Phytochemical Constituents, Antioxidant, Cytotoxic, and Antimicrobial Activities of the Ethanolic Extract of Mexican Brown Propolis

Propolis is a complex mixture of natural sticky and resinous components produced by honeybees from living plant exudates. Globally, research has been dedicated to studying the biological properties and chemical composition of propolis from various geographical and climatic regions. However, the chemical data and biological properties of Mexican brown propolis are scant. The antioxidant activity of the ethanolic extract of propolis (EEP) sample collected in México and the isolated compounds is described. Cytotoxic activity was evaluated in a central nervous system and cervical cancer cell lines. Cytotoxicity of EEP was evaluated in a C6 cell line and cervical cancer (HeLa, SiHa, and CasKi) measured by the 3-(3,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium (MTT) assay. The antibacterial activity was tested using the minimum inhibitory concentration (MIC) assay. Twelve known compounds were isolated and identified by nuclear magnetic resonance spectroscopy (NMR). Additionally, forty volatile compounds were identified by means of headspace-solid phase microextraction with gas chromatography and mass spectrometry time of flight analysis (HS-SPME/GC-MS-TOF). The main volatile compounds detected include nonanal (18.82%), α-pinene (12.45%), neryl alcohol (10.13%), and α-pinene (8.04%). EEP showed an anti-proliferative effect on glioma cells better than temozolomide, also decreased proliferation and viability in cervical cancer cells, but its effectiveness was lower compared to cisplatin.


Introduction
Propolis also known as "bee glue" is a nontoxic hive product accumulated by bees from diverse plants containing compounds such as flavonoid aglycones, phenolic acids and their esters, phenolic aldehydes, alcohols, ketones, sesquiterpenes, coumarins, steroids, amino acids, and inorganic compounds. It functions in sealing holes, cracks, reconstruction, and smothering the inner surfaces of the beehive. Propolis and its extracts have application in treating diseases due its anti-inflammatory, antioxidant, antibacterial, antimycotic, antifungal, antiulcer, anticancer, and immunomodulatory properties [1,2]. Egyptians, Greeks, Romans, Chinese, Arabs, and Incas have traditionally used it as an

Antioxidant Activity
The EEP antioxidant activity was assessed using two different 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 × (A control − A sample )/A control , where A control and A sample are the absorbance. The IC 50 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 (A control − A sample )/A control . The IC 50 was calculated from the scavenging activities (%) versus concentrations of the respective sample curve.

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 reagent 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).
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 m/z. 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].

Cell Culture
Rat C6 glioma cell and human cervical cancer cell lines (HeLa, SiHa, and CaSki) were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were routinely maintained in Dulbecco's modified Eagle´s medium (DMEM) supplemented with fetal bovine serum (Gibco BRL) with 5% for C6 cells and 10% for cervical cancer cells, and incubated at 37 • C in an atmosphere comprising 5% CO 2 and 95% air at high humidity. Cells were harvested with 0.025% trypsin and 0.01% EDTA (Gibco BRL).
The effect of EEP, cisplatin, and temozolomide on the proliferation of cells were evaluated using the MTT assay (3-(3,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium), which is based on the reduction of a tetrazolium salt in metabolically active cells. The procedure was as follows. Viable cells were seed into 96-well plates in 100 µL per well of DMEM culture medium at a density of 3 × 10 3 for C6 cells, 2 × 10 4 for CaSki cells, and 1 × 10 4 cells for HeLa and SiHa cells. After treatment, the medium was removed and the MTT solution was added to each well, followed by 1 to 2 h in a humidified atmosphere containing 5% CO 2 at 37 • C. The absorbance of the samples was measured spectrophotometrically at λ 570 nm using a microtiter plate ELISA reader. Results are expressed as the percentage of MMT reduction.
C6 cells were exposed for 72 h with 2 to 200 µg/mL of EEP, since it is the time used to make the exposure with temozolomide (first line treatment used for glioblastoma), whereas HeLa, SiHa, and CaSki cells were exposed for 24 h with 15 to 500 µg/mL of EEP; after that time, cell viability was quantified using the MTT assay. Temozolomide (250 µM) and cisplatin (5-320 µM) were used as a control. The concentration of drugs to reach 50% growth inhibition (IC 50 ) was obtained from the survival curves. The experiments were conducted in triplicate in independent experiments. Values are expressed as the mean ± SEM of at least three independent experiments. SigmaPlot 12.3 software (Systat Software, Santa Clara, CA, USA) was used.

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 agent that had restricted growth to a level <0.05 at 660 nm after incubation at 37 • C for 16-24 h.

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 reagent 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 reagent [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].

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 IC 50 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 IC 50 value the higher antioxidant activity. The EEP GUA-4 showed an antioxidant activity (IC 50 = 67.9 µg/mL) comparable to the reference ascorbic acid (IC 50 = 43.2 µg/mL) and tenfold lower than Trolox (IC 50 = 6.3 µg/mL) and seven-fold lower than quercetin (IC 50 = 9.9 µg/mL). While caffeic acid (12) showed the highest activity (IC 50 = 5.9 µg/mL) along with ferulic acid (10) (IC 50 = 9.9 µg/mL) and syringic acid (11) (IC 50 = 9.8 µg/mL). The lowest antioxidant activity corresponds to 5-methylchrysin ether (9) (IC 50 = 112.9 µg/mL) and 5-methyl-pinobanksin ether (5) (IC 50 = 98.4 µg/mL).

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 IC 50 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 IC 50 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 . From this information, we emphasized that EEP and compounds possesses anti-cancer effects against cancer cell lines.

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].

Conclusions
In the present study, we have isolated twelve (1-12) components and identified 40 volatile compounds in Mexican propolis. The current study revealed the presence of antioxidant, antimicrobial, and cytotoxic phytochemicals [galangin (3); ferulic acid (10); syringic acid (11); and caffeic acid (12)] in EEP. It is concluded that EEP can be a potential addition in pharmaceutical products for the improvement of human health by contributing in the antioxidant defense system fighting against the production of free radicals. México, being a megadiverse country, has numerous numbers of propolis differing in chemical composition. However, unfortunately it is still unexplored.