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Phytochem Anal. 2014 Mar-Apr;25(2):127-33. doi: 10.1002/pca.2478. Epub 2013 Sep 23.

Preparative isolation of six anti-tumour biflavonoids from Selaginella doederleinii Hieron by high-speed counter-current chromatography.

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Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou, 350004, China.



Biflavonoids are the primary constituents of Selaginella doederleinii Hieron, to which different bioactivities have been attributed, including anti-cancer, anti-inflammatory, anti-oxidant, anti-fungal and anti-virus activity. However, effective methods for separation of these compounds are not currently available.


To develop a high performance and bioassay-guided method for preparative isolation of biflavonoids from S. doederleini via high-speed counter-current chromatography (HSCCC).


The anti-proliferation effects of four fractions (70% ethanol, petroleum ether, dichloromethane and acetic ether extracts) of S. doederleinii on five human cancer cells were monitored. The dichloromethane and acetic ether extracts showed good cytotoxicities to the studied cancer cell lines, guiding the subsequent separation. Two solvent systems composed of n-hexane:ethyl acetate:methanol:water (1:2:1.5:1.5, v/v) and n-hexane:ethyl acetate:methanol:water (3:2:3:2, v/v) were developed for separation of the active fractions, respectively. Identification of the biflavonoids was performed by EI-MS(n) , (1) H- and (13) C-NMR.'


Under the optimised conditions, 12.6 mg amentoflavone (91.4%), 6.6 mg robustaflavone (90.4%), 7.5 mg 2'', 3''-dihydro-3', 3'''-biapigenin (98.2%) and 7.3 mg 3', 3'''-binaringenin (90.3%) from acetic ether extract (500 mg) and 6.3 mg heveaflavone (93.5%) and 5.3 mg 7, 4', 7'', 4'''-tetra-O-methyl-amentoflavone (94.5%) from dichloromethane extract (200 mg) were obtained, respectively. The anti-proliferation effects of the six biflavonoids on the five human cancer cells were further verified.


The study provides methodological references for simultaneously preparative isolation of several bioactive biflavones from the herbal family of Selaginella. It is the first report discovering 2'', 3''-dihydro-3', 3'''-biapigenin and 3', 3'''-binaringenin from this herb and describing their cytotoxicities to human cancer cell lines.


Activity-guided preparative isolation; HSCCC; Selaginella doederleinii Hieron; biflavonoids; cytotoxicity

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