Combination of FCPC and NMR for the activity-guided isolation of skin whitening natural agents

Greek flora is characterized by high biodiversity, consisting of 6300 species of which 950 are endemic providing an ideal source for the discovery of bioactive compounds [1]. Bioactive natural products have proven to be a significant backbone in drug discovery. Additionally, the mainstream in Natural Products Chemistry is moving away from the conventional isolation of plants' secondary metabolites to the targeted isolation of the bioactive ones. This project aims to identify natural compounds with skin whitening properties via the development of a high-throughput platform based on Fast Centrifugal Partition Chromatography (FCPC) and NMR methodologies combined with biological activity for the direct detection of bioactive compounds prior to any isolation. For this purpose, 600 plants from a unique in-house plant-library comprising around 32% of the Greek flora were selected and their extracts were prepared. The extracts were tested in vitro for their tyrosinase inhibition activity and the most promising ones were selected for further elaboration. Specifically, the extracts of Morus alba, Veratrum album and Paeonia mascula were fractionated with FCPC utilizing a step-wise solvent system consisted of Heptane/Ethyl acetate/Butanol/Methanol/Water resulting in 45 fractions per extract. The fractions were assayed for their tyrosinase inhibition potential and were profiled by means of NMR spectroscopy. Biological activity was statistically correlated with the spectral data through a newly developed Hetero-Covariance Approach (HetCA) [2], followed by the Statistical TOtal Correlation SpectroscopY (STOCSY) [3] analysis resulting in the identification of several whitening agents, such as 2,4,3'-trihydroxydihydrostilbene from M. alba, oxyresveratrol from V. album and 1,2,3,4,6-pentagalloyl-β-D-glucose from P. mascula. The correlation of the spectral profiling of a rational fractionation with biological data has proven to be a powerful tool for the expeditious discovery of bioactive natural products reducing significantly the workload.

Acknowledgements: The research leading to these results has received funding from the Agrocos Project FP7-KBBE-2009 – 3 Programme number 245336.

Keywords: FCPC, NMR metabolic profiling, Hetero-Covariance Approach, anti-tyrosinase activity, activity-guided isolation.

References:

[1] Dimopoulos P, Raus T, Bergmeier E, Constantinidis T, Iatrou G, Kokkini S, Strid A, Tzanoudakis D. Vascular Plants of Greece. An annotated checklist. Botanic Garden and Botanical Museum Berlin-Dahlem & Hellenic Botanical Society, Berlin & Athens, 2013, 372 pp.

[2] Aligiannis N, Halabalaki M, Chaita E, Kouloura E, Argyropoulou A, Benaki D, Kalpoutzakis E, Angelis A, Stathopoulou K, Antoniou S, Sani M, Krauth V, Werz O, Schütz B, Schäfer H, Spraul M, Mikros E, Skaltsounis L. Heterocovariance based metabolomics as a powerful tool accelerating bioactive natural product identification, submitted to Chemistry Select; May 2016.

[3] Cloarec O, Dumas ME, Craig A, Barton RH, Trygg J, Hudson J, Blancher C, Gauguier D, Lindon JC, Holmes E, Nicholson JK. Statistical total correlation spectroscopy: An exploratory approach for latent biomarker identification from metabolic ¹H NMR data sets. Anal Chem 2005; 77: 1282 – 1289