Category: Preclinical Development
Purpose: Cassava, Manihot esculenta Crantz, is a most important starch-rich crop capable of being grown under conditions of severe (several months of) drought. As well as being vitally important for food security, as cassava is the major staple diet of 500 million people across Africa, Asia, and South America, cassava roots are also a valuable raw material as a source of biologically active hydroxycoumarins and of starch. Both products play key roles in the pharmaceutical industry as bioactives, e.g., antimicrobial, antifungal, anticancer, and other pharmacological effects, and as excipients.1,2 The biosynthesis of the key compound scopoletin is not yet fully understood.1,2 Our research project is important as it seeks to determine whether post-harvest physiological deterioration (PPD), a major problem in cassava storage on harvesting, can be controlled in order to extend the shelf life of cassava. PPD causes a rapid discolouration in starch-rich cassava roots making them unsightly and unmarketable with negative economic impacts for those involved in its production, distribution, and consumption. The specific objectives are to study the biosynthetic pathways existing under PPD in cassava leading to the production of bioactive hydroxycoumarins and to analyse such bioactives. Different isotopically labelled cinnamic acids will be designed, synthesized, and fed to cassava roots and their hydroxycoumarin products isolated and purified to homogeneity. These products will report on the metabolic processes taking place in the biosynthesis of hydroxycoumarins in cassava roots under PPD.
Methods: A modified Knoevenagel-Doebner condensation3 and isotope exchange reactions were used to synthesize the isotopically labelled hydroxycinnamic acids as potential intermediates for hydroxycoumarin production in cassava under PPD. The isotope exchange reactions were done as follows: d7-cinnamic acid, 0.5 mL labelled water (H217O, H218O) and conc. HCl (10 µL) in acetonitrile (1 mL) were kept at 70 °C for 4 days. Synthesized isotopically labelled intermediates, cinnamic-2-D, 3-13C acid and cinnamic-2-D, 3-13C, D acid; d7-cinnamic-17O2 acid and d7-cinnamic-18O2 acid were dissolved in 4% aq. Na2CO3. The pH of the solution was adjusted to 7.5 with 1 M HCl. Cassava roots were peeled, cubed, fed with the labelled intermediates and then maintained under controlled conditions, 20 °C and 80-90% relative humidity (RH) for PPD to occur. The cassava cubes were then extracted with ethanol and chromatographically purified products were studied with a variety of spectroscopic and spectrometric methods. NMR spectra including: 1H, 13C, COSY, HSQC, HMBC, NOESY were recorded on a Bruker Avance III 500 MHz spectrometer at 25 oC. MS analysis was achieved using a High Resolution Electrospray Ionisation Time of Flight (HR-ESI-TOF) instrument from Bruker (Germany) coupled to an Ultimate High Performance Liquid Chromatograph (UHPLC) from Thermo Fisher Scientific with a pre-packed C18-RP column Acquity UPLC BEH (1.7 µm, 2.1 x 50 mm) from Waters (USA). Aliquots (10 µL) of each sample were injected using an RS auto-sampler, with gradient elution of methanol/water as the mobile phase at a flow rate of 0.3 mL/min. The data obtained were processed and analysed using Data Analysis 4.3 software from Bruker (Germany).
Results: Fractions purified and collected from HPLC were analysed by LC-MS. This showed analytes corresponding to scopoletin-13C and scopolin-13C and scopoletin-13C,D and scopolin-13C,D, scopoletin-17O and scopolin-17O and scopoletin-18O and scopolin-18O respectively. These differently labelled hydroxycoumarin analytes confirm the existence of different biosynthetic pathways in M. esculenta. The order of o-hydroxylation, E-Z isomerisation followed by lactonisation is also observed leading to the loss of deuterium on carbon-2 for cassava chips fed with cinnamic-2-D,3-13C acid and cinnamic-2-D,3-13C, D acid. Furthermore, the feeding of cinnamic acid with 17O and 18O carboxylic acid enriched precursors led to the incorporation of only one of each enriched atom, showing that the lactone is also fixed enzymatically from air in the compounds of interest.
Conclusion: Cassava is an important staple crop in drought prone countries around the world. It is a rich source of biologically important hydroxycoumarins which are produced along a number of biosynthetic pathways as demonstrated using our isotopically-labelled precursor studies. We acknowledge financial support from the Schlumberger Foundation under Faculty for the Future, and the University of Bath.
1. Blagbrough, I.S., Bayoumi, S.A.L., Rowan, M.G. and Beeching, J.R. Phytochem. 2010, 71, 11940-1951 DOI: 10.1016/j.phytochem.2010.09.001
2. Gnonlonfin, G.J.B., Sanni, A. and Brimer, L. Crit. Rev. Plant Sci. 2012, 31, 47-56 DOI: 10.1080/07352689.2011.616039
3. Robbins, R.J. and Schmidt, W.F. J. Label. Compd. Radiopharm. 2004, 47, 797-806.