Total phenolic and flavonoid contents
Total phenolic (TP) and flavonoids (TF) contents of Algerian propolis extracts were determined using Folin-Ciocalteu and aluminium nitrate methods. TP and TF of various propolis extracts ranged from 5.36 ± 1.14 to 187.16 ± 4.83 GAE µg/mg and 0.38 ± 0.41 to 63.53 ± 1.28 QE µg/mg, respectively. Methanol extract was found to be the richest extract on both phenolic and flavonoid contents (Table 1).
Total phenolic and flavonoid contents of Algerian propolis extracts
Total phenolic EGA (µg/mg)
Total flavonoid EQ (µg/mg)
5.36 ± 1.14
79.16 ± 2.66
119.94 ± 4.83
187.16 ± 4.83
0.38 ± 0.41
16.25 ± 0.21
60.34 ± 1.39
63.53 ± 1.28
The methanol extract exhibited a higher TP and TF values compared to propolis collected from Khanchla and Ghardaia (TPC: 14.23–4.93 GAE µg/mg and TFC: 3.45–1.94 QE µg/mg, respectively) (Rebiai et al. 2014). TP and TF amounts obtained in this study were similar to those reported for propolis collected from Fundão Region in Portugal (151.00 GAE µg/mg) (Moreira et al. 2008) and Korean propolis collected from Yeosu (212.7 GAE µg/mg) (Choi et al. 2006). But the reported values were lower compared to Chinese samples collected from Hebei (302 GAE µg/mg) (Ahn et al. 2007) and Hubei (299 GAE µg/mg) (Kumazawa et al. 2004). The chemical composition of different propolis has been widely investigated. It was found to be very complex and varies according to the geographical region, climate or plant type (Mercan et al. 2006; Yasar et al. 2016). This chemical diversity is very important for biological and medicinal properties of propolis such as antibiofilm, antimicrobial, antioxidant, anticancer, antiallergic and antiviral (Daikh et al. 2020; Yasar et al. 2016; Yildirim et al. 2016; Mercan et al. 2006). The most abundant biologically active components of propolis are flavonoids and phenolic acids. Moreover, therapeutic activity of propolis depends largely on polyphenol contents with particular flavonoids (Campos et al. 2015; Boulechfar et al. 2019). Among the biological activities linked to phenolic and flavonoids composition, antibacterial, antibiofilm and anticancer activities are largely documented. In addition, some researchers stated that propolis with rich content in aromatic acids (etc. caffeic acid and ferulic acid) and flavonoids (etc. chrysin and galangin) exhibited a strong antibacterial, antibiofilm and/or anticancer effect (Patel, 2016; Ahangari et al. 2018).
Biofilm inhibition activity
The biofilm inhibitory effects of Algerian propolis was determined against S. aureus ATCC 29213 by using different concentration (10 to 800 µg/mL). Results of the antibiofilm screening assay are given in Fig. 1. All tested extracts showed antibiofilm effect on staphylococcal biofilm. In general, the biofilm inhibition efficiency of extracts increased with the increasing concentration of propolis indicating that the biofilm inhibition activity of propolis acts in a dose-dependent manner. Similar results were found in study where Polish propolis showed dose-depended antibiofilm activity (Wojtyczka et al. 2013).
Methanol and chloroform extracts exhibited the highest potency compared to petroleum ether and ethyl acetate extracts against bacterial biofilm production. The minimum and maximum inhibition percentages of methanol extract were found as 55% (50 µg/mL) and 92% (200 µg/mL), respectively. The highest activity was achieved at concentrations of 400 µg/mL (82%) and 500 µg/mL (83%) in chloroform extract. Compared to methanol, chloroform extract inhibited only 55% of the bacterial biofilm at a concentration of 200 µg/mL. Antibiofilm assay revealed that about 800 µg/mL of extract was required for the high inhibition of petroleum ether (67%). However, the ethyl acetate extract decreased the biofilm inhibition by ~ 64% and 59% at 400 and 600 µg/mL concentrations, respectively. Our results showed that methanol extract was the most effective extract, undouptly due to its flavonoid content known to have antibacterial properties. As indicated by Kothari (2014), methanol is the most suitable solvent for the extraction of flavonoids. Furthermore, the high antibiofilm effect of propolis collected from the mountains of Kafkas and Ural is due to their rich flavonoid content (Bryan et al. 2015a). It is worth noting that methanol extract also acts at very low concentrations compared to other tested extracts.
Our previous results about antibiofilm activity of five different Algerian propolis on eight bacterial strains were quite similar to the results of this study. Several factors were investigated for their potential influence on biofilm formation (Daikh et al. 2020). All of these results revealed that the used solvent during extraction in association with propolis origin and the tested bacterial strains significantly affect biofilm formation. Moreover, caffeic and ferulic acids were pointed to be responsible of the observed antibiofilm. We also reported that the high antibiofilm effect of the methanol extract of Algerian propolis was due to the higher content of ferulic and caffeic acid compared to other extracts. Caffeic acid was also reported to exhibit a biofilm activity by Luis et al. (2014). Bioactive natural compounds not only increase the permeability of the bacterial membrane, but they also reduce metabolic activity, mobility and membrane transport (Veloz et al. 2019). Stan et al. (2016) found that propolis extracts had a strong inhibition on clinical S. aureus strains adherence to the cellular substrate. Several possible mechanisms were suggested to understand the antibacterial and biofilm inhibition efficacy of propolis. Propolis can affect cell membrane permeability leading to a reduction in the production of adenosine triphosphate and can compromise bacterial mobility and other activities (Almuhayawi, 2020). Both activities could be due to the synergic effects of phenolics, in particularly flavonoids such as Galangin, Pinobanksin and Pinocembrin, and other propolis constituents (Castaldo and Capasso, 2002; El-Guendouz et al. 2016).
Fluorescent microscopy analysis
The bacterial biofilm structure, untreated (control group) and treated with propolis, was examined by Fluorescence Microscope (Fig. 2). It is clearly seen that the biofilm structure of S. aureus is compact and dense ensemble, and each community is interconnected by water channels (Fig. 2A and 2B). As seen in Fig. 2C-2J, the decrease in the amount of bacterial biofilm treated with propolis was significant compared to control group. Contrary of control biofilm, biofilm community and their water channels degraded after treatment with propolis. Moreover, the decrease in number of live cells (bright) and the increase in number of dead cells (opaque) were also showed. Particularly, the effect of methanol extract on biofilm structure was more pronounced (Fig. 2I and 2J).
The biofilm is viscous and has high water content. The structure of the biofilm is quite complex and connected to each other by water channels which provide nutrient and material accessibility to the deepest regions of the biofilm (Archer et al. 2011). Vikram et al. (2010) reported that flavonoids not only inhibited biofilm production of E. coli and reduced the virulence of Vibrio harveyi, but also modulated cell-cell communication. They also notified that among the tested flavonoids, naringenin showed nonspecific inhibition in auto inducer-mediated cell-cell signalling (Vikram et al. 2010). As indicated by Bryan et al. (2015b), complete inactivation of S. aureus and E. coli biofilms after 18 h of Russian propolis treatment was observed that was confirmed by the confocal microscopy. Moreover, the treatment with propolis caused lysis of cells and the decomposition of the cell walls was predominately caused by organic compounds in Russian propolis according to microscopic images (Ambi et al. 2017). In similar another study, the microscopic studies confirmed the antibiofilm action of propolis (Grecka et al. 2020). According to our microscopy images, the deterioration of biofilm structure showed that the cell-cell communication was disrupt. Particularly during our microscope examination, we observed that the water channels between the cell assemblages were interrupted, and the density of these channels was also less compared to the control. Thus, we assumed that the absence of water channels after treatment with propolis was evidence of lethal effect of propolis on bacterial cells. Furthermore, it was more and more pleasing for us that the results of fluorescence microscopy and antibiofilm activity confirmed each other.
The FTIR spectrum of S. aureus represented in Fig. 3 reveals the characteristic features of protein with the presence of strong band at 3288.76 cm-1 related to NH of amide A (Sionkowska et al. 2004; Kochan et al. 2020). Band at 2918.68 cm-1 could be attributed to amide B. In addition, the presence of bands at 1638.26 cm-1, 1550.05 cm-1 and 1240.81 cm-1 is characteristic of functional group of amide I, II and III respectively. According to Davis and Mauer (2010), picks located between 1500 − 1200 cm-1 are both characteristic bands of fatty acid bending vibrations and proteins. While picks located between 1200 − 900 cm-1 are attributed to carbohydrates in cell walls.
The analysis of FTIR spectrum of cells reveals the presence of a band at 1400 cm-1 characteristic of fatty acid and amino acids and in general attributed to C = O symmetric stretching of COO- of the cited components. In addition, the presence of a band at 720 cm-1 assigned to C-H of > CH2 of fatty acids and proteins is also observed. Band observed at 1080.31 cm-1 is assigned to DNA, RNA and phospholipids.
After treatment with chloroform (Cl), ethyl acetate (EA) and methanol (ME) extracts of Algerian propolis, all the mentioned bands remained and had the same intensity indicating no change on S. aureus proteins constitution (Fig. 3C, 3D, 3E). On the contrary, the treatment with petroleum ether extract caused a decrease of the intensity of protein bands (Fig. 3B). Additionally, bands at (2918.68 cm-1, 1550.05 cm-1 and 1240.81 cm-1) disappeared, concomitantly with the reduction of the intensity of the band appearing at 1638.26 cm-1 and increasing the band at 3288.76 cm-1 suggesting a change of secondary structure of S. aureus proteins and probably their denaturation.
FTIR spectra of Cl (Fig. 3C), EA (Fig. 3D) and ME (Fig. 3E) extracts seem practically superimposable, which means that the three tested extracts present the same chemical profile. The three tested extracts present characteristic bands of aliphatic compounds which appeared as strong band at 3283.76-3284.69 cm-1 and 2900 cm-1. Band at 2850 is cm-1 is attributed to hydroxyl groups. Moreover, aromatic compounds and flavonoids are also present. The presence of such aromatic components could be detected by the presence of bands between 1650 − 1600 cm-1 and 1550 − 1400 cm-1 (Oliveria et al. 2016; Anjos et al. 2015; Afonso et al. 2020). Peaks at 1170.5-1171.41 cm-1 and 1234.64-1242.40 cm-1 are assigned to stretching vibrations of C-O and C-O-C in alcohols, esters and sugars present in propolis (El-Guendouz et al. 2016).
On the contrary, PE extract spectra are qualified by the presence of fatty acids with the presence of characteristic bands of C-H asymmetric stretching bands appearing near 2930 and 2870 cm-1. Two other bands appearing around 1640 cm-1 and 1480 cm-1 could be attributed to the presence of aliphatic acids (El-Guendouz et al. 2019).
The cytotoxic efficacy of different propolis extracts was determined in breast cancer cell line (MDA-MB-231) using MTT assay. To assess the cytotoxic effect, cells were treated with various concentrations of propolis extracts for 24 hours. The extracts showed cytotoxic activity in a dose-dependent manner. EC50 values were found in a range from 97 to 117 µg/mL (Fig. 4).
Most active extract was found to be ethyl acetate extract, showing an EC50 value of 97 µg/mL. The cytotoxic effect of propolis extracts was in the following order: Ethyl acetate ˃ Chloroform ˃ Petroleum ether ˃ Methanol. Our results are in accordance with those reported in the literature. Xuan et al. (2014) showed that ethanolic extract of Chinese propolis had cytotoxic effects on MCF-7 and MDA-MB-231 with a dose and time-dependent manner. Same results were reported by Milošević-Đorđević et al. (2015) for two Serbians propolis samples. In addition, Turkish propolis was also showed cytotoxic effect on MDA-MB-231 cells and the highest antiproliferative activity was obtained at 10 mg/ml concentration for 24 and 72 h (Uçar and Değer, 2019).
Natural products such as propolis contain many bioactive substances with cytotoxic and anticancer effects on different cancer cells. Caffeic acid phenyl esters (CAPE) are the most common compounds in propolis and it was inhibited MDA-MB-231 cell proliferation (Chang et al. 2017). Moreover, its cytotoxic effect was proven against human myeloid cancer cells (Jin et al. 2008). Algerian propolis is rich in biologically active phytochemicals and has a good immunomodulatory effect (Soltani et al. 2017). Another study showed that Algerian propolis inhibited lung cancer cells proliferation and induced apoptosis (Kebsa et al. 2018). Furthermore, the inhibitory effect of Algerian propolis on lung cancer cell proliferation was confirmed with in vitro tests (Brihoum et al. 2018). Algerian propolis was also reported to reduce melanoma tumor progression/dissemination and to extend mice life span. Galangin was pointed to significantly reduce melanoma cell proliferation and to induce autography/apoptosis dependently (Benguedouar et al. 2015).
Western blot analysis
Western blot analysis was performed to determine the effect of propolis on the level of proteins involved in apoptosis and cell cycle. As represented in Fig. 5, the densitometry analysis of the Caspase 3 protein level indicated that the expression was increased significantly 1.42-, 1.35-, 1.35- and 1.53-folds in PE, Cl, EA, ME extracts with respect to control, respectively (p < 0.05). Although PE and Cl extracts decreased the level of Bax protein (2- and 1.69-folds, respectively), EA and ME extracts increased Bax level (2.19- and 2.15-folds, respectively). The results of densitometry analysis of the Bcl-2 shown in Fig. 5 indicated that Bcl-2 expression was decreased 1.56-, 1.44-, 2.38- and 2.43-folds in PE, Cl, EA, ME extracts treated MDA-MB-231 cells (p < 0.05). Similarly, to Bcl-2, CDK-4 protein level was decreased significantly with propolis extracts treatment (2.86-, 1.13-, 2.85- and 3.44-folds, respectively, p < 0.05). While, P53 protein level was increased significantly in PE, Cl, EA, ME extracts treated cells (1.58-, 1.57-, 3.17- and 2.1-folds, respectively).
Results demonstrated that Algerian propolis caused a cell growth inhibition on the tested cells due to induction of apoptosis and cell cycle arrest. Intrinsic apoptotic pathway was induced by propolis treatment due to induction of caspase-3 and 9 activities (Kebsa et al. 2018). Algerian propolis ethanol extract was also reduced the proliferation of LNCaP cells by arresting cell cycle at G0/G1 and inducing apoptosis (Zabaiou et al. 2019).
Real time PCR
The effects of propolis treatment on gene expression analysis of Caspase3, Bax, Bcl-2, CDK-4 and P53 were revealed in MDA-MB-231 cells. Real Time-PCR results of mRNA levels were represented in Fig. 6. Caspase3 mRNA level was increased 1.49-, 1.62-, 10.41-, 28.44-folds in PE, Cl, EA and ME extracts treated cells with respect to control, respectively (p < 0.05). Although, PE and Cl extracts decreased the level of Bax mRNA (8.6- and 6.98-folds, respectively). While PE and ME extracts increased Bax mRNA level (1.34- and 3.91-folds, respectively). On the other hand, Bcl-2 mRNA levels were decreased 2.08-, 1.36-, 1.81- and 3.63-folds due to extract treatment. P53 mRNA level was increased 2.27-, 3.16-, 11.79- and 24.16-folds because of PE, Cl, EA, ME extracts treatment (Fig. 6, p < 0.05). Similarly, to protein level, CDK-4 mRNA level was decreased significantly (5.02-, 1.38-, 2.79- and 1.34-folds, respectively).
Our results are in contradiction with those reported in the literature. Cuban propolis caused to decrease the expression of apoptosis-related genes such as Caspase3, P53, p21 and Bax in MDA MB-231 cells. Whereas, Bcl-2, Bcl-XL, Noxa and Puma expression was not changed (Frión-Herrera et al. 2019). Brazilian propolis caused reduction of mitochondrial membrane potential in A549 cells by overexpression of Bax and Noxa and suppression of the Bcl-XL. While, the expression level p53, Caspase3, bcl-2 genes remained unchanged, p21 expression was increased (Frión-Herrera et al. 2015). Brazilian propolis and caffeic acid, one of the main phenolic acids present in propolis, were also tested on Hep-2 cells. Both products induce apoptosis. Only propolis downregulates P53 expression. Caspase-3 expression was increased due to propolis treatment and that was correlated with induction of both early and late apoptosis due to propolis and caffeic acid treatment (da Silva et al. 2017).
Apoptosis analysis of Mila extracts
In this study, Annexin-V was used to analyse apoptosis. For quantification Arthur image-based cytometer was used. H2O2, was used as a positive control. As shown in Fig. 7, after 24 hours of application with extracts, apoptotic cell proportions were increased significantly. The ratio of apoptotic cells in propolis treatment was 6 to 10-folds higher than the control ones. These results confirmed that propolis extracts caused apoptosis and had a cytotoxic effect. Also, in all treated cells, there was a statistically significant decrease in the percentage of live cells compared to the control ones. Our results are in accordance with previous research on Chinese propolis. Xuan and co-workers studied the effects of Chinese propolis on Annexin A7 (ANXA7), P53, Reactive oxygen species levels, NF-𝜅B p65 and mitochondrial membrane potential in MCF-7 and MDA-MB-231 (Xuan et al. 2014). They found that propolis caused significant induction of ANXA7 expression, NF-𝜅B p65 and ROS level. Chinese propolis increased the tumor suppressor proteins (p21CIP1 and p53) in HCT116 cell line and induced apoptosis (Ishihara et al. 2009). Similarly, Turkish propolis increased in protein level associated with P53, Bax and P21 (CDKN1A) genes in MCF-7 cells and its activity was also confirmed by western blot (Misir et al. 2020).