*2.9. Antibacterial Activity*

The in vitro antibacterial activity of EEP and compounds **1**–**4**, **10**–**13**, and **15**–**17** were determined using a broth microdilution test as recommended by Clinical and Laboratory Standards Institute (New York, NY, USA) M7-A11 for bacteria [20]. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the test agen<sup>t</sup> that had restricted growth to a level <0.05 at 660 nm after incubation at 37 ◦C for 16–24 h.

#### **3. Results and Discussion**

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

The total polyphenol contents for the selected propolis samples were found to be 178.9 ± 5.7, 198.4 ± 4.1, 167.6 ± 7.8, and 246.3 ± 3.2 mg GAE/g of dry extract for GUA-1, GUA-2, GUA-3, and GUA-4, respectively. It should be noted that the Folin-Ciolcateau reagen<sup>t</sup> was reported in the literature as not specific to only phenols and could react with other reducing compounds that could be oxidized by the Folin reagen<sup>t</sup> [21]. The total flavonoid contents were 64.32 ± 5.75, 58.34 ± 2.8, 77.45 ± 6.9, and 87.5 ± 1.9 mg QE/g of dry extract for GUA-1, GUA-2, GUA-3, and GUA-4, respectively. Flavonoids can form complexes with aluminum chloride to yield a yellow solution. Valencia et al. [22] has been reported values of 629.6 ± 9.9 mg GAE/g of dry extract for TPC and 185.9 ± 3.2 mg QE/g of dry extract and TF in a propolis from Sonora, México. The most important flavonoids isolated in this propolis sample were pinocembrin, pinobanksin 3-acetate, and chrysin. The results also confirm the influence of the geographic region and the season of collection for the quality and properties of the propolis [2].

#### *3.2. Antioxidant Activity Assays*

The EEP was evaluated for its ability to quench the DPPH., which is one of the few stable organic nitrogen radicals and bears a purple colour. This assay is based on the measurement of the loss of DPPH• after reaction with samples. It is considered as the prior mechanism involved in the electron transfer. The IC50 value is a parameter widely used to measure the antioxidant activity of test samples. It is calculated as the concentration of antioxidants needed to decrease the initial DPPH concentration by 50% [23]. Thus, the lower IC50 value the higher antioxidant activity. The EEP GUA-4 showed an antioxidant activity (IC50 = 67.9 μg/mL) comparable to the reference ascorbic acid (IC50 = 43.2 μg/mL) and tenfold lower than Trolox (IC50 = 6.3 μg/mL) and seven-fold lower than quercetin (IC50 = 9.9 μg/mL). While ca ffeic acid (12) showed the highest activity (IC50 = 5.9 μg/mL) along with ferulic acid (10) (IC50 = 9.9 μg/mL) and syringic acid (11) (IC50 = 9.8 μg/mL). The lowest antioxidant activity corresponds to 5-methylchrysin ether (9) (IC50 = 112.9 μg/mL) and 5-methyl-pinobanksin ether (5) (IC50 = 98.4 μg/mL).

There are reports of the DPPH scavenging capacity, in terms of IC50, for pinocembrin (**1**), chrysin (**2**), galangin (**3**), isorhamnetin (**7**), ferulic acid (**10**), syringic acid, (**11**) and ca ffeic acid (**12**) [23–26]. For alpinetin (**4**) only the percentage of scavenging activity of DPPH is reported [27]. Nevertheless, the information available for dillenetin (**6**), 5-methyl-pinobanksin ether (**5**), 5-methylchrysin ether (**9**), and 5-methylgalangin ether (**8**) is scarce. Thus, the present work stands for the first report of the IC50 as a measurement of their antioxidant activity (Table 2).

In the ABTS assay, the antioxidant activity is measured as the ability of test compounds to decrease the color reacting directly with the radical ABTS•<sup>+</sup> [28]. Ferulic acid (**10**), syringic acid (**11**), ca ffeic acid (**12**), and chrysin (**2**) showed the lowest IC50 (Table 2). The EEP, 5-methylchrysin ether (**9**), and 5-methyl-pinobanksin ether (**5**) showed weak antioxidant activity (Table 2).

The analysis of their chemical structures can explain the weak antioxidant activity of the methylated flavonoids. According to Procházková et al. [29] the catechol structure in the B-ring, 2,3-double bond in conjugation with a 4-oxo function in the C-ring and the hydroxyl groups in meta position in ring-A. On the other hand, the activity of phenolic acids lies on the presence of two *o*-hydroxyl groups in the aromatic ring.


**Table 2.** Scavenging ability of ethanol extract of propolis (EEP) GUA-4 and compounds **1**–**12**.

#### *3.3. Cytotoxicity of EEP on Cancer Cells*

Cytotoxicity was expressed as the percentage grow inhibition of C6, HeLa, SiHa, and CaSki cells treated with EEP. In all the cases, EEP shows a cytotoxicity concentration-depended manner. In Table 3, we show the IC50 value of EEP over four cancer cell lines. Those results show that EEP restricts glioblastoma cells (C6 cell cancer line) proliferation in vitro as efficiently as temozolomide (reference drug), whereas, for cervical cancer cell lines, it requires a higher concentration of the EEP compared to cisplatin.

There are a few studies of beneficial properties of Mexican propolis. Li et al. [11] reported that three of the 39 compounds isolated from the methanolic extract of Mexican propolis exhibited a potent cytotoxic effect in a colon, melanoma, lung, cervix, and fibrosarcoma cancer cell lines. Li et al. reported the isolation of flavonoids from methanolic extract of Mexican propolis, and one of them revealed significant cytotoxic effect against pancreatic human cancer cell line with IC50 values of 4 μM [10]. Other studies described the potent cytotoxic activity of galangin (**3**); ferulic acid (**10**); syringic acid (**11**); and caffeic acid (**12**) against different cancer cell lines [22]. Interestingly, a few researchers reported that these compounds could be useful for therapeutic treatments. For example, Benguedouar et al. [30] reported that ethanolic extract of Algerian propolis (EEP) and galangin (**3**) decreased the number of B16F1 melanoma cells in vitro compared to control. Celinska-Janowicz et al. [31] state that the ethanolic extract of propolis isolated abundant polyphenolic compounds such as ferulic acid (**10**) and caffeic acid (**12**) revealed pro-apoptotic activity on human tongue squamous carcinoma cells (CAL-27). From this information, we emphasized that EEP and compounds possesses anti-cancer effects against cancer cell lines.


**Table 3.** Cytotoxic effect of EEP on several cancer cell lines.

## *3.4. Antibacterial Activity*

As shown in Table 4, antimicrobial screening against four oral pathogens revealed that compounds **1**, **3**, **4**, **12**, and **16** were inhibitory to the growth of *Streptococcus mutans*, *Streptococcus oralis*, *Streptococcus sanguinis,* and *Phorphyromonas gingivalis*. Among these, compounds **1**, **3**, **4**, and **12**, were either equally or more potent than their respective crude extract of origin (Table 4). Earlier in vitro studies have shown that the Sonoran ethanolic extract of propolis exhibited antibacterial activity against *E. coli* (ATCC 25922) and *S. aureus* (ATCC 6538P). The propolis constituents CAPE, pinocembrin, pinobanksin 3-*O*-acetate, and naringenin exhibited significant inhibitory activity on the growth of *S. aureus*. CAPE exhibited the maximum inhibitory effect on the bacterial growth (CAPE (MIC 0.1 mmol/L), pinocembrin (MIC 0.4 mmol/L), pinobanksin 3-*O*-acetate (MIC 0.8 mmol/mL), and naringenin (0.8 mmol/L)). None of the propolis constituents influenced the growth of *E. coli* at any of the tested concentrations [32].


**Table 4.** Antimicrobial activity of ethanolic extract and compounds **1**–**4**, **10**–**13**, **15**–**17**, and chlorhexidine digluconate (CHX) a.

a positive control; b minimum inhibitory concentration.

#### *3.5. Chemical Composition of EEP GUA-4*

The EtOH-soluble extract of sample GUA-4 was fractionated by chromatography on a VLC column and by Sephadex LH-20, giving 12 known compounds (**1**–**12**). The compounds were identified as pinocembrin (**1**, 405.4 mg) [33], chrysin (**2**, 126.3 mg) [34], galangin (**3**, 56.7 mg) [33], alpinetin (**4**, 15.6 mg) [35], 5-methyl-pinobanksin ether (**5**, 16.8 mg) [36], dillenetin (**6**, 12.3 mg) [37], isorhamnetin (**7**, 24.6 mg) [38], 5-methylgalangin ether (**8**, 9.8 mg) [39], 5-methylchrysin ether (**9**, 16.3 mg) [40], ferulic acid (**10**, 24.9 mg) [33], syringic acid (**11**, 6.7 mg), and caffeic acid (**12**, 8.9 mg) [33] (Figure S1) by means of 1D (Table S1) and 2D NMR spectral analysis. The presence of pinocembrin (**1**), chrysin (**2**), galangin (**3**), ferulic acid (**10**), syringic acid (**11**), and caffeic acid (**12**) sugges<sup>t</sup> that its main botanical source are a species of *Populus* typical of the country, such as *Populus mexicana* Wesmael, *Populus guzmanantlensis* Vazques and Cuevas and *Populus simaroa* Rzedowski. Although flavonoids without B-ring substituents appear common in temperate propolis from both the northern and southern hemispheres, in this study we report for the first time the presence of dillenetin (**6**) and isorhamnetin (**7**) with B-ring substitution as constituents of Mexican poplar propolis. To the best of our knowledge, this is the first report of dillenetin (**6**) as a propolis constituent. 5-methylpinobanksin (**5**), alpinetin (**4**), isorhamnetin (**7**), 5-methylgalangin ether (**8**), and 5-methylchrysin ether (**9**) were previously identified by diode array detection and ESI mass spectrometry (LC-DAD-ESI-MS) as constituents of Italian, Portuguese, and Czech propolis [41,42].
