*2.3. Propolis Samples*

Beekeepers kindly provided four raw propolis samples from Silao and Irapuato, Guanajuato, México (Table 1). The samples were harvested using traps in 2018 and 2019 and were frozen and stored at −20 ◦C until analysis.


**Table 1.** Location of recollection, harvesting method, and weight recollected.

## *2.4. Antioxidant Activity*

The EEP antioxidant activity was assessed using two di fferent assays in vitro: DPPH and ABTS. Both methods were modified and translated into 96-well plates. Each test was done in three replicates.

Radical scavenging activity (RSA) for DPPH was evaluated according to the method described in [11]. Briefly, an ethanolic solution of 0.208 mM was added to 0.1 mL of different concentrations of extracts and pure compounds. The 96-well plate was maintained in a dark at room temperature for 20 min and the absorbance was recorded at 540 nm. The RSA was calculated as: RSA = 100 × (Acontrol − Asample)/Acontrol, where Acontrol and Asample are the absorbance. The IC50 values were calculated from the relationship curve of RSA versus concentrations of the respective sample curve.

The ABTS test was performed according to the methodology previously reported in [13,14] and slightly modified. The RSA of the ABTS radical was calculated using the following equation: % inhibition = 100 (Acontrol − Asample)/Acontrol. The IC50 was calculated from the scavenging activities (%) versus concentrations of the respective sample curve.

#### *2.5. Total Phenol and Total Flavonoid Content*

In this paper, we used spectrometric procedures for the quantification of the total phenolic and flavonoids content in propolis. The total phenol content in extracts was adapted from the method described by Singleton and Rossi [15]. The total flavonoid content was determined using the aluminum chloride reagen<sup>t</sup> and the method described by Marquele et al. [16]. The total phenol content was expressed as mg equivalents of gallic acid/g of dry extract of propolis (EEP). The total flavonoid content was expressed as mg equivalents of quercetin/g of dry extract of propolis (EEP).

#### *2.6. Extraction and Isolation of Compounds 1–12 from EEP GUA-4*

The air-dried and powdered propolis samples GUA-4 were extracted with ethanol for up to one week and the resultant extract was concentrated in vacuo. A portion of ethanol-soluble extract (10.3 g) was subjected to a silica gel vacuum column chromatography (VLC) and eluted with a gradient mixture of dichloromethane–acetone (1:0 → 0:1) to give eight pooled fractions (F2–F8). Fractions F2, F4, and F7 showed the best antioxidant activity. Fraction F2 was chromatographed over a Sephadex LH-20 column and eluted with methanol to yield **1** (40 mg). Fraction F4, eluted with dichloromethane-acetone (9:1), was chromatographed over a Sephadex LH-20 column, using methanol as eluent, to give six fractions. Fraction F4-3 (100 mg) was separated by TLC with dichloromethane–acetone (99:1), followed by TLC with dichloromethane–acetone (98:2), to give **2** (20 mg) and **3** (65 mg). Fraction F5 was rechromatographed on a Sephadex LH-20 column using methanol as solvent to give six subfractions (F5-1: 10 mg; F5-2: 12.3 mg; F5-3: 17.5 mg; F5-4: 15.7 mg; F5-5: 25.3 mg; F5-6: 18.1 mg). Subfraction F5-1 yielded crystals of 4. F5-5 (25.3 mg) yielded crystals of 5 (9.0 mg). Mother liquor was subjected to a preparative thin-layer chromatography (PTLC) with acetone-toluene (5:95) to give 6. Subfraction F5-6 (18.1 mg) yielded crystals of 7 (11.0 mg). Fraction F7 was chromatographed on silica gel using a hexane–EtOAc gradient system to give five fractions (F7-1: 13.9 mg; F7-2: 517.7 mg; F7-3: 28.4 mg; F7-4: 16.7 mg; F7-5: 32.8 mg; F7-6: 56.8 mg; F7-7: 45.1 mg). Subfraction F7-1 was identified as 8. Subfraction F7-2 was chromatographed by RP-MPLC, using H2O–CH3CN (1:0 → 0:1) to afford five subfractions (F7-2.1: 125.3 mg; F7-2.2: 275.1 mg; F7-2.3: 67.8 mg; F7-2.4: 37.0 mg; F7-2.5: 12.3 mg). Subfraction F7-2.3 was identified as 9. Subfractions F7-2.4, F7-2.5, and F7-2.6 were dissolved in CH2Cl2–MeOH and left overnight to give crystals of 10, 11, and 12, respectively.

#### *2.7. Head Space Solid Phase Microextraction (HS-SPME), GC-MS Analysis, and Identification of Volatile Components*

The HS-SPME was performed using a Stableflex® fiber 50/30 μm DVB/CAR/PDMS (1 cm) as described in our previous work [17].

The analysis of volatile compounds was carried out on a GC Agilent 6890 N (Agilent Technology, Santa Clara, CA, USA) series gas chromatograph coupled to a LECO time of flight mass spectrometer (LECO Corporation, St. Joseph, MI, USA). The volatile compounds were separated on a 5% diphenyl-95% dimethyl polysiloxane (30 m × 0.18 mm i.d.; 0.18 μm film thickness) capillary column (Bellefonte, PA, USA). The carrier gas was helium with a flow rate of 1 mL/min and split ratio of 1:50. The column initial temperature was 40 ◦C. It was then raised to 300 ◦C with a rate of 20 ◦C/min and was held for 5 min. The ionization electron energy was 70 eV and the mass range scanned was 40–400 *<sup>m</sup>*/*<sup>z</sup>*. The injector and MS transfer were set at 300 and 250 ◦C, respectively. The volatile constituents of propolis were identified by co-injection of the sample with standard samples when available; based

on their Kovats Index, calculated in relation to the retention times of a series of alkanes (C-8–C-20), in comparison with those of the chemical compounds gathered by Adams [18] and by comparing their MS fragmentation patterns with those of pure compounds in the spectral database of the National Institute of Standards and Technology (NIST) [19].
