
42	Cd, Cr, Pb	FAAS	[179]

FAAS—flame atomic absorption spectrometry; GFAAS—graphite furnace atomic absorption spectrometry; AS–atomic absorption spectrophotometry.

). Lead and cadmium are the toxic metals found in most conducted researches. Other toxic compounds found were As, Cr, and Cd. Hodel et al. [14] found As, Cd, and Pb in 26.3%, 5.2%, and 73.9% of the 19 samples evaluated.

Substances originating from plants as raw material for propolis production (resins) can be sources of contamination, as mentioned above. Researchers have shown that the primary botanical sources (Baccharis dracunculifolia) for the production of propolis can accumulate cadmium [180], turning this plant into a possible source of contamination. Other sources of this toxic metal can be agricultural inputs, such as fungicides, used in plantations in Santa Catarina (SC) and Rio Grande do Sul (RS) [31,34,181,182], where Cd was found in pollen samples.

Heavy metals in Argentinian raw propolis have been detected concentrations in ppm of Zn (15.2), Pb (7.73), and Cu (2.45) [183]. Although, in the research by González–Martín et al. [162], in Chilean and Spanish processed propolis have been reported heavy metals, such as, Cr (17.7), Ni (7.01), Cu (6.44), Zn (6.44), and Pb (7.21 ppm).

In Brazil, the maximum levels for metals in crude propolis are not defined in this regulation, but it states that inorganic contaminants, such as Cd, As, and Pb, should not be present in propolis in higher amounts as defined by RDC regulation number 42 by ANVISA for honey [184].

In Mexico, publications on heavy metals in propolis are limited to that of Montiel et al. [185], where the highest concentrations were reported in ppm in an agricultural area Pb (4.75), in urban area Cd (3.87), and in rural area Cr (4.74), all in the altiplano region of Mexico. Compared with the Brazilian propolis, these results are higher in Cd, but Cr and Pb present lower results [177,179]. The Mexican and Brazilian regulations do not specify permissible limits for heavy metals or pesticides. Notwithstanding, in Latin America, the Cuban, Salvadoran, and Argentine regulations specify that permissible limits for lead and arsenic are a maximum of 2 and 1 ppm, respectively. Both Mexican and Brazilian propolis have been current samples that exceeded the limits of Pb [186,187].

5. Conclusions

The differences in scientific production between the two countries regarding the same product are notorious. Both countries’ geographic and ecosystem differences provide propolis with different chemical profiles and well studies in terms of chemical markers (in the case of Brazil). These chemical composition characteristics of propolis help in its commercial valorization. The biological potential was explored differently in terms of forms of extraction and application of the obtained extracts. In terms of antimicrobial activity, the number of species studied was higher with Brazilian propolis (all types and forms of extraction) and its cytotoxic activity, considering the variety of cells tested. The majority of the biological studies in both countries present findings very promising to combat important health problems, however, data in both countries indicate that more research is still needed in order to determine the optimal concentrations of propolis or its components, period of intake or type of extract to use before it can be administered to humans. In terms of contamination, information is still incipient, as propolis is considered an environmental bioindicator, so it needs to be better studied. New studies are needed in all areas to value propolis from a commercial and a scientific point of view.