Analysis of plant secondary metabolism using stable isotope‐labelled precursors

Special issue of Phytochemical Analysis on NMR-based analytical techniques. open access articleIntroduction Analysis of biochemical pathways typically involves feeding a labelled precursor to an organism, and then monitoring the metabolic fate of the label. Initial studies used radioisotopes as a label and then monitored radioactivity in the metabolic products. As analytical equipment improved and became more widely available, preference shifted the use stable ‘heavy’ isotopes like deuterium (2H)‐, carbon‐13 (13C)‐ and nitrogen‐15 (15N)‐atoms as labels. Incorporation of the labels could be monitored by mass spectrometry (MS), as part of a hyphenated tool kits, e.g. Liquid chromatography (LC)–MS, gas chromatography (GC)–MS, LC–MS/MS. MS offers great sensitivity but the exact location of an isotope label in a given metabolite cannot always be unambiguously established. Nuclear magnetic resonance (NMR) can also be used to pick up signals of stable isotopes, and can give information on the precise location of incorporated label in the metabolites. However, the detection limit for NMR is quite a bit higher than that for MS. Objectives A number of experiments involving feeding stable isotope‐labelled precursors followed by NMR analysis of the metabolites is presented. The aim is to highlight the use of NMR analysis in identifying the precise fate of isotope labels after precursor feeding experiments. As more powerful NMR equipment becomes available, applications as described in this review may become more commonplace in pathway analysis. Conclusion and Prospects NMR is a widely accepted tool for chemical structure elucidation and is now increasingly used in metabolomic studies. In addition, NMR, combined with stable isotope feeding, should be considered as a tool for metabolic flux analyses

Activity of Antioxidants from Crocus sativus L. Petals: Potential Preventive Effects towards Cardiovascular System.

The petals of the saffron crocus (Crocus sativus L.) are considered a waste material in saffron production, but may be a sustainable source of natural biologically active substances of nutraceutical interest. The aim of this work was to study the cardiovascular effects of kaempferol and crocin extracted from saffron petals. The antiarrhythmic, inotropic, and chronotropic effects of saffron petal extract (SPE), kaempferol, and crocin were evaluated through in vitro biological assays. The antioxidant activity of kaempferol and crocin was investigated through the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay using rat cardiomyoblast cell line H9c2. The MTT assay was applied to assess the effects of kaempferol and crocin on cell viability. SPE showed weak negative inotropic and chronotropic intrinsic activities but a significant intrinsic activity on smooth muscle with a potency on the ileum greater than on the aorta: EC50 = 0.66 mg/mL versus EC50 = 1.45 mg/mL. Kaempferol and crocin showed a selective negative inotropic activity. In addition, kaempferol decreased the contraction induced by KCl (80 mM) in guinea pig aortic and ileal strips, while crocin had no effect. Furthermore, following oxidative stress, both crocin and kaempferol decreased intracellular ROS formation and increased cell viability in a concentration-dependent manner. The results indicate that SPE, a by-product of saffron cultivation, may represent a good source of phytochemicals with a potential application in the prevention of cardiovascular diseases

Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic chalcones: Overcoming cisplatin resistance in CYP1B1-overexpressing lines

© 2017 Elsevier Masson SAS The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1, mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j (DMU2139) have been identified from two series of synthetic pyridylchalcones. They inhibit human CYP1B1 enzyme bound to yeast-derived microsomes (Sacchrosomes™) with IC 50 values of 10 and 9nM, respectively, and show a very high level of selectivity towards CYP1B1 with respect to the IC 50 values obtained with CYP1A1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 Sacchrosomes™. Both compounds also potently inhibit CYP1B1 expressed within ‘live’ recombinant yeast and human HEK293 kidney cells with IC 50 values of 63, 65, and 4, 4nM, respectively. Furthermore, the synthesized pyridylchalcones possess better solubility and lipophilicity values than ANF. Both compounds overcome cisplatin–resistance in HEK293 and A2780cells which results from CYP1B1 overexpression. These potent cell-permeable and water-soluble CYP1B1 inhibitors are likely to have useful roles in the treatment of cancer, glaucoma, ischemia and obesity

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one, a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in Sacchrosomes™ and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC50 values of 58 and 65 nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10-fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent