Same analytical method for both (bio)assay and zone isolation to identify/quantify bioactive compounds by...

Area of Science:

• Analytical Chemistry
• Natural Product Chemistry
• Spectroscopy

Background:

• Direct combination of High-Performance Thin-Layer Chromatography (HPTLC) with Nuclear Magnetic Resonance (NMR) spectroscopy is hindered by differing sensitivity requirements.
• HPTLC requires sufficient isolated compound zones for subsequent offline NMR detection (HPTLC//NMR).
• A need exists for a streamlined method for bioprofiling and isolating bioactive zones from complex botanical mixtures.

Purpose of the Study:

• To develop a straightforward HPTLC-based procedure for both bioprofiling and isolation of bioactive zones from multicomponent mixtures.
• To demonstrate the method's capability in identifying and quantifying coeluting structural isomers, such as ursolic (UA) and oleanolic (OA) acids.
• To validate the developed HPTLC-EDA-Vis//¹H qNMR-PULCON procedure as a reliable alternative for analyzing botanical extracts.

Main Methods:

• HPTLC-effect-directed analysis (EDA) was employed to reveal bioactive compounds in various botanical extracts (Lamiaceae and Rosaceae families).
• A case study focused on separating and identifying coeluting structural isomers (UA, OA) using HPTLC-Vis and subsequent ¹H NMR.
• Quantification was performed using the Pulse Length-based Concentration (PULCON) methodology (HPTLC//¹H qNMR-PULCON), with results corroborated by deconvoluted ¹H or 2D ¹H-¹³C HSQC NMR spectra.

Main Results:

• The HPTLC-EDA-Vis//¹H qNMR-PULCON method successfully identified and quantified bioactive compounds, including challenging coeluting structural isomers UA and OA.
• Distinct ¹H NMR signals (allylic H-18 protons) allowed differentiation and quantification of UA and OA.
• A high correlation (R² = 0.9718) was observed between HPTLC-Vis densitometry and HPTLC//¹H qNMR-PULCON quantification, with a mean isolation rate of 82% for UA and OA.

Conclusions:

• The developed HPTLC-EDA-Vis//¹H qNMR-PULCON procedure is a straightforward, eco-friendly, and reliable method for bioprofiling and analyzing bioactive compounds in botanical extracts.
• This technique effectively addresses the challenge of coeluting structural isomers, offering a significant advancement over traditional bioactivity-guided fractionation.
• The method's simplicity, use of NMR calibration over extended periods, and low solvent consumption make it a practical alternative for researchers.