title: Effective Method of Purification of Betulin from Birch Bark: The Importance of Its Purity for Scientific and Medicinal Use.
abstract: A new and relatively simple method for purification of betulin from birch bark extract was developed in this study. Its five purification steps are based on the differential solubility of extract components in various solvents and their crystallization and/or precipitation, on their affinity for Ca(OH)2 in ethanol, and on the affinity of some impurities for silica gel in chloroform. In addition, all used solvents can be simply recycled. Betulin of more than 99% purity can be prepared by this method with minimal costs. Various observations including crystallization of betulin, changes in crystals during heating, and attempt of localization of betulin in outer birch bark are also described in this work. The original extract, fraction without betulinic acid and lupeol, amorphous fraction of pure betulin, final crystalline fraction of pure betulin and commercial betulin as a standard were employed to determine the antiproliferative/cytotoxic effect. We used WST-1 tetrazolium-based assays with triple negative breast cancer cell line BT-549. The decrease in cell survival showed clear relationship with the purity of the samples, being most pronounced using our final product of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549 clearly showed the importance of purity of betulin for biological experiments and, apparently, for its medicinal use.
authors.affiliation: Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Medical Biochemistry, CZ-50003, Hradec Králové, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Šiman
authors.affiliation: Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Medical Biochemistry, CZ-50003, Hradec Králové, Czech Republic.
authors.forename: Alžběta
authors.initials: A
authors.lastname: Filipová
authors.affiliation: University hospital Hradec Králové, Department of Research and Development, CZ-50005, Hradec Králové, Czech Republic.
authors.forename: Alena
authors.initials: A
authors.lastname: Tichá
authors.affiliation: Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Medical Biochemistry, CZ-50003, Hradec Králové, Czech Republic.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Niang
authors.affiliation: Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Medical Biophysics, CZ-50038, Hradec Králové, Czech Republic.
authors.forename: Aleš
authors.initials: A
authors.lastname: Bezrouk
authors.affiliation: Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Medical Biochemistry, CZ-50003, Hradec Králové, Czech Republic.
authors.forename: Radim
authors.initials: R
authors.lastname: Havelek
mesh_terms: D029662:Betula / Q000737:chemistry*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003460:Crystallization; D006801:Humans; D024301:Plant Bark / Q000737:chemistry*; D064726:Triple Negative Breast Neoplasms / Q000188:drug therapy / Q000473:pathology; D014315:Triterpenes / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology / Q000627:therapeutic use; D000094062:Betulinic Acid
chemical_list: D014315:Triterpenes; C002503:betulin; D000094062:Betulinic Acid