# Comparison of the Biological Potential and Chemical Composition of Brazilian and Mexican Propolis
## 3 Discussion
### 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 CO\({}_{2}\) (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 CO\({}_{2}\) 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-CO\({}_{2}\) 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-CO\({}_{2}\) extracts have been compared to ones obtained with ethanol using maceration and UAE. The lower polarity of SC-CO\({}_{2}\) 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-CO\({}_{2}\) to flavonoids [70; 71], artemisinin C, and p-coumaric acid from green Brazilian propolisis [34; 72] has been detected. SC-CO\({}_{2}\) 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 (Figures 2-5). Figure 2: Antimicrobial activity of Brazilian propolis against different microorganisms. (**A**) Several microbial species inhibited by different propolis extracts: Ethanolic (Et) and Supercritical (Sc). (**B**) Distribution of the Minimal Inhibitory Concentrations (MICs) found in the 13 studies evaluated. Frequency: number of MICs found in that interval. EtGP, ScGP, EtRP, ScRP, EtBP and ScBP. Figure 4: Antibacterial activity of Brazilian propolis against Gram-positive and negative bacteria. Distribution of the MICs found in the 13 studies utilized. (**A**) Gram-positive bacterial species (**B**) Gram-negative bacterial species. Frequency: number of times a MICs was found in that interval. EtGP, ScGP, EtRP, ScRP, EtBP, and ScBP. Gram-positive microorganism: _Staphylococcus_ (spp. _aureus_ ATCC 25923, 25913, 3359, _epidemidis_ 25/4, 194/2), _Enterococcus_ spp, _Enterococcus faecalis_, _Corynebacterium pseudotuberculosis_, _Eubacterium lentum_, _Peptostreptococcus anaerobius_, _Streptococcus_ (_mutans_, _sobritus_, _pygenes_ 93007, 75194, _sanguinis_, _salivarius_), _Actinomyces_ naslundi_, _Lactobacillus casei_, _Paenibacillus larvae_.