Evaluation of the Potential of Brazilian Red Propolis Extracts: An Analysis of the Chemical Composition and Biological Properties

Abstract

The optimized extraction process of natural matrices such as propolis that results in extracts with significant compounds has been one of the main needs of the industry. The aim of this work was to analyze the content of the active components of Brazilian red propolis extracts previously treated with ultrasound, as well as to evaluate in vitro their performance regarding antioxidant capacity and against bacteria and tumor cells. The results of the chromatographic analysis showed the influence of ultrasound treatment for higher yields of formononetin and kaempferol. However, just a higher content of these two components was not enough to interfere with higher concentrations of phenolic compounds and flavonoids among the extracts. The ten extracts obtained showed activity against two bacterial strains, and eight of them showed >70% cytotoxicity against five neoplastic cell lines. These results demonstrated the influence of ultrasound technology as a pretreatment in obtaining the ethanolic extracts of propolis, increasing the possibility of the applicability of Brazilian red propolis in different areas.

1. Introduction

The production of propolis is considered to be an instinctive behavior of honey bees, such as the species Apis mellifera, especially since it is a fundamental substance for the construction and preservation of the hive as well as for encapsulating the carcass of invaders [1,2]. These applications are possible due to its intrinsic constitution, where vegetal balm, wax, essential and aromatic oils, pollen as well as other substances are usually found at concentrations of 50%, 30%, 10%, 5% and 5%, respectively; these are usually brought by the bees after interaction with the local flora [3,4,5]. Thus, several studies show that the composition of propolis depends on the type of local vegetation the bees come into contact with [6,7,8]. Therefore, the vegetation of origin has a direct influence on the composition of the propolis, causing different types to be found around the globe [9,10,11].

In this context, propolis from Brazil has specific chemical compounds when compared to other countries, in particular due to plant diversity and the diversity of bee species, which in Brazil are mostly a cross of European and African species [12,13]. Based on physical-chemical analysis, fourteen types of propolis have been found in Brazil [14,15,16]; among these types, red propolis, originally from the mangrove forests in northern and northeastern Brazil has been one of the most exploited in recent years [14]. The coin vine (Dalbergia ecastaphyllum), a typical tree of the northeastern coastal strip, has an exudate tinged with red that is responsible for the color of this variety of propolis [17,18,19]. However, a study published in 2014 showed that another plant species plays an important role in providing resin for the composition of Brazilian red propolis [20]. The Brazilian red propolis produced in the northeast region of Brazil, specifically in the state of Alagoas, was included in this study, and has a certified designation of origin (geographic indication) for the scientific validation of its particular chemical composition [21,22].

The prominence with regard to red propolis has been associated with the broad spectrum of its biological properties, for example, antimicrobial [23], wound-healing [24], anti-inflammatory [25], anticancer [26], antioxidant [27], immunomodulating [28] and antiparasite [29] activities, which are related to its unique chemical composition. However, the biological action of red propolis is mostly associated with the presence of kaempferol and formononetin, as well as other isoflavones, which act synergistically with the other substances [27,30]. Formononetin has a fundamental role in this context, and is therefore an important biomarker of red propolis [31,32,33], whereas kaempferol presents broad applications in Chinese traditional medicine owing to its antioxidant activity [34,35]. Vestitol and neovestitol, as well as other chemical compounds from red propolis, were analyzed for antitumor activity, revealing their ability to cause genetic damage to these cells [36].

An important point is that the consumption of raw propolis may not be suggested, as it may have potential contaminants [7,37]. Therefore, a purification process of the samples is recommended, which is capable of preferentially removing the waxy material and preserving the polyphenol portion, which is considered the compound class that contributes the most to propolis’ biological effects when compared with its other components [38]. Therefore, the infusion method (the sample is immersed in a solvent for a long period) has been widely used by the industry to obtain chemical compounds with biological properties, presenting limitations and a low yield. Isolated ethanol extraction is not sufficient for obtaining extracts with high concentrations of compounds with biologically active functions [39,40,41]. With the aim of better using matrices rich in phytochemical substances, ultrasound technology (also called sonication) is an alternative for pretreatment during the extraction process.

The ultrasound-assisted extraction method provides high extraction yield compared with other methods, especially considering the labor and time requirements, and demonstrates better yield and selectivity of strategic compounds [42,43]. Different studies show that ultrasound is a key technology for reaching a balance between chemical extraction and a “green” or sustainable method (that does not produce polluting waste). It is currently being applied to several processes in the food industry [44,45,46], which allows for a more complete extraction with high reproducibility, and it is characterized by the decreased use of organic solvent, simplified processing, and the higher purity of the final product [47]. It is important to highlight that the literature is limited with regard to the utilization of the ultrasound technique to obtain propolis extracts, especially considering the types of Brazilian propolis.

In this context, the purpose of this study was to evaluate the impact of ultrasound technology using several temperatures and times in the pretreatment process to obtain Brazilian red propolis extracts. For this purpose, the extracts were obtained under different conditions in regard to their chemical characteristics, considering also the evaluation of their antioxidant, antimicrobial and cytotoxic in vitro properties.

2. Materials and Methods

2.1. Materials

Acetic acid (Synth, Diadema, SP, Brazil), methanol and DMSO (dimethyl sulfoxide) (Sigma-Aldrich Chemical Co.-St. Louis, MO, USA), all with high purity for high performance liquid chromatography (HPLC) and ethanol P.A. (Anhydrol) were selected. Filter with regenerated cellulose membrane (0.45 μm) (SLCR025NS, Millipore Co., Bedford, MA, USA) was also used. The standards formononetin (CAS number 485-72-3), gallic acid (CAS number 149-91-7), kaempferol (CAS number 520-18-3), quercetin (CAS number 117-39-5) and 2,2-diphenyl-1-picrylhydrazyl (DPPH^●) (CAS number 1898-66-4) were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

2.2. Red Propolis: Obtaining and Processing

The sample of red propolis used in this study was acquired from an apiary in the city of Marechal Deodoro (Alagoas, Brazil). The sample was processed in a mill (Cadence Brasil) in order to obtain a diameter between 52 and 92 μm, thus facilitating the extraction process and the uniformity of the material. The sample was kept at −10 °C in inert atmosphere conditions (N₂) in a fractional form in order to avoid the oxidation of the material [48].

2.3. Obtaining Ethanolic Extracts

According to the methodology of Chen et al. [49], propolis and 80% ethanol (1:12.5 m/v) were homogenized and exposed to ultrasound technology in an ultrasonic bath (135 W, 50–60 Hz, Quimis, RMS, Diadema, SP, Brazil) with different conditions of sonication and temperature (

Table 1. Process conditions for obtaining red propolis extracts with or without ultrasound.

Sample Names	Temperature (°C)	Ultrasound Time (min)
E:UT (control this study) *	Ambient (±25 °C)	Not applied
E:25−10	25	10
E:25−20	25	20
E:25−30	25	30
E:50−10	50	10
E:50−20	50	20
E:50−30	50	30
E:75−10	75	10
E:75−20	75	20
E:75−30	75	30

* Conventional extraction as it is currently employed in the industry.

). After, the samples were kept in infusion for seven days with manual and periodic shaking, and after this period they were centrifuged (Fanem—206-BL, Brazil) (5000 RPM/11 min) and filtered using qualitative filter paper (80 g/cm³). The concentration of the extracts was carried out in an oven with air circulation at 45 °C (Quimis, Q314M222, Diadema, SP, Brazil) until mass normalization. A control sample called untreated extract (E:UT) was obtained under the same conditions, but without the application of ultrasound.

2.4. Chromatographic Analysis of Extracts

To identify and quantify kaempferol and formononetin, methanolic solutions of propolis extracts (10 mg·mL⁻¹) obtained under different processing conditions were prepared. Before injection, samples were filtered on a 0.45 μm cellulose membrane filter (Millipore). The chromatograph used was an HPLC LC-20AT interfaced with an automatic injector and diode array detector (DAD) SPD-M20, both from Shimadzu (Kyoto, Japan). The chromatographic separation was adapted as proposed by Salgueiro and Castro [50] and Cabral et al. [51]. The column used was a NUCLEODUR^® 100-5 C18 EC (150 × 4 mm × 5 μm) coupled with ZORBAX Eclipse Plus C18 pre-column (4.6 × 12.5 mm) (Agilent, Santa Clara, CA, USA).

The injection volume was 20 μL with a flow of 1 mL·min⁻¹ at room temperature (25 ± 2 °C). The elution gradient was composed of mobile phase of acetic acid (5%) and methanol (solvent B). The total run time was 42 min, being 0 to 35 min with 0−92% solvent B, 35 to 40 min with 92−0% solvent B; and 40 to 42 min (0% B). The DAD detection was set with a range of 190 to 800 nm, and the chromatographic acquisition between 300 and 320 nm. For the identification of the compounds, the ultraviolet spectrum and the retention time of the samples and the standards were compared (

Table 2. Identification and quantification conditions by HPLC-DAD of the compounds formononetin and kaempferol in red propolis extracts obtained by different conditions.

Standards	Retention Time (min)	Wavelength (nm)	Concentration Range (mg·L−1)	DL (mg·g−1)	QL (mg·g−1)
Formononetin	19.46	300	0.5–12.5	0.31	1.02
Kaempferol	17.53	320	0.5–12.5	0.12	0.41

DL = detection limit; QL = quantification limit.

). In order to ensure reliable results, the validation followed the methodologies of the National Institute of Metrology, Quality and Technology (INMETRO) [52] and the National Health Surveillance Agency (ANVISA) [53]. This analysis was performed according to the parameters of selectivity, linearity, accuracy, detection and quantification limits. The chromatographic run and method validation were performed with other phenolic compounds (standards) already reported or not in propolis samples (results not shown—total of eleven compounds). However, only formononetin and kaempferol met the limit of detection and limit of quantification.

2.5. Total Phenolic Compounds in the Red Propolis Extracts

The phenolic content was quantified based on reaction with the Folin-Ciocalteu reagent [54] with reading in a spectrophotometer (PerkinElmer, LAMBDA 25 UV/Vis Systems, Washington-USA) at 765 nm, proposed by Singleton and Rossi [55] and Singleton et al. [56]. The quantification was expressed in milligram equivalents of gallic acid per gram of sample (mgGAE·g⁻¹) from a calibration curve (R² = 0.9994, y = 0.0096x − 0.0311) obtained from the analysis of aqueous solutions of the gallic acid standard with known concentration (12 to 200 µg·mL⁻¹) in the identical conditions of quantification of the samples.

2.6. Flavonoid Contents of the Extracts

According to Meda et al. [57], the flavonoid fraction was measured, with adaptations, in a spectrophotometer (PerkinElmer, LAMBDA 25 UV/Vis Systems, Washington, DC, USA) at 415 nm. The same procedure was followed using aqueous solutions of quercetin with known concentrations (from 5 to 105 µg·mL⁻¹) in order to obtain an analytical curve (R² = 0.9994, y = 0.0271x − 0.014). The flavonoid concentration was reported in milligram equivalents of quercetin per gram of sample (mgQE·g⁻¹).

2.7. Antioxidant Activity of the Extracts

To measure the antioxidant activity of the extracts, the DPPH (2,2-diphenyl-1-picrylhydrazyl) reactive method was used according to Brand-Williams et al. [58] and Molyneoux [59], with adaptations. Six dilutions (10 to 85 μg·mL⁻¹) were prepared and reacted in test tubes with ethanolic solution (EtOH 99%) of 0.004% DPPH^●. Quantification of the reduction of DPPH^● radical activity was established at 517 nm in a spectrophotometer (PerkinElmer, LAMBDA 25 UV/Vis Systems, Washington, USA).

The capacity to sequester the radical in free form was demonstrated as the percentage of inhibition of radical oxidation and was calculated based on Equation (1). The IC₅₀ (concentration required of the extract to inhibit 50% of the DPPH^● radical) was measured with the equation of the straight line generated by analyzing the six concentrations of each sample.

% Sequestering = 100 − [(final absorbance of the sample × 100)/absorbance of the blank]

(1)

2.8. Antimicrobial Activity of Extracts

The determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the extracts for Gram-positive and Gram-negative bacterial models, Escherichia coli (ATCC 1775) and Staphylococcus aureus (ATCC 25,923), respectively, followed methodology widely used in the scientific field [60,61]. The strains were kindly provided by Oswaldo Cruz Institute Bacterial Culture Collection (Oswaldo Cruz Institute, FIOCRUZ, Manguinhos, Rio de Janeiro, Brazil). The bacteria were seeded on brain heart infusion agar (BHI), incubated at 37 °C/24 h to prepare the inoculum. To determine the MIC, the initial inoculum was 1–2 × 10⁵ CFU·mL⁻¹; the extract concentrations ranged from 1600 to 3.125 μg·mL⁻¹. The assays were performed in triplicate for each concentration of the extract tested.

2.9. In Vitro Cytotoxicity of Extracts

The cytotoxicity (in vitro) of the extracts from this study was evaluated against human (Homo sapiens) tumor cell lines of leukemia, prostate carcinoma, glioblastomas and colon carcinoma: cell lines HL-60, PC3, SF295, SNB19 and HCT-116, respectively, all from the National Cancer Institute (USA). The lines were cultivated in RPMI 1640 supplemented with penicillin/streptomycin antibiotic solution (1%) and fetal bovine serum (FBS) (10%) (Gibco^®, Life Technologies, Carlsbad, CA, USA). The cells were incubated at 37 °C with 5% CO₂ (Thermo Scientific, Waltham, MS, USA). Trypsin (0.25%) was used to reduce cell adhesion to the walls of the culture bottle.

The cytotoxic potential of the extracts against tumor cell lines was estimated using the method with MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium) salt, as described by Mosmann [62] and Amaral et al. [63]. For formazan quantification in viable cells (0.1 × 10⁶ cells·mL⁻¹), absorbance (at 595 nm) was measured by means of a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc., Packard, ON, Canada). The observed values correspond to the inhibitory concentration with 100% of its maximum effect.

2.10. Statistical Analysis

For statistical evaluation, analysis of variance (ANOVA) and Tukey’s test (95% confidence level) were applied to evaluate the significant differences (p < 0.05) between the means obtained for each assay. For this, the GraphPad Prism software, version 9.2, (San Diego, CA, USA) was used.

3. Results and Discussion

3.1. Phenolic Compounds, Flavonoids and Antioxidant Activity of the Red Propolis Extracts

The composition of propolis is completely variable, which can impact on standardization processes and use within the medical/pharmaceutical field. The chemical composition of propolis from various sources can result in distinct biological activities; however, these activities can be the same among different samples, according to studies from our group [8,41,64,65,66]. A complex and difficult activity would be the worldwide standardization of propolis [67]; therefore, a minimum composition of total phenolic compounds and flavonoids is of considerable importance to define the standard of the obtained material, regardless of geographic origin, type of propolis or extraction method used. The Technical Regulation of Identity and Quality of Propolis from the Ministry of Agriculture, Livestock and Supply is an attempt by the Brazilian government to maintain and regulate the quality of propolis products in the domestic market [68]. According to this document, the minimum total phenolic content in the extract should be 5%, while for flavonoids the minimum content should be 1%. The characterization of the compounds of sample chemicals is important for quality control and thus standardization, since it allows comparative assays, especially between biological assays, to be performed [43].

The analysis of the phenolic compound and flavonoid contents and the antioxidant action for the red propolis extracts obtained according to Table 1 are presented in Figure 1. The phenolic-containing compounds ranged from 210.22 ± 21.66 (sample E:25−30) to 235.88 ± 40.07 mgGAE·g⁻¹ (sample E:25−10) across the samples, without any significant differences (p > 0.05). Values between 77.89 ± 2.53 mgQE·g⁻¹ (sample E:50−20) and 104.79 ± 22.5 mgQE·g⁻¹ (sample E:25−10) were found when the flavonoid content was analyzed, whereas the antioxidant activity (IC₅₀) ranged from 38.28 ± 2.23 (sample E:25−30) to 59.71 ± 1.21 µg·mL⁻¹ (sample E:50−20), with significant differences (p < 0.05) between the control extract (untreated) and the treated extracts.

In general, all the samples presented total phenolic compound values that were higher than 200 mgGAE·g⁻¹, without significant differences (p > 0.05). Therefore, no influence was observed in relation to the use of different temperature conditions and the duration of exposure to the ultrasound with regard to the total phenolic compound contents in the extracts. Likewise, when the control was compared with the samples treated with ultrasound (regardless of the type of treatment employed), there was no significant difference in the total phenolic content, which shows that the application of this technology does not influence the total content of these compounds directly.

Studies on Brazilian red propolis (source: Sergipe region, Brazil) report values from 151.55 ± 1.95 to 300.00 ± 0.01 mgGAE·g⁻¹ in extracts obtained using different extraction methods by employing organic solvents and/or supercritical fluids [48,69]. Values similar to the ones in this study were informed by Alencar et al. [14] (232.00 ± 22.30 mgGAE·g⁻¹) for red propolis samples of the same origin, but when using different extraction conditions, e.g., a solvent proportion four times higher relative to the amount of sample used in this work. Red propolis from the same geographic region was also studied by Cabral et al. [51] and Machado et al. [48], who reported values lower than those used in this study for phenolic compounds in the extracts obtained by conventional extraction (ethanol extraction) and extraction with supercritical CO₂, respectively. It should be noted that the variations in the final concentrations of phenolic compounds may be correlated to differences in the extraction process, as well as the seasonality and geographic origin of the samples [66,70,71].

Concerning the differences identified for the flavonoids and antioxidant activity (DPPH), an increase of up to 7% in the flavonoid concentration and a reduction of 23% in the IC₅₀ were observed in the antioxidant action evaluates, when comparing the samples obtained with the application of ultrasound and the control (Figure 1). Therefore, the inclusion of ultrasound pretreatment was effective for finding red propolis extracts with higher antioxidant activities when considering the lower IC₅₀ values determined here.

The extracts obtained under the same temperature conditions, regardless of their duration of ultrasound exposure, did not present significant differences in regard to the flavonoid content, showing that prolonged exposures do not contribute to an increased yield of these compounds. At a temperature of 25 °C (the same temperature used to obtain the control extract), it was observed that the application of ultrasound did not significantly contribute to an increased total flavonoid content in the extracts. Thus, the increase in the extraction of these compounds may be associated to the application of low temperatures associated with the use of adequate solvents, given the sensitivity of these compounds to high temperatures [72]. The flavonoid content is important information that should be obtained for propolis extracts because it is a quality control parameter within the regulations of countries such as Brazil, Argentina and Switzerland [68]. Flavonoid values lower than the ones in this work were determined by Mendonça et al. [73] and Andrade et al. [38] for red propolis extracts treated with ultrasound that were collected from the same geographic region (0.31 ± 0.01 and 31.48 ± 0.50 mgQE·g⁻¹, respectively).

Regarding the antioxidant activity, all the extracts obtained at 25 °C (samples E:25−10, E:25−20 and E:25−30), regardless of the duration of the ultrasound pretreatment, presented the lowest IC₅₀ values and, consequently, the highest antioxidant activities, given that the lower the effective concentration is, the lower the quantity of extract requisite to oxidize 50% of the DPPH^● radicals available in the reaction (Figure 1) [74]. These results indicate that the higher the concentration of flavonoid presence in the extracts is, the higher the antioxidant activity of the evaluated sample (as represented by a lower IC₅₀), suggesting that these compounds are answerable for the antioxidant activity of propolis [75,76,77]. Freires et al. [78] evaluated the red propolis extracts obtained from ethanolic extraction (native to northeastern Brazil) and reported higher IC₅₀ values (44 to 90 µg·mL⁻¹).

Exposure to ultrasound may help to improve the release of intracellular compounds because the cells are easier to rupture, increasing the quantitative availability of these compounds [79]. As mentioned, the variation in the total content of antioxidant compounds obtained when compared with the results from other studies may be related to the geographic origin, seasonality, or extraction method. However, when reviewing studies on red propolis extracts of the same geographic origin obtained through other extraction methods [48,73], the application of ultrasound may be the factor that is responsible for obtaining extracts with higher antioxidant activities (Figure 1). In general, the extracts obtained at 25 °C presented the highest values for the investigated parameters, which explains the fact that the use of high temperatures contributes to the reduction in yield for these compounds of interest.

3.2. Quantification of Compounds by HPLC

The HPLC method (or other chromatographic techniques) has been frequently used for the determination of the profile of chemical constituents of propolis, since it allows its quantification and separation; however, its high cost makes its application as a routine activity not recommended [80,81]. The intrinsic complexity of propolis was demonstrated in the large number of chromatographic peaks obtained from the HPLC method; however, it was noted that the peaks found showed good resolution between them, with a high likelihood of identifying new compounds (Figure 2). Several chemical classes, such as isoflavonoids, prenylated benzophenones, terpenes and flavonoids, have been found in previously researched Brazilian red propolis samples [38,82]. However, the marked presence of isoflavonoids in its composition makes the red propolis from the state of Alagoas in Brazil an atypical case of chemical composition [83].

Concerning the identification and quantification of the chemical species of interest in the samples (red propolis extracts) (Table 2), only formononetin was identified in all the obtained extracts, and kaempferol was found and quantified in eight of the ten samples studied here. It was not possible to quantify the kaempferol in the control sample (E:UT) and in the E:25−30 sample given that this compound was present at a concentration below the quantification limit (result confirmed by repeating the analysis in triplicate). The values of these two components (formononetin and kaempferol) in the obtained extracts are shown in Figure 3. Therefore, formononetin was the most abundant chemical species found in the extracts, and its profile was confirmed by the HPLC method [84,85].

Formononetin concentrations ranged from 4.54 ± 0.01 to 8.36 ± 0.25 mg·g⁻¹ (samples E:UT and E:50−20, respectively), with significant differences between the extracts and the control. Being a biomarker particular to Brazilian red propolis [86], formononetin was present in significant amounts in all extracts, independent of extraction conditions. The kaempferol concentration ranged from 0.53 ± 0.07 to 1.04 ± 0.02 mg·g⁻¹ (samples E:75−30 and E:50−20, respectively), with significant differences (p < 0.05) between the extracts in which the compound was found. Among the extracts studied, sample E:50−20 showed the better concentrations of the considered compounds, with significant differences (p < 0.05) when compared with the control, thus showing that the application of an ultrasound pretreatment at a temperature of 50 °C is efficient at increasing formononetin extraction by approximately 60% and allows for significant kaempferol extraction. The efficiency of the pretreatment with ultrasound technology for the improved yield of these compounds can also be confirmed through a comparative analysis of the control (E:UT) and the extracts obtained at the same temperature and over different exposure durations (E:25−10, E:25−20 and E:25−30), with significant differences (p < 0.05) between the extracts and the control.

For the extracts obtained at 75 °C, regardless of the duration of exposure to the ultrasound, there were no significant differences in the formononetin yield. However, the maximum concentration at this temperature for kaempferol was obtained at the lowest exposure time (E:75−10), which may forecast the degradation of this compound upon more prolonged exposure at higher temperatures, owing to its thermal sensitivity [87]. In general, prolonged exposure negatively affects the yields of the studied compounds (Figure 3). Almeida et al. [21] also showed the complexity of the chromatograms obtained for red propolis extracts from the equivalent geographic region, and they confirmed the occurrence of isoflavonoids, including formononetin and the flavonol kaempferol. Batista et al. [17] also identified and quantified formononetin in Brazilian red propolis extracts from the equivalent geographic region that were processed with ultrasound technology at room temperature (25 °C), with exposure for 1 h, and they observed lower values for this compound when compared with sample E:50−20 (25% lower), which may indicate that a temperature of 50 °C is optimal for extracting this component. Reis et al. [27] also identified formononetin as the main compound in Brazilian red propolis extracts from the same geographic region.

Kaempferol is a compound with high antioxidant activity, but it is present at low concentrations in red propolis [27]. Barbarić et al. [88] detected kaempferol in only three European propolis samples among the twenty studied, with a concentration range between 0.0697 and 0.2931 mg·g⁻¹. Importantly, this compound was not identified in samples of Cuban red propolis extracts from different regions [20].

Results similar to those in this work were described by other authors when comparing the yields of the chemical species of interest from several natural matrices when ultrasound and conventional extraction were applied, showing that extraction with ultrasound pretreatment is more selective and thus generates products with higher biological value [89,90,91,92,93]. Therefore, among the parameters evaluated in this study, although it does not show the highest antioxidant activity, sample E:50−20 was the sample that presented the highest content of the formononetin and kaempferol (the compounds of interest), thus showing the greater biological potential of this extract when it was obtained under the tested conditions. It should be noted that different studies indicate that formononetin presents different important biological activities, e.g., acting in a positive manner against distinct tumor cell lines [94,95]. Thus, the antioxidant property of an extract cannot be related only with the occurrence of phenolic compounds and/or flavonoids, since other classes of compounds can also support this activity [96].

3.3. Determination of the Antimicrobial Activity

Substantial advances have been made in the antimicrobial study of red propolis extract, making it an interesting alternative to be potentially applied against a range of microorganisms [97,98]. This is implied in the growing quantity of publications on the extracts of red propolis obtained by different extraction methods and their bioactive compounds isolated and differentiated in recent years [99].

Table 3. Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) (µg·mL⁻¹) of the Brazilian red propolis extracts obtained under different conditions.

Sample	Staphylococcus aureus ¹		Escherichia coli ²
	MIC	MBC	MIC	MBC
E:UT	11	750	46	187
E:25−10	6	>1000	46	93
E:25−20	11	>1000	93	187
E:25−30	11	>1000	23	93
E:50−10	23	23	93	750
E:50−20	11	11	46	>1000
E:50−30	23	23	93	>1000
E:75−10	23	23	23	>1000
E:75−20	23	23	93	750
E:75−30	23	>1000	93	187

¹ ATCC 25923; ² ATCC 1775.

presents the MIC and MBC values found for the ten Brazilian red propolis extracts obtained in this study. All extracts showed activity against S. aureus (representative Gram-positive) and E. coli (representative Gram-negative) bacteria; however, this parameter depended on the extraction patterns applied. The bacterial growth of the controls chosen in the study was not affected. Because the sample that was not treated with ultrasound (E:UT) presented lower MIC and MBC values compared with some of the treated extracts (including under the same temperature conditions for MBC), we cannot claim that the ultrasound treatment was efficient at increasing the antimicrobial potential of the extracts against the studied bacteria for some of the tested temperatures.

The MIC for S. aureus ranged from 6 to 23 µg·mL⁻¹, whereas the variation for E. coli ranged from 23 to 93 µg·mL⁻¹ (Table 3). To obtain whole antimicrobial properties, investigations to determine the MBC were conducted for all the studied extracts. The MBCs for S. aureus and for E. coli varied between 11 and >1000 µg·mL⁻¹ and between 93 and >1000 µg·mL⁻¹, respectively (Table 3). The literature suggests a promising MIC of a raw extract of a natural product is less than 500 μg·mL⁻¹, thus causing robust investigations to be conducted to clarify its mechanism of action [100,101]. Hence, regardless of the given parameters, the low MIC values observed for the ten red propolis extracts show that the study subjects are promising antimicrobial compounds.

In general, when the extraction conditions in use are compared, the extracts presenting higher antimicrobial activities were those that exhibited the highest antioxidant activities, with high total contents of phenolic compounds and flavonoids (Figure 1). The extracts with higher active concentrations against S. aureus and E. coli were E:50−20 and E:25−30, respectively, as represented by the lower MIC and MBC values. Among all the conditions tested here, four extracts did not show active bactericidal action against S. aureus, and three did not show activity against E. coli (as represented by MBC > 1000 µg·mL⁻¹). As observed in other studies and confirmed in this experiment, the extracts tested here showed significant activity against the Gram-positive strain (S. aureus), in contrast to the Gram-negative strain (E. coli). This behavior can be explained by the bacterial cell wall, which, due to its structural differences between Gram-positive and Gram-negative bacteria, may have facilitated the action of propolis in the case of Gram-negative bacteria [102,103]. Similar results were obtained in other studies investigating red propolis extracts with other origins [24,104].

The antimicrobial activity mechanism of propolis is multifactorial and may be credited to the presence of some bioactive compounds, in particular flavonoids [105]. The antimicrobial action of propolis has been associated with different mechanisms that are essential for survival and proliferation, such as inhibition of nucleic acid synthesis, impacts on energy metabolism, biofilm formation and cytoplasmic membrane fluidity [106,107]. Because they present a wide diversity of components, the possibility of synergistic action among chemical species found in red propolis, such as vestitol, neovestitol, isoliquiritigenin and galagin (not tested in this study), cannot be ruled out as a potential explanation for its antimicrobial activity [108,109,110,111,112,113]. However, Neves et al. [33] identified the correlation between antimicrobial activity against S. aureus and P. aeruginosa and the flavonoid formononetin present in the ethanolic extracts of Brazilian red propolis originated in the state of Pernambuco.

The MIC values for red propolis extracts are reported in different studies, with variations between 3.8 and 100 μg·mL⁻¹ for S. aureus [14,21,113] and between 6.3 and >1000 μg·mL⁻¹ for E. coli [48,114]; propolis is thus considered to be a strong antibacterial agent based on the classification by Freires et al. [78]. The influence of the extraction method on the antimicrobial activity and the potential of the red propolis extracts from Alagoas was also demonstrated by other studies in our research group [41,48]. It is also noteworthy that different authors have reported the influence of seasonality on the antimicrobial activity of propolis, which makes studies using samples from the same region even more relevant [115,116,117].

Therefore, the antimicrobial data for the samples of red propolis extracts analyzed here propose that these extracts present a relevant potential action against foodborne microorganisms, and they may thus constitute an important preservative to add to food formulations [118,119,120,121]. Nedji et al. [122] demonstrated the potential effect of propolis when it is applied in the food and pharmaceutical industries, owing to the antimicrobial effect presented by this matrix against some bacteria and fungi of interest in drugs and foods. For example, Morsy et al. [123] demonstrated the positive effect of red propolis as a food additive through oral administration in sheep, which resulted in improved biochemical parameters. It is noteworthy that among the biological properties of red propolis, its antimicrobial activity has the highest relevance, which means this work can provide support to clarify this potential application.

3.4. Determination of Antitumor Activity In Vitro

Cancer is a major public health concern, with estimates of increasing incidence, prevalence and death rates in the coming years [124]. Within this perspective, the search for new molecules capable of acting in anticancer therapy has been one of the main targets of the pharmaceutical industry, especially those of natural origin [125]. Considering the period from 1981 to 2019, about 25% of all anticancer medications were based on natural products [126], which reinforces the importance of studies with propolis within this perspective. The extracts of red propolis have presented a great relevance in recent years in relation to their antiproliferative action against tumor lines [127].

The MTT viability test was applied to determine the cytotoxic activity of the ten red propolis extracts on distinct tumor cell lines (HL-60 leukemia, HCT-116 colon carcinoma, SF295 and SNB19 glioblastoma, as well as PC3 prostate carcinoma). The results presented here show that the majority of the extracts (tested at a 50 µg·mL⁻¹ concentration) changed the cell viability of the tested lines (Figure 4), with a significant decrease in the cell concentration (p < 0.05).

The percentage of inhibition varied according to the tumor lineage and the extract analyzed, where for the HCT-116 line the range was between 1.73 and 100% for samples E:UT and E:50−20, respectively; for the PC3 line the range found was from 2.16 to 100.00% for samples E:UT and E:25−20, respectively; for the SF295 line the range was from 5.21 to 100.00%, for samples E:25−30 and E:25−20, respectively; and from 67.89 to 97.52%, samples E:25−10 and E:25−20, respectively, for line SNB19, with significant differences among samples (p > 0.05). For the HL-60 line, regardless of the conditions, all the extracts presented a percent inhibition that was higher than 99%, without showing significant differences between the analyzed samples (p < 0.05) (Figure 4a–e).

In general, the extracts with the highest concentrations of formononetin and kaempferol (E:50−10, E:50−20 and E:50−30) were those presenting higher cell growth inhibition for the tested lines, thus suggesting that these compounds may be associated with the strong cytotoxic action of red propolis against the five neoplasm lines tested in this study. Therefore, it was observed that the extracts obtained at a temperature of 50 °C were those that showed the greatest cell proliferation inhibition, thus showing the importance of using an adequate temperature, together with the addition of ultrasound, to find extracts with higher biological potential. Different authors have highlighted the antitumor activity of red propolis by comparing it with other types of propolis, as it has been shown to have superior cytotoxic action against different tumor cell lines [19,41,48].

Novak et al. [128] evaluated red propolis extracts and their fractions that were rich in formononetin in the inhibition of hematological tumor cells, and they observed the strong antitumor activity due to the presence of this flavonoid. Almeida et al. [21] and Cavendish et al. [84] associated the biological properties of the red propolis extracts with the presence of formononetin, whereas other studies showed that formononetin, in synergy with xantocimol, confers strong antitumor action to this matrix [20,129]. Some studies indicate that the antiproliferation action of this type of propolis may be associated to the mechanism of cell cycle arrest and apoptosis [130,131]. Frozza et al. [69] analyzed the cytotoxicity of red propolis extract against human epidermoid carcinoma of the larynx (Hep-2) and human cervical adenocarcinoma (HeLa). They obtained an IC₅₀ of approximately 80 µg·mL⁻¹, thus showing the potential of the studied extract. Santiago et al. [132] showed that the cytotoxic action against cancer cells of red propolis extract might be associated with the immunomodulatory action presented by its components.

Thus, studies that demonstrate the component profiles of propolis extracts, such as our study, become increasingly relevant. In this context, Vukovik et al. [133] analyzed the cytotoxicity of 11 flavonoids isolated from propolis against two types of tumor cell lines (MDA-MB-231 breast and HCT-116 colon), and observed that about 55% of the isolated compounds induced cytotoxic reactions in the tested cells.. Li et al. [134] investigated the cytotoxic action of several classes of flavonoids isolated from red propolis (42 compounds), including formononetin, against different tumor cells, and they found that the compounds (2S)-7-hydroxy-6-methoxy flavanone and (3S)-mucronulatol were the ones that presented the highest antiproliferation activities, supporting the use of these flavonoids as antitumor agents.

The results obtained here show the high biological potential of the Brazilian red propolis, which thus constitutes an important resource for the development of pharmaceutical products. The study of its isolated compounds for the evaluation of their therapeutic potential needs to be extended to show, for example, the mechanism of action against the tested cell lines and more studies in vivo. The results of our work have exposed new data about the antitumoral activity of the red propolis from Brazil and confirmed the presence of the compound specifcs. Propolis has been used as an adjuvant in cancer therapy and we were able to confirm the biological importance of propolis. Thus, propolis has presented important characteristics capable of increasing chemotherapeutic action, besides mitigating possible adverse events in patients under treatment [70,135].

4. Conclusions

The influence of ultrasound technology in association with different temperatures in obtaining ethanol extracts of red propolis with high biological potential was demonstrated. The extracts obtained under different processing conditions presented significant differences (p > 0.05) in regard to their antioxidant activity, formononetin and kaempferol concentrations, antimicrobial activity and cytotoxicity. It was noted that the extracts obtained from a temperature of 25 °C showed a better yield when it came to the concentration of flavonoids and phenolic compounds, and in addition in the antioxidant action through the analysis of the DPPH^● radical scavenging. However, the extracts submitted to a temperature of 50 °C showed the highest concentration of formononetin and kaempferol, two key components in the composition of red propolis. These results suggest that other components present in propolis are part of the composition of the phenolic and flavonoid class, as well as influencing a possible antioxidant action, which demonstrates the complexity of this natural matrix. Formononetin was found in all the studied extracts, whereas kaempferol was detected above the quantification limit in only eight extracts. All the extracts showed high antibacterial actions against S. aureus and E. coli strains. The cytotoxicity of 80% of the extracts obtained was >75% against HCT-116, PC3, SNB19 and HL-60 tumor cell lines. In general, the sample with the greatest biological potential was E:50−20, considering that it presented high antiproliferation effects against the tested tumor cell lines (HL-60, HCT-116, PC3, SF295 and SNB19), in addition to relevant antioxidant and antibacterial activities, which were related with elevated concentrations of kaempferol and formononetin.