Integrated HPTLC-based methodology for the tracing of bioactive compounds in herbal extracts by employing multivariate chemometrics. The case study of anti-tyrosinase agents from Morus alba

The contribution of naturally derived products for drug discovery purposes is undisputed. The implementation of high-throughput screening (HTS) technologies has allowed the rapid and effective investigation of the chemical composition and pharmacological properties of a large number of plant extracts against several biological targets. The scope of this study was the discovery of plant derived tyrosinase inhibitors utilizing a HTS approach and the introduction of an integrated methodology for the rapid identification of bioactive compounds during bioassay-guided procedures.

An extended extract library encompassing 3600 extracts from approximately 150 families was generated from plants originated from six regions with intense biodiversity and subsequently was screened for tyrosinase inhibition. Chemometric tools supported the development of a novel integrated HPTLC-based procedure for the tracing and targeted isolation of bioactive compounds in active extracts. Fractions resulted from CPC separation of Morus alba extract, the most prominent agent identified during anti-hyperpigmentation screening, were assayed for tyrosinase inhibition potential and analyzed with HPTLC. Multivariate data analysis tools enabled the tracing of compounds that contributed to the appearance of a tyrosinase inhibitory effect in active fractions. Two methodologies were developed for the generation of the dataset; one based on chromatogram binning and the second based on manual peak picking. Targeted isolation of compounds indicated to contribute best to the anti-hyperpigmentation activity was performed and IC₅₀ values were estimated. Both methodologies were found capable to trace the components (e.g. oxyresveratrol, trans-dihydromorin, 2,4,3'-trihydroxydihydrostilbene) that exhibit the highest bioactivity in the mixture. All steps of the experimental procedure implemented techniques that afford essential key elements for application in HTS procedures for drug discovery purposes.