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DOI: 10.1055/s-0036-1596365
Combined “omics” techniques for targeted investigation on CYP450 roles during bioactive quinonemethide triterpenes biosynthesis: Maytenus spp. case study
Publication History
Publication Date:
14 December 2016 (online)
Maytenus ilicifolia Mart. ex Reissek (Celastraceae) is part of Brazilian flora. It occurs as denser populations in sub-tropical climate [1] and is known as “espinheira santa”. It is applied for several diseases, such as antiulcerogenic [1]. Root barks of Maytenus spp. accumulate quinonemethide triterpenes (QT) which are promising anticancer agents [2]. Our current efforts aimed to furnish conclusive data on the ocurrence of QT in biogeographycally distinct samples within the genus. Metabolomics data had determined QT for M. gonoclada, M. aquifolium and M. ilicifolia. 1D TOCSY-NMR search for QT spin systems [7.04 dd (J AB =7.5; J AC =1.5; 6.37 d (J AB =7.5)) and HPLC-DAD retention time of 11.7 min for maytenin and 15.1 min for pristimerin [Shimadzu LC-10 AD, SPD – M 10 AVP detector, PhenomenexTM Gemini C18 column [5 µm (250 × 4,6 mm)] kept at 35 °C; MeOH:H2O:H3PO4 (80:20:1) isocratic mode, 0,8mL min-1; UV-Vis > 254nm < 420nm} were positive for these compounds. Furthermore, determination of QT in the samples led us to QT rich extracts. They were used in a two steps purification process: (1) pTLC of raw extracts and (2) recrystallization for each band (MeOH:ACN:H2O). Aliquots of total yielding material (34 mg maytenin; 90 mg pristimerin) were used in wound healing tests and cytotoxicity assay. Maytenin inhibited celular migration in the same order as colchicine (60%), and presented substantial cytotoxicity (IC50= 18.3 ± 1.6µM) against the breast cancer cell line MDA-MB-231. Samples provided integral RNA (Qiagen RLCTM protocol) for transcriptomics investigation. Primer designing relied on functional survey of oxidoreductases acting onto pentaciclic saturated carbon skeletons for functionalization of these skeletons. Clans CYP71 and CYP85 oxidoreductases were displayed as central players for this reaction [3]. In conclusion, our ongoing investigation contributes to integrate phytochemistry and biochemistry in order to shed light in key steps of bioactive compounds biosynthesis.
Acknowledgements: Authors would like to acknwoledge FAPESP for fellowship (2014/14067 – 2) and grant [CIBFar (2013/07600 – 3)]. We thank MMA-ICMbio for allowing the harvests. M.F., A.D.A would also like to thanks CNPq for their fellowships.
Keywords: Quinonemethide triterpenes, Celastraceae, anticancer compounds, TOCSY, cDNA.
References:
[1] Angiosperm Phylogeny Group. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III". Bot J Linn Soc 2009; 161: 105 – 121
[2] Costa PM, Ferreira PMP, Bolzani VS, Furlan M, Santos VAFFM, Corsino J, Moraes MO, Costa-Lotufo LV, Montenegro RC, Pessoa C. Antiproliferative activity of pristimerin isolated of Maytenus ilicifolia (Celastraceae) in human HL-60 cells. Toxicol in Vitro 2008; 22: 854 – 863
[3] Christoffersen RE, Percival FW, Bozak KR. Functional and DNA sequence divergence of the CYP71 gene family in higher plants. Drug Metabol Drug Interact 1995; 12: 207 – 219