Ethyl Acetate Fraction of Teucrium Polium Extract Abolishes Human Umbilical Vein Endothelial Cells (HUVEC) Tubulogenesis in Collagen Bed through Suppression of Cell Proliferation/VEGF Secretion
Angiogenesis has essential role in growth and metastasis of tumors. Development of therapies aimed to suppress angiogenesis using medicinal plants is one of the effective approaches for prevention/treatment of cancer. The current study was performed to investigate in vitro anti-angiogenic effect of Teucrium Polium (TP) extract and its fractions. The aerial part of Teucrium Polium was powdered and extracted with 50% ethanol. The extract was fractionated in to aqueous (AQ), n-butanol (BU), ethyl acetate (EA) and n-hexane (HE) fractions. Anti-angiogenic effect of TP was examined on human umbilical vein endothelial cells (HUVECs) in three-dimensional collagen matrix. The endothelial cells form capillary-like branches that can be visualized using phase contrast microscope and the number of tube-like structures can be quantified as a measure of in vitro angiogenesis. Furthermore, anti-proliferative and vascular endothelial growth factor(VEGF )suppressive effect of TP as important factors in the process of angiogenesis were assessed using3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)and quantitative ELISA, respectively. Based on our findings, among the TP fractions, EA fraction showed the highest inhibitory activity on angiogenesis. This fraction with IC50: 68 µg/mL, inhibited angiogenesis at 60 µg/mL. The crude extract and other fractions of TP inhibited angiogenesis in a dose-dependent manner at doses higher concentrations than EA fraction, significantly.TP extract and EA fraction were able to inhibit proliferation of HUVEC and inhibited VEGF secretion in a dose dependent manner. The ethyl acetate fraction at 60 µg/ml inhibited VEGF secretion perfectly. Our data indicated that ethyl acetate fraction of Teucrium Polium could be a potential candidate for the prevention of angiogenesis in cancer and other related disorders. However, this suggestion needs more quantitative and in vivo investigations for confirmation.
 Mansouri K, Khodarahmi R, Ghadami SA. An in vitro model for spontaneous angiogenesis using rat mesenteric endothelial cells: possible therapeutic perspective for obesity and related disorders. Pharm Biol 2013;51(8): 974-980.
 Tahergorabi Z, Khazaei M.A review on angiogenesis and its assays.Iran J Basic Med Sci 2012; 15(6): 1110-26.
 Huang D, Lan H, Liu F, Wang S, Chen X, Jin K, et al. Anti-angiogenesis or pro-angiogenesis for cancer treatment: focus on drug distribution. Int J clinExp Med 2015;8(6):8369-8376.
 T. Edgar L, B. Hoying J, Utzinger U, J. Underwood C, Krishnan L, K. Baggett B, et al. Mechanical interaction of angiogenicmicrovesselswith the extracellular matrix. J Biomech Eng. 2014; 136(2): 0210011–02100115. Madanecki P, Kapoor N, Bebok Z, Ochocka R, Collawn J , Bartoszewski R. Regulation of angiogenesis by hypoxia: the role of microRNA.Cell MolBiol Lett 2013; 18(1):47-57.
 Seyfi P, Mostafaie A, MansouriK,Arshadi D, Mohammadi-Motlagh HR,Kiani A. In vitro and in vivo anti-angiogenesis effect of shallot (Allium ascalonicum): A heat-stable and flavonoid-rich fraction of shallot extract potently inhibits angiogenesis. ToxicolInVitro 2010; 24(6): 1655-1661. Lu K, Bhat M, Basu S. Plants and their active compounds: natural molecules to target angiogenesis. Angiogenesis 2016; 19(3): 287-95.
 Min K, Kwon TK. Anticancer effects and molecular mechanisms of epigallocatechin-3-gallate.Integr Med Res 2014;3(1): 16-24.
 Keshavarz M, Mostafaie A, MansouriK,Bidmeshkipour A, Motlagh HR, Parvaneh S.In vitro and ex vivo antiangiogenic activity of salvia officinalis.PhytotherRes 2010; 24(10): 1526-1531.
 Shakiba Y, Mansouri K, Mostafaie A.Anti-angiogenic effect of soybean kunitz trypsin inhibitor on human umbilical vein endothelial cells.Fitoterapia 2007;78(7-8): 587-589.
 TabatabaeiYazdi F, AlizadehBehbahani B.Antimicrobial effect of the aqueous and ethanolicTeucriumpolium L.extracts on gram positive and gram negative bacteriainvitro. J ParamedSci 2013;4(4):56-62.
 Stankovic MS, Curcic MG, ZizicJB,Topuzovic MD, Solujic SR, MarkovicSD.Teucrium plant species as natural sources of novel anticancer compounds: antiproliferative, proapoptotic and antioxidant properties. Int J MolSci 2011; 12(7): 4190-4205.
 YazdanparastR, Esmaeili MA, AshrafiHelanJ. Teucriumpolium extract effects pancreatic function of streptozotocin diabetic rats: a histopathological examination. Iran BiomedJ 2005;9(2): 81-85.
 TepeB, Degerli S, ArslanS,Malatyali E, Sarikurkcu C. Determination of chemical profile, antioxidant, DNA damage protection and antiamoebic activities of Teucriumpolium and Stachysiberica. Fitoterapia 2011; 82(2): 237-246.
 Elmasri WA, Hegazy ME, Mechref Y, W.Pare P. Structure-antioxidant and anti-tumor activity of Teucriumpolium phytochemicals. Phytochem Lett 2016; 15: 81-87.
 Mousavi SM, NiazmandS,Hosseini M,Hassanzadeh Z, Sadeghnia HR, Vafaee F et al. Beneficial effect of Teucriumpolium and Metformin on diabetes- induced memory impairment and brain tissue oxidative damage in Rats. Int J AlzheimersDis 2015; 2015:493729.
 Sharififar F, Dehghn-NudehG, Mirtajaldini M.Major flavonoids with antioxidant activity from Teucriumpolium L. Food Chem 2009; 112(4): 885-888.
 Kandouz M, Alachkar A, Zhang L, Dekhil H, Chehna F, Yasmeen A, et al. Teucriumpolium plant extract inhibits cell invasion and motility of human prostate cancer cells via restoration of the E-cadherin/catenin complex. J Ethnopharmacol 2010;129(3):410-5
 Eskandary H, Rajabalian S, Yazdi T, Eskandari M, Fatehi K, Ashraf Ganjooei N. Evaluation of cytotoxic effect of Teucriumpolium on a new glioblastoma multiform cell line (REYF-1) using MTT and soft agar clonogenic assays. Int J Pharmacol 2007; 3(5): 435-437.
 Bahramikia S, YazdanparastR.Phytochemistry and medicinal properties of TeucriumpoliumL.(Lamiaceae). Phytother Res 2012; 26(11): 1581-1593.
Menichini F, Conforti F, Rigano D, Formisano C, Piozzi F, Senatore F. Phytochemical composition, anti-inflammatory and antitumour activities of four Teucrium essential oils from Greece.Food Chem 2009; 115 (2):679-686.
 Doleckova I, Rarova L, Gruz J, Vondrusova M, Strnad M, Krystof V. Antiproliferative and antiangiogenic effect of flavone eupatorin, an active constituent of chloroform extract of Orthosiphonstamineus leaves. Fitoterapia 2012; 83(6): 1000-1007.