The chemical composition of propolis is highly dependent on the conditions of the location and the chemical constituents of the botanical source (Bankova et al. 2018). One production center of BP is the southern region of Brazil. Forty-four samples of BP from Paraná and the Santa Catarina States were analyzed to identify the regional identity of these locations. Their compounds were identified and quantified by comparing their chemical profile with authentic standards (Machado et al. 2021). According to this study, the BP from Southern Brazil is chemically characterized by caffeoyl-quinic acids (range, 11.14 − 21.45 mg/g), p-coumaric acid derivatives (6.27 − 12.17 mg/g), flavonols (9.35 − 23.55 mg/g), followed by benzoic acid derivatives (3.18 − 7.45 mg/g), and dihydroflavonols (0.17 − 4.25 mg/g).

Phenolic compounds already reported in Brazilian green propolis have been found on standardized BP extracts, like p-coumaric acid (1), drupanin (2), artepillin C (3), and baccharin (4), revealing similarity on phenolic profile between these two propolis types (Fonseca et al. 2011). According to Rodrigues et al. (2016), besides those traditional green propolis chemical markers, BP can also present lower concentrations of propenoic and cinnamic acid. Other phenolic compounds like chrysin, pinocembrin, galangin, caffeic acid phenylethyl ester, and pinobanksin-3-O-acetate have also been detected by HPLC–UV-ESI–MS analysis in Brazilian propolis samples (Fabio et al. 2019).

An RP-HPLC–DAD–ESI–MS/MS was used to chemically characterize a BP sample from Paraná (Araújo et al. 2020). The raw material was submitted to extraction and partition with different solvents in a Soxhlet system, revealing the prevalence of some classes of compounds in the fractions. In all fractions, artepillin C, baccharin, and 3-hydroxy-2,2-dimethyl-8-prenyl chromane-6-propenoic acid were identified, although the hexane fraction was enriched with propenoic and cinnamic acid derivatives like drupanin; the hexane:ethyl acetate fraction presented propenoic and cinnamic acid derivatives, and flavonoids as kaempferol and quercetin. In comparison, the ethyl acetate fraction was rich in flavonoids, chlorogenic acids, and quinic acid esterified by one or more units of cinnamic, p-coumaric, caffeic, or ferulic acids. The methanol and aqueous fractions contained quinic and caffeoyl acid derivatives.

Furthermore, diterpenes as isocupressic acid (5), (E)-communic acid (6), (Z)-communic acid (7), and abietic acid (8) have been isolated from samples of BP from Paraná State (Tazawa et al. 2016). The presence of the 15-acetoxyisocupressic acid and an unreported diterpene, the rel-(5S,6S,8R,9R,10S,18R,19S)-18,19-epoxy-2-oxocleroda-3,12(E),14-triene-6,18,19-triol 18,19-diacetate 6-benzoate, were reported on BP ethanolic extract (Santos et al. 2021).

Capillartemisin A and caffeoylquinic acid derivatives like 3,4-di-O-E-caffeoylquinic acid, the 3,5-di-O-E-caffeoylquinic acid, O-hexosyl-caffeoyl dihydrocaffeate, 4,5-di-O-E-caffeoylquinic acid, and O-E-coumaroyl-caffeoylquinic acid were detected in Minas Gerais (Southeast region) BP by LC-DAD-MS analysis (Dembogurski et al. 2018). Brown propolis from Mato Grosso also furnished acetylisocupressic acid, dihydro-p-coumaric acid (9), caffeic acid (10), and aromadendrin (Fernandes et al. 2019). Analysis by UPLC-MS of the hydroalcoholic extract of BP from Rio Grande do Sul (South region) revealed the presence of rutin, chlorogenic acid, ferulic acid, and caffeic acid (Waller et al. 2017).

Another area of production of BP is the northeast region. A sample from Bahia State was submitted to extraction with hexane, methanol, and dichloromethane, and the chemical profile was assessed by CG-MS (Santos et al. 2017). The hexane fraction was composed of pentadecane, hexadecane, heptadecane, and tricosane. Methyl cinnamate and sitosterol cinnamate were isolated from the hexane fraction, and ananixanthone was isolated from the dichloromethane fraction.

The GC–MS chromatographic analysis of BP from Piauí (Northeast region) displayed the triterpenes lupeol, germanicol, β-amyrin, hop-22-(29)-en-3-one, and lupenone for hexanic fraction, and for dichloromethane fraction, the compounds 2,3-dihydroxybenzofurane, lupeol, and dodecanoic acid. In ethyl acetate fraction, p-coumaric acid and 3,5-dihydroxybenzoic acid were identified (Santana et al. 2014).

Samples from Paraná (South) and Ceará (Northeast) state were submitted to the acid/n-butanol hydrolysis method to detect proanthocyanidins and their quantification by precipitation with BSA (bovine serum albumin). All the samples had a positive reaction for proanthocyanidins with low tannin content values (between 0.6 and 1%) but without a complete chemical characterization (Mayworm et al. 2014).

The volatile oil from BP has been characterized by several studies, showing differences in the chemical profile of propolis from different locations. Headspace solid-phase micro-extraction (HS-SPME) and GF-MS analysis helped identify more than 315 volatile compounds in BP from Bahia, Minas Gerais, Paraná, and Sergipe States (Olegário et al. 2019). Terpenes were the predominant class of compounds in all samples, followed by the aldehydes.

The sesquiterpenes β-caryophyllene (11) and humulene were the most abundant compounds in the BP sample from Bahia state (Olegário et al. 2019); acetophenone, (R)-α-pinene, and ( +)-δ-cadinene (12) were predominant in the Paraná sample; d-limonene and nonanal were the major compounds in the Sergipe state sample (Northeast region).

Brown propolis from Minas Gerais sample displayed sesquiterpenes (33.62%), oxygenated sesquiterpenes (26.98%), and oxygenated monoterpenes (18.99%) (Ribeiro et al. 2021a, b, c). Quantification differences were observed for samples from this state, although the sesquiterpenes β-caryophyllene and α-copaene (13) were predominant. 1,8-Cineol (14), terpineol-4-ol (15), nerolidol (16), spathulenol (17), δ-cadinene, aromadendrene (18), γ-muurolene (19), and the alkyl-phenylketone, acetophenone have also been reported (Lima et al. 2019; Olegário et al. 2019; Ribeiro et al. 2021a, b, c; Símaro, et al. 2021).

The volatile oil of BP from Mato Grosso do Sul displayed (E)-caryophyllene, δ-cadinene, spathulenol, α-copaene, (E)-nerolidol, and aromadendrene, with the prevalence of viridiflorene and trans-α-bergamotene (Fernandes et al. 2015).

One of the major bottlenecks in propolis studies is the elucidation of its botanical sources. The botanical source visited by bees is directly related to the chemical composition of propolis, impacting its pharmacological properties. Many publications on propolis do not describe its type, botanical sources, and even its chemical composition, making it difficult to standardize this medicinal product.

Several approaches are aiming to find the probable botanical source of propolis under study in the literature. One of the approaches is to observe bees in the field, as Apis mellifera bees collect red exudates from Dalbergia ecastaphyllum to produce red propolis, which was confirmed by the chemical similarity between the plant exudate and propolis (Daugsch et al. 2008). Another approach is the identification of chemotaxonomic markers and correlating them with botanical species near the hives, which led Ccana-Ccapatinta et al. (2020) to describe Symphonia globulifera as the source of benzophenones in Brazilian red propolis. Some researchers use palynology for botanical identification of propolis, but bees visit many plants for nectar collection and a few plants for resin collection, making it challenging to identify the botanical source for propolis production (Freitas et al. 2011). Currently, the metabolomics associated with techniques such as UPLC-MS/MS has shown to be an essential tool in propolis’s chemical prospection to identify its origin.

The botanical origins of some Brazilian propolis were well-established, as Baccharis dracunculifolia for green propolis and Dalbergia ecastaphyllum and Symphonia globulifera for red propolis. Several plants have been described as responsible for their composition regarding brown propolis, as Pinus spp., B. dracunculifolia, Eucalyptus spp., and Araucaria angustifolia (Freitas et al. 2011; Frota et al. 2021; Ribeiro et al. 2021a, b, c; Santos et al. 2021; Serafim et al. 2022).

Baccharis dracunculifolia, popularly known as “alecrim-do-campo,” is largely distributed in South America from Southeastern Brazil to Argentina and Uruguay (Ribeiro et al. 2018). It is the primary plant source of Southeastern Brazilian propolis, called green propolis, because of its color. Green propolis contains high levels of prenylated p-coumaric acids, mainly artepillin C and baccharin, and the volatile compounds nerolidol and spathulenol, all found in B. dracunculifolia (Beserra et al. 2021; Bernardes et al. 2022).

Due to its geographic location, B. dracunculifolia is found mainly in BP samples collected in the southeastern region of Brazil, and there are other possible associated botanical sources, making it brown. The phenolic acids of B. dracunculifolia and its volatile components nerolidol and spathulenol are described in several BP (Dembogurski et al. 2018; Araújo et al. 2020; Ribeiro et al. 2022). Compounds not described for Baccharis are usually identified in phytochemical studies, thus evidencing the participation of other plants in the production of brown propolis (Beserra et al. 2021).

Diterpenic acids reported in Brazilian Southeast BP are also found in conifers species as Araucaria angustifolia. Some phytochemical studies of Brazilian brown propolis confirmed A. angustifolia as the primary plant source (Sartori et al. 2021; Ribeiro et al. 2021a, b, c; Santos et al. 2021). In a previous work published by our research group, we reported the isolation of diterpenes from Araucaria sp. Brazilian brown propolis. During the collection of propolis samples in the field, bees collected the exudates from the A. angustifolia trunk for propolis production (Santos et al. 2021). The bees collect A. angustifolia exudate, store it in the corbicula of the left leg, and take it to the hive (Fig. 1). Araucaria’s participation was confirmed later through the phytochemical study of this propolis (Santos et al. 2021).

Collection of exudates of Araucaria angustifolia by Apis mellifera bee in União da Vitória (Paraná state, Brazil)

The participation of A. angustifolia in the chemical composition of propolis is mainly due to the presence of acid diterpenes, such as 13-epi-cupressic acid, abietic acid, and communic acid (Santos et al. 2021; Tazawa et al. 2016). From volatile compounds, A. angustifolia presents the sesquiterpene germacrene-D and the diterpenes hibaene and phyllocladene as significant components of its volatile oil (Brophy et al. 2000). The anti-inflammatory and antimicrobial biological properties are attributed to diterpenes. Many diterpenes isolated from BP and A. angustifolia possessed antimicrobial activity (Bankova et al. 1999; Ribeiroet al. 2021a, b, c).

Diterpenes from Pinus spp. and Eucalyptus spp. are also found in BP. Almost all isolated diterpenes from a Brazilian Southeast BP sample were reported in Pinus resin, as 19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxodehydroabietic acid, dehydroabietic acid, communic acid, and isopimaric acid. Pinoresinol and matairesinol lignans were also isolated from the same propolis sample (Ribeiro et al. 2021a, b, c). These lignans are described as major compounds in A. angustifolia knots resin and are also described as major compounds in Pinus taeda resin (Eberhardt et al. 1993).

Many BBP contain α-pinene and β-pinene as major compounds of their volatile fraction. These compounds are chemical markers of Pinus volatile oil, corroborating this plant as a botanical source for some BBP (Ioannou et al. 2014). Eucalyptus spp. also contributes with flavonoids and glucopyranoside compounds for BP composition (Freitas et al. 2008).

Determination of total phenolic and flavonoid contents in propolis samples has been widely used to determine biological properties (Sawaya et al. 2011), mainly using spectrophotometric assays. Usually, total phenolic content is measured by Folin–Ciocalteau’s method, and flavonoid content is measured by the AlCl3 complexation method (Machado et al. 2021). However, these methods do not specify each compound in the class of phenolic and flavonoids, being not selective. It is crucial to identify the compounds responsible for the biological activities to guarantee the quality of propolis and its products. The development of validated analytical methods is mandatory to guarantee selectivity, accuracy, and precision in quantifying compounds (Machado et al. 2021).

Several analytical methods have been developed to analyze raw propolis and its commercial products. Many of them aim to identify the chemical components with biological activities, mainly phenolic compounds used as biomarkers/standards (Fabio et al. 2019). It is challenging to develop analytical methods for propolis analysis because it bears a complex matrix demanding different methods’ approaches to analyze all the compound classes present in propolis (Pavlovic et al. 2020). For propolis’ polyphenol analysis, thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE) are the most used methods. HPLC coupled with photodiode array detector (DAD) is beneficial for polyphenol analysis, but HPLC coupled with a mass spectrometer (HPLC–MS) has gained space in propolis analysis, and it allows the identification of compounds in complex matrices (Fabio et al. 2019).

Besides phenolic analysis, propolis volatile compound analysis is essential, and solid-phase microextraction (SPME) with GC coupled to the mass spectrometer (GC–MS) is a good choice for this class of compounds (Pavlovic et al. 2020). Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (HS-SPME GC–MS) allows the study of several samples. Furthermore, HS-SPME has the advantage of avoiding using solvents, except when the matrix effect interferes in analysis (Burzynski-Chang et al. 2018).

There are some qualitative and quantitative methods reported in the literature for BP analysis, using the above-cited techniques. The qualitative methods are used only for chemical characterization, and there are reported quantifying method samples, even without validation. Most of the methods are not validated and usually show incomplete information about compound quantification. This review did not include the qualitative and classical spectrophotometric methods for phenolic and flavonoid analysis.

HPLC has been the most used equipment for quantitative method development, varying the type of detector and mass analyzer apparatus to determine the chemical composition of ethanol extracts from different types of Brazilian propolis, considering their predominant botanical origin (Salomão et al. 2008). Fonseca et al. (2011) quantified phenolic compounds by HPLC–MS in brown and green Brazilian propolis from São Paulo. Tazawa et al. (2016) discovered a novel diterpene in BP from the state of Paraná, using 1D- and 2D-NMR analyses and identified five more diterpenes. All the six compounds were quantified in the sample by ultra-performance liquid chromatography (UPLC) using calibration curves without validation.

Rodrigues et al. (2016) used HPLC–DAD to quantify prenylated phenolic acids and phenolic acids in brown and green Brazilian propolis samples from Paraná and Minas Gerais, respectively. Waller et al. (2017) detected 17 compounds in a BP sample from the state of Rio Grande Sul using HPLC–MS, in which the compounds were characterized by their UV and mass spectra. They used external standards calibration curves to quantify p-coumaric acid, rutin, chlorogenic acid, ferulic acid, and caffeic acid.

Mayworm et al. (2014) quantified tannins in different types of Brazilian propolis. Tannic acid was used as a reference for determining tannin content using the precipitation method with bovine serum albumin (BSA). Brown propolis samples contained tannins in a low quantity compared with other propolis types.

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