RESUMO
Viscum album L. (Santalaceae) is an important medicinal plant traditionally used to treat several diseases, including cancer therapy. This paper provides detailed morpho-anatomical characteristics of the leaves, stems and berries of Viscum album subsp. album growing as hemi-parasite on the branches of Malus domestica (Suckow) Borkh. (Rosaceae) to aid species identification and botanical characterization. Additionally, for the first time, microchemical analyses of all tissues and Energy Dispersive X-Ray Spectroscopy analyses of the calcium oxalate crystals are provided for the first time. The plant features leathery presents green leaves with parallel veins, small yellow unisexual flowers in 3-flowered cymes, and the dioecious inflorescences usually consist of three flowers, with female flowers generating white fleshy berries, in which a seed is embedded in the mucilaginous mesocarp, normally containing two embryos. Anatomically, the analyzed leaves were isobilateral and amphistomatic, and showed straight anticlinal epidermal cell walls, thick cuticles with epicuticular wax crystalloids, and paracytic stomata. The midrib is flat on both sides and has a single vascular bundle, whereas the strongly shortened petiole is concave-convex in shape and contains five bundles. The stems show a primary structure with a ring of nine vascular bundles enclosing the pith. Calcium oxalate druses and cubic and quadrangular prisms were observed in different plant parts. The results of this study provide new microscopy information that can help in the authentication of mistletoe raw materials.
Assuntos
Erva-de-Passarinho , Plantas Medicinais , Viscum album , Viscum album/química , Oxalato de Cálcio , Extratos VegetaisRESUMO
Viscum album L. is a semi-parasitic plant with antitumor activity attributed to theaqueous extracts. However, European V. album ethanolic extracts (VAE) have also demonstrated invitro activity in tumor models. Aims: Evaluate the metabolic profiles of fifty VAE harvested duringsummer and winter seasons and their antitumor activity through 2D and 3D models. Methodology:VAEwerepreparedbymacerationfrom:V.albumsubsp.albumgrowingonMalus domestica,Quercus sp.and Ulmus sp.; V. album subsp. austriacum from Pinus sylvestris; V. album subsp. abietis from Abies alba.Chemical analyses were performed through liquid chromatography coupled with high resolutionmass spectrometry and Partial Least Squares Discriminant Analysis (PLS-DA) was performed in theMetaboanalyst 4.0. The antitumor potential of the selected VAE was evaluated in 2D and 3D models(MDA-MB-231 cancer cells) by MTT, crystal violet and glycolytic pathway analysis. Results anddiscussion:Thefirst3principalcomponentsinPLS-DAexplained60%and40%ofdatavariationin positive andnegativemodesrespectively.Threegroupswereformedandshowedchemicalsimilarityamong V. album subspecies. The compounds responsible for group separation were tentativelyidentifiedas:pinobankasinornaringenin hexoside;isorhamnetin-3-hexoside,meglutolanddifferent aminoacids.ThesummerVAEat0.5%v/vinducedhighercytotoxicdamagethanthewinterpreparations, and Abies alba and Quercus sp. VAE promoted 49% and 42% reduction of tumorviability in 3D model (72h incubation), respectively. MDA-MB-231 glycolytic pathway in 2D modelshowed a decrease in the glucose consumption and extracellular lactate production. Also, PFK (6-phosphofructo-1-kinase)andPK(Pyruvatekinase)activitieswereinhibitedbyAbiesalbaandQuercus sp. VAE at 48h of incubation. Conclusion: VAE extracts showed different metabolomes andthe glycolytic pathway should be an important target involved in the inhibition of tumor growth bytheseextracts
Assuntos
Tintura Mãe , Viscum album , Metaboloma , AntineoplásicosRESUMO
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.