Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts

Background Viscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album “mother tinctures”, MT) prepared from different host trees. Methods Samples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD. Results Chemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3. Conclusion Our results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

The European Medicines Agency reported the traditional use of different V. album ethanolic extracts to treat cardiovascular disease [11]. Additionally, Poruthukaren et al. [12] described a reduction in blood pressure after V. album ethanolic use for 12 weeks.
Nevertheless, the antitumor effects of ethanolic V. album extracts in biological systems have been described. In vivo studies have shown the anticancer activity of alcoholic and glycerine V. album extracts by stimulating immune mechanisms and inhibiting tumor cell proliferation [13]. The simultaneous application of V. album ethanolic extract and doxorubicin increases the toxicity in Ehrlich tumor cells, opening up the possibility of exploring a tentative synergy between single chemical compounds and complex herbal extracts [14,15]. In addition, the in vitro research performed with these extracts highlighted the apoptotic mechanisms and cell growth reduction [16,17] involved in melanoma, leukemia [16], and human cervix adenocarcinoma tumor [17] death. Panossian et al. [18] suggested that phenylpropanoids detected in V. album ethanolic extracts had antitumor activity through the inhibition of protein kinase C.
Furthermore, a recent study performed with V. album ethanolic extracts showed tumor cell cycle arrest and apoptotic death in in vitro models. Chemical analysis of these extracts identified compounds such as caffeic acid, chlorogenic acid, sakuranetin, isosakuranetin, syringenin 4-O-glucoside, syringenin 4-O-apiosyl-glucoside, alangilignoside C and ligalbumoside A [16].
It is widely known that many metabolites isolated from European mistletoe are not produced by the plant itself but are due to host tree metabolism [19], supporting the importance of understanding the subspecies of European mistletoe and the influence of the host tree. This work shows, for the first time, the in vitro anti-proliferative effects and the chemical composition of ethanolic extracts produced from different V. album host trees.

Plant growth and harvest
The green, unripe berries, leaves and stems of three European subspecies of V. album L. were harvested in July 2016 in natural habitats in Switzerland ( Fig. 1a-g). The following V. album subspecies were collected from five different host trees: V. album ssp. album growing on Malus domestica (VAM; Fig. 1c), Quercus robur (VAQ; Fig. 1d) and Ulmus carpinifolia (VAU; Fig. 1e); V. album ssp. abietis growing on Abies alba (VAA; Fig. 1f); and V. album ssp. austriacum growing on Pinus sylvestris (VAP; Fig. 1g). From each host tree, at least five bushes of the same age containing the same parts of the plant were submitted to solvent extraction as follows: first and second youngest leaves, berries, and first and second youngest stems ( Fig. 1a- Preparation of V. album mother tinctures All solvents and reagents exhibited analytical purity quality. The fresh material (5 g) was fragmented into segments smaller than 5 cm long and dried in an oven at 105°C for 2 h, following the Brazilian Homeopathic Pharmacopeia [20] and the French Pharmacopeia [21]. Once the percentage of solid residue of each fresh plant had been established, the total volume of mother tincture (MT), as well as the volume and the concentration of ethanol used in the maceration process, was determined. Next, the maceration extractive process was conducted over a period of 3 weeks at room temperature in 80% w/w ethanol. To increase the efficiency of the extraction process, all V. album ethanolic solutions were shaken by hand for 60 s twice a day. After 3 weeks, the macerates were filtered and kept at 20 ± 5°C. The final MT concentrations were 40 to 50% v/v [21].

Thin layer chromatography
Thin layer chromatography (TLC) was performed as specified in the monograph of V. album in the French Pharmacopeia [21]. Samples (10-20 μL/band) were applied as 10 mm bands with a 10 mm track distance onto the plate by a capillary 10 mm from the lower edge of the plate. TLC separations were achieved in a chamber (210 × 100 mm) saturated with distilled water:methanol: glacial acetic acid:dichloromethane at 2:3:8: as a mobile phase. The bands were visualized under UV light (365 nm) before and after spraying with NP/PEG as the revealing solution.

Determination of total flavonoid content
The concentration of total flavonoids as rutin equivalents was determined spectrophotometrically in the UV region (360 nm) in comparison with the standard curve of rutin absorption, adapted from Rolim et al. [22].

HPLC viscotoxin analysis
Before HPLC analysis, all mother tincture samples were purified by solid phase extraction (Bakerbond, carboxylic acid, wide pore SPE column) to separate the VT from mother tincture impurities. For this, the SPE column was previously washed with methanol and water and subsequently equilibrated with 5 mL of a 200 mM ammonium solution. Aliquots from 0.5 to 2 mL of each mother tincture were added to the column and the pH was adjusted to 7.0-7.5. The samples were eluted Original picture of a Viscum album bush. 1 First youngest leaves, 2 first youngest stems, 3 second youngest leaves, 4 green unripe berries, 5 second youngest stems; b Viscum album schematic figure. 1 First youngest leaves, 2 first youngest stems, 3 second youngest leaves, 4 unripe berries, 5

LC-HRMS analysis
Samples were prepared as described in the V. album monograph with modifications [21]. UHPLC Dionex Ultimate 3000 coupled to a Q Exactive Plus Orbitrap mass spectrometry system (Thermo Fisher Scientific, Germany), equipped with an electrospray ionization (ESI) source operating in negative ion mode at a voltage of 2.9 kV was employed for the chemical analysis. Separations were performed on a reversed-phase column (Thermo Syncronis C18 50 mm × 2.1 mm; 1.7 μm). Water-formic acid 0.

Cell lines and culture conditions
The following cell lines were used: MOLT-4 (human acute lymphoblastic leukemia cell line), Yoshida (mouse sarcoma cell line) and non-tumor NIH/3 T3 (mouse embryonic fibroblasts cell line), obtained from ATCC, Rockville, MD, USA (Yoshida and MOLT-4) and from the German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany (NIH/3 T3). All cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, and 1% penicillin-streptomycin in a humidified atmosphere with 5% CO 2 at 37°C. Cell lines were maintained in exponential growth, and cells from sub-confluent monolayers (Yoshida and NIH/3 T3) were harvested by trypsin-EDTA to carry out the experiments.

Cell viability assay
Cell viability was evaluated by the WST-1 colorimetric methodology. Briefly, 90 μL of each cellular suspension containing 5 × 10 4 cells/mL was pre-cultured in 96-well plates. After 24 h, 90 μL of V. album mother tinctures, pre-diluted in cellular culture medium, were added at concentrations varying from 0.05 to 0.5% v/v. Cellular viability rates were measured after incubation for 4 and 24 h by the addition of 20 μL of WST-1 to each well. The absorption at 450 nm and 650 nm against a background control was measured after 3 h of incubation at 37°C in the dark in a multiwell plate reader. Since ethanol was used as the solvent for the extraction of tinctures, its effect on cell viability was also evaluated using the same MT concentrations. The percentage of viable cells was calculated in relation to control cells (untreated and treated with ethanol solvent) using mean values from at least three independent experiments, and the calculation was performed in triplicate. The IC50 was calculated using GraphPad 5 Software.

Statistical analysis
In vitro experiments were performed at least three different times, and results were analyzed by ANOVA with Dunnett's post hoc test using GraphPad 5. P values < 0.05 were considered statistically significant.

Results and discussion
Chemical analysis of V. album ethanolic extracts TLC plates of ethanolic extracts showed orangeyellowish bands at 365 nm UV light after spraying with NP/PEG reagent, and this result is typical of flavonoid compounds. Each MT exhibited one fluorescent blue spot with a Rf (retention factor) value similar to that of the chlorogenic acid standard (Rf 0.60). According to ANSM [21], chlorogenic acid is a marker of V. album species, and its identification is important to assure the quality of the vegetal material. Łuczkiewics et al. [23] also identified chlorogenic acid in alcoholic extracts of V. album, corroborating the results found in this work.
Determination of the total flavonoid content of the mother tincture Table 1 shows the flavonoid concentration expressed as mg/g of plant fresh weight (mg/g fw) after ethanolic extraction. The highest concentration was detected in V. album growing on Quercus robur (9.67) and Malus domestica (6.30). Pietrzak et al. [24] observed an increase in the flavonoid content of V. album ssp. abietis extracted by a mixture of polar organic solvents with water. Additionally, Pietrzak et al. [25] determined that the flavonoid content in methanol extracts from V. album growing on different host trees ranged from 0.270 to 0.428 mg/g. The host species and the organ harvested influenced the chemical composition of mistletoe [8].

HPLC viscotoxin analysis
Quantitative analysis (Table 1) of VT isoforms A1, A2, A3, B and 1-PS in ethanolic extracts showed that V. album ssp. abietis contains predominantly viscotoxin A3, whereas V. album ssp. austriacum contains viscotoxins A2 and 1-PS, which were not detected in V. album ssp. album. The three European subspecies of V. album could be distinguished on the basis of their VT composition, and this result is in accordance with literature data [26,27]. However, the total content of VT is greater in aqueous preparations than in hydroalcoholic preparations when the viscotoxin proportions are compared for the same V. album ssp. [27].

LC-HRMS analysis
This analysis was performed to obtain an overview and compare the chemical compounds of the different V. album MTs prepared. The major peaks present in the LC-HRMS chromatograms were tentatively identified, and this compound identification may help in the standardization of the V. album extracts used in the in vitro analysis. Regarding the chemical complexity, a total of seven compounds (1-7, Fig. 2 [28]. Quinic acid has already been described in the alcoholic extract of V. schimperi [29].  [30], who also used a reverse-phase C18 column. Chlorogenic acid has already been reported in the ethanol extract of V. album [16,23] and is important to assure the quality of the vegetal material.    [35].

Cell viability assay
The toxicity of the alcoholic vehicle was first evaluated by WST assay in Molt-4 and Yoshida tumor cells, and no cellular toxicity was observed (Fig. 3a-b). Therefore, the next tests with MT were performed with vehicle concentrations between 0.05 and 0.5% v/v. Molt-4 (Fig. 3a) and Yoshida cells (Fig. 3b) exhibited approximately the same sensitivity to the tinctures, with MT from Abies alba (VAA) being the most effective, with IC50 values of 0.07 ± 0.01% v/v and 0.05 ± 0.03% v/ v for Molt-4 and Yoshida, respectively. The non-tumor cells (3 T3) were less sensitive with IC50 values of 1.60 ± 0.48% v/v after incubation with VAA. VAP did not exhibit toxicity in Molt-4 cells at any concentration tested (Fig. 3a) and exhibited only slight toxicity in Yoshida cells (p < 0.05; concentration range from 0.35-0.50, Fig. 3b).
The anti-proliferative assay highlighted the antitumor potential of MT within 4 h of incubation ( Fig. 4a-b), in which a significant reduction in viability of approximately 90% was detected (0.5% v/v VAA, VAM, VAQ; p < 0.0001).
Flow cytometry was used to characterize the mechanism of cell death induced by V. album MT after 4 and 24 h of incubation (Fig. 5a-b). The number of necrotic cells increased proportionally to the MT concentration and to the incubation time, with a very similar profile for both cell lines, except in response to VAP. In Molt-4 cells, 0.5% v/v VAA, VAM and VAQ induced 61, 31 and 21% necrotic cell death, respectively, after 4 h of incubation (Fig. 5a top). Similar percentages of necrosis were observed in Yoshida cells (66, 47 and 35%), respectively, with the same MT concentrations. The necrotic effects were more evident after 24 h, supporting the necrotic potential of Abietis, Malus and Quercus MT (Fig. 5b  bottom).
The present data suggest that the antitumor activity of MT from V. album ssp. abietis may be related to its higher content of VT A3 in addition to the presence of small molecules, as discussed below. Indeed, Schaller et al. [26] demonstrated that the viscotoxin A3 of V. album aqueous extract was more cytotoxic than other VT isoforms when the Yoshida sarcoma cell line was evaluated [26]. Additionally, Coulon et al.  [36] attributed the highest antitumor activity of viscotoxin A3 to its physicochemical characteristics, in which the hydrophobic residues and the net liquid charge were able to increase the cell membrane interaction. In addition, in vitro studies revealed that methanol extracts of mistletoe berries and ethanolic tinctures from whole V. album decreased the proliferation of colon cancer cells and viability of the murine melanoma lineage in a dose-dependent manner, respectively [16,25]. These cellular alterations were attributed to modifications of mitochondrial activity and the cell cycle, among other cellular damage. Both studies showed that the polyphenolic composition of these V. album extracts was involved in the antitumor activity.
Regarding the seven compounds tentatively identified in this study, compound 1 was able to promote cytotoxicity in squamous cell carcinoma 4 (SCC-4). Compounds 2, 3 and 4 were chlorogenic acid isomers formed by quinic and cinnamic acid esterification [37]. Recently, the antitumor potential of chlorogenic acid was described in in vitro and in vivo models [38,39]. Additionally, Pan et al. [40] demonstrated that a type of quercetin-di-hexoside (quercetin 3,4′-di-O-glucoside) was an effective inhibitor of the growth of HepG2, PC3 and HT29 cells, corroborating the antitumor potential of V. album ethanolic extracts.
Pinobanksin or naringenin pentoside-hexoside (compound 6) presented a higher peak intensity in V. album extracts prepared with V. album ssp. abietis when compared to the other ethanolic extracts. The antitumor potential of these flavanones was previously described in tumor cell lines [41,42]. In addition, the metabolomics analyses performed by our group with 50 different V. album samples harvested in winter and summer seasons from the same habitat in Switzerland also confirmed the importance of compound 6 in subspecies sample differentiation (data not shown).

Conclusion
The present study shows the antitumor potential of V. album tinctures in in vitro models. The cell death mechanism involved necrotic effects depending on the influence of the host tree, time and dose. Mother tincture from V. album ssp. abietis growing on Abies alba was the most effective, probably because of the higher content of VT A3, while MT from V. album ssp. austriacum growing on Pinus sylvestris exhibited only a slight antitumor effect. Additionally, tumor cells were more sensitive than normal fibroblasts, suggesting that V. album MT has promising antitumor potential. The small molecules identified in MT were quinic acid, three isomers of chlorogenic acid, quercetin-di-hexoside, pinobankasin or naringenin pentoside-hexoside and lyoniresinol-hexoside. Further studies using in vitro and in vivo models in addition to stability assessments, should be performed to stimulate the development of new pharmaceutical formulations containing V. album ethanolic extracts.