Propolis from Poland versus propolis from New Zealand - chemical composition and antiproliferative properties on glioblastoma cell lines.

Several studies have previously reported that propolis and its ingredients inhibit glioma cancer cell lines. The chemical composition and antiproliferative activity of propolis from Poland (PPE) and propolis from New Zealand (MPE) were compared in this study. The chemical composition was investigated by gas chromatography-mass spectrometry. Antiproliferative activity of PPE and MPE was determined by a cytotoxicity test and DNA binding by 3 H-thymidine incorporation on Human Diffuse Astrocytoma cell line (DASC) derived from a patient with a Grade II glioma and glioblastoma multiforme T98G and LN-18 cell lines from American Type Culture Collection. The chemical composition of both propolis was comparable, with marginal differences in the amount of some compounds. Flavonoids and chalcones, of which pinocembrin, pinobanksin, pinobanksin 3-acetate, chrysin and galangin showed the highest level, were the main components of both examined propolis (PPE–49.4% and MPE–52.1%). The performed cytotoxicity test showed powerful activity of PPE and MPE propolis on DASC, T98G and LN-18 cells. The degree of the antiproliferative activity was similar in the case of both propolis (viability after 72 h for 30 µg/mL ranged from 22.0% to 51.6% and proliferation inhibition after 72 h approximately was from 18.6% to 75.6%). These results are the first to show that propolis from Poland and propolis from New Zealand have a strong cytotoxic and antiproliferative effect on DASC (Grade II glioma) derived from a patient and glioblastoma multiforme T98G and LN-18 cell lines. This activity may be associated with the high content of polyphenolic compounds in both propolis. These findings suggest that Polish and New Zealand propolis shows promising anticancer activity in the treatment of glioblastoma. However, further studies are required.

The injector worked at 250˚C in the split (1:50) mode. The initial column temperature was 50˚C, rising to 310˚C at 5˚C/min and the higher temperature was maintained for 15 min. MSD detector acquisition parameters were as follows: transfer line temperature 280˚C, MS Source temperature 230˚C and MS Quad temperature 150˚C. The EIMS spectra were obtained at the ionisation energy of 70 eV. The MSD was set to scan 41-600 a.m.u. Following the integration, the fraction of each component in the total ion current was calculated. Hexane solutions of C 10 -C 40 n-alkanes were separated under the above conditions. Gas chromatographic linear programmed retention indices (I T ) were calculated on the basis of the retention times of the n-alkanes hexane solution and separated components of the extract samples.
To identify the separated components, two independent analytical parameters were used: mass spectra and calculated retention indices. The mass spectrometric identification of non-derivatised components was performed with an automatic system for GC-MS data processing supplied by the NIST 14 library (NIST/EPA/NIH Library of Electron Ionization Mass Spectra). The mass spectra and retention indices of the components registered in the form of TMS derivatives were compared with those presented in a recently published database [9] and a private mass spectra library. Identification was considered reliable if the results of the computer search of the mass spectra library were confirmed by experimental RI values, i.e. if their deviation from the published database values did not exceed ± 10 u.i. (the average quantity of inter-laboratory deviation for non-polar stationary phases).

Total phenolic content analysis
Total phenolic content (TPC) was measured using the Folin-Ciocalteu colorimetric method (FC).
Absorbance versus a prepared blank was read at 760 nm using Cintra 3030 (GBC Scientific Equipment, Australia). The results were expressed as milligrams of gallic acid equivalent (GAE) per gram of a dry extract. The concentration of samples equalled 2 mg/mL (extract dissolved in 70% ethanol). Assays were performed in triplicate. Data were expressed as mean ± SD.

Cell culture
The study was performed using Diffuse astrocytoma steam-like cells (DASC) and glioblastoma multiforme (T98G and LN-18) cell lines. DASC cell line was derived from a 43-year-old patient with diffuse astrocytoma (Grade II), which was described in our previous research [10]. The study was approved by the local Ethics Committee [10]. T98G and LN-18 were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in a humidified incubator at 37 °C and 5% CO 2 atmosphere, in MEM (DASC and T98G) or DMEM (LN-18) supplemented with 10% heat inactivated FBS; 100 U/mL penicillin and 0.1 mg/mL streptomycin. Subconfluent cells were detached with a trypsin-EDTA solution in PBS and counted in a Neubauer hemocytometer.

Cytotoxicity assay
Cell viability was measured using an MTT assay, as previously described for glioma cells [10]. The effects of PPE and MPE extracts on DASC, T98G and LN-18 cell lines were studied after 24 h, 48 h and 72 h of the treatment. The cells were cultured in a humidified incubator at 37 °C and 5% CO 2 atmosphere; in MEM or DMEM supplemented with 10% heat inactivated FBS; 100 U/mL penicillin and 0.1 mg/mL streptomycin. Doses of propolis (10,20,30,50, 100 µg/mL) were selected in our previous experiments [8]. Cells at a density of 1 × 10 5 cells/mL were seeded onto 96-well plates at a volume of 200 µl per well and grown for 22 h at 37˚C in a humidified 5% CO 2 incubator. The data were expressed as a percentage of the control (0.1% DMSO). DNA synthesis assay [ 3 H]-thymidine assays were performed to study DNA synthesis in the cells after the treatment, as described in our previously published study [10]. The cells were seeded (1.5 × 10 5 cell/well) on 24well plates in MEM or DMEM supplemented with 10% heat inactivated FBS; 100 U/mL penicillin and 0.1 mg/mL streptomycin and exposed to the treatment medium containing DMSO (0.1% -control), PPE and MPE (30 µg/mL). The cells were cultured for 44 h prior to adding 0.5 µCi of 3 H-thymidine per well. After 4 h of incubation, the medium was removed and the cells were washed twice with cold 0.05 M Tris-HCl and 5% trichloroacetic acid, then scraped and transferred to a scintillation cocktail.
The level of [ 3 H]-thymidine incorporated in the newly synthesised DNA strand was assessed by a scintillation counter in relation to the number of cells proliferating during the S phase of the cell cycle.

Statistical analysis
All data were analysed using Dell Inc. (2016). Dell Statistica (data analysis software system), version 13. software.dell.com. The results were expressed as mean ± SD and statistically compared to the control. Values were tested for a normal distribution using the Shaphiro-Wilk test. Differences between two groups were analyzed using Student's t-test or U Mann-Whitney test. P < 0.05 was considered to be statistically significant.

Results And Discussion Chemical composition of Polish and Manuka propolis
The complex chemical composition of propolis is associated with the quality of the resinous materials gathered by honey bees from different floral sources available around the hive, which has a direct impact on the quality and bioactivity of propolis. In this study, more than 100 individual compounds in PPE and more than 150 compounds in MPE were identified. A list of these constituents is presented in Table 1. Flavonoids and chalcones were the main components of both examined propolis (PPE-49.4% and MPE-52.1%) ( Table 2). The main representatives of this group of compounds in PPE and MPE were, respectively, pinocembrin (8.16% and 14.64%), pinobanksin (4.25% and 4.70%), pinobanksin 3acetate (11.27% and 9.21%), chrysin (5.33% and 5.73%), galangin (8.95% and 9.60%) and their derivatives. (Table 1). These compounds are characteristic of propolis originating from bud exudates of Populus nigra [11,3]. Our analysis also confirmed research results published by other authors who have demonstrated that New Zealand propolis has very high levels of pinocembrin and pinobanksin-3-O-acetate [1]. Cinnamic acid derivatives such as esters: 3-methyl-2-bytenyl (E)-caffeate, benzyl (E)caffeate, benzyl (E)-p-coumarate, 2-phenylethyl p-coumarate, benzyl (E)-ferulate, CAPE, cinnamyl (E)p-coumarate and others were the second significant group of compounds in PPE and MPE (19.8% and 14.5%) ( Table 2). Considerable quantities of aromatic acids were present in both studied propolis extracts, although propolis from Poland (PPE-18.3%) contained twice as great a quantity of aromatic acids as propolis from New Zealand (MPE-7.8%) ( Table 2). The main representatives of this group were p-coumaric acid, (E)-ferulic acid and (E)-caffeic acid. PPE contained high levels of p-coumaric acid (9.80%) ( Table 1). TPC determination confirmed that PPE and MPE are rich in phenolic compounds. Their levels were calculated to be 243.7 ± 9.0 in PPE and 245.6 ± 5.9 mg GAE/g in MPE (Table 3). Other authors have demonstrated higher or lower TPC in propolis. The values ranged from 14.6 to 150.8 mg GAE/g in Polish propolis [12] and from 99 ± 4.0 to 775 ± 8.5 mg GAE/g in Manuka propolis [13]. TPC value depended on the extraction method utilised.
Comparison of the chemical composition of the tested propolis revealed that both PPE and MPE had similar quantities of the identified active components and the total content of phenols, which is consistent with the classification of propolis from New Zealand as the "Poplar" type. According to Kumazawa et al. [14], comparison of the antioxidant activity and composition, total phenol and flavonoid content in individual samples of ethanolic extracted propolis from 14 countries showed that New Zealand-sourced propolis was similar in composition to propolis from Bulgaria, Uzbekistan and Hungary, and to propolis from three South American countries: Chile, Uruguay and Argentina.   Cytotoxicity and antiproliferative activity Chemical compounds present in propolis offer powerful bioactive protection against pathogens and are therefore used by bees to immunise the hive environment [15]. For this reason, they may also serve as a significant source of bioactive substances for pharmaceutical purposes. A number of research studies have focused on the potential utilisation of propolis phenolic compounds in the development of new anti-cancer drugs [16,17]. Our previous study revealed that Polish propolis has strong cytotoxic and antiproliferative activity and, additionally, cooperates with (TMZ) synergistically, enhancing its growth-inhibiting activity against glioblastoma U87MG cell line through the reduction of NF-κB activity [8]. In this study, cytotoxicity and antiproliferative activity was determined using DASC for the dose 30 µg/mL, it was 77.9 ± 4.3% and 81.3 ± 4.0% after 24 h, 58.6 ± 0.3% and 63.4 ± 7.8% after 48 h, and 47.0 ± 3.2% and 51.6 ± 8.1% after 72 h for PPE and MPE, respectively (Fig. 1A,B,C). A significant, but lower than 10%, difference (p < 0.05) in the reduction of DASC cells treated with PPE in comparison to those treated with MPE was observed for the 100 µg/mL concentration after 48 h (approximately 7%) (Fig. 1B) and for 20, 50, 100 µg/mL concentrations after 72 h (8.4%, 6.9%, 3.0%, respectively) (Fig. 1C). For T98G cell line we observed a stronger significant reduction in cell numbers  (Fig. 1D,E,F). Interestingly, dose-dependent decreases in T98G cells viability were observed after 24, 48 and 72 h but only for the 10-50 µg/mL dose range. After treatment 100 µg/mL dose we observed "reflection effect" because decrease viability was smaller than for 50 µg/mL dose. A significant, difference (p < 0.05) in the reduction of T98G cells treated with PPE in comparison to those treated with MPE was observed for the 50 µg/mL concentration after 24 h (Fig. 1D), for 10, 20, 30, 50 µg/mL concentration after 48 h (Fig. 1E) and for 20, 50, 100 µg/mL concentrations after 72 h (Fig. 1F) (Fig. 1H), for, 50 and 100 µg/mL concentration after 48 h (Fig. 1I).
Interestingly, significantly stronger cytotoxic effect on LN-18 cells was observed after treatment with PPE than MPE.  [26]. Chrysin induces apoptosis in cancer cells by the activation of caspases, suppression of antiapoptotic proteins such as IAP, c-FLIP, PI3K/Akt signal pathway, inhibition of IKK and NF-kB activity [27]. In our previous study we demonstrated that natural bee products such as bee bread, royal jelly and honey extract showed varied activity against U87MG and SVGp12 cell lines. Furthermore, the use of these bee products may increase the cytotoxic effect of TMZ on U87MG and SVGp12 cell lines. We also observed that U87MG cells were sensitive to natural bee products, but no impact of natural bee products on DASC cells was noted [10].

Conclusions
Summarising, these results are the first to show that propolis from Poland and propolis from New

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the Local Ethical Committee (R-I-002/346/2008).  The results are presented as a percentage of control. All statistical analyses were performed using Student-t test (significant changes: *p<0.05 vs control).