Countercurrent Separation of Natural Products: An Update

This work assesses the current instrumentation, method development, and applications in countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC), collectively referred to as countercurrent separation (CCS). The article provides a critical review of the CCS literature from 2007 since our last review (<i>J. Nat. Prod.</i> <b>2008</b>, <i>71</i>, 1489–1508), with a special emphasis on the applications of CCS in natural products research. The current state of CCS is reviewed in regard to three continuing topics (instrumentation, solvent system development, theory) and three new topics (optimization of parameters, workflow, bioactivity applications). The goals of this review are to deliver the necessary background with references for an up-to-date perspective of CCS, to point out its potential for the natural product scientist, and thereby to induce new applications in natural product chemistry, metabolome, and drug discovery research involving organisms from terrestrial and marine sources

Analysis and Purification of Bioactive Natural Products: The AnaPurNa Study

Based on a meta-analysis of data mined from almost 2000 publications on bioactive natural products (NPs) from >80 000 pages of 13 different journals published in 1998–1999, 2004–2005, and 2009–2010, the aim of this systematic review is to provide both a survey of the status quo and a perspective for analytical methodology used for isolation and purity assessment of bioactive NPs. The study provides numerical measures of the common means of sourcing NPs, the chromatographic methodology employed for NP purification, and the role of spectroscopy and purity assessment in NP characterization. A link is proposed between the observed use of various analytical methodologies, the challenges posed by the complexity of metabolomes, and the inescapable residual complexity of purified NPs and their biological assessment. The data provide inspiration for the development of innovative methods for NP analysis as a means of advancing the role of naturally occurring compounds as a viable source of biologically active agents with relevance for human health and global benefit

Natural Deep Eutectic Solvents: Properties, Applications, and Perspectives

As functional liquid media, <u>n</u>atural <u>d</u>eep <u>e</u>utectic <u>s</u>olvent (NADES) species can dissolve natural or synthetic chemicals of low water solubility. Moreover, the special properties of NADES, such as biodegradability and biocompatibility, suggest that they are alternative candidates for concepts and applications involving some organic solvents and ionic liquids. Owing to the growing comprehension of the eutectic mechanisms and the advancing interest in the natural eutectic phenomenon, many NADES applications have been developed in the past several years. However, unlike organic solvents, the basic structural unit of NADES media primarily depends on the intermolecular interactions among their components. This makes NADES matrices readily influenced by various factors, such as water content, temperature, and component ratio and, thus, extends the metabolomic challenge of natural products (NPs). To enhance the understanding of the importance of NADES in biological systems, this review focuses on NADES properties and applications in NP research. The present thorough chronological and statistical analysis of existing report adds to the recognition of the distinctiveness of (NA)­DES, involves a discussion of NADES-related observations in NP research, and reportes applications of these eutectic mixtures. The work identifies potential areas for future studies of (NA)­DES by evaluating relevant applications, including their use as extraction and chromatographic media as well as their biomedical relevance. The chemical diversity of natural metabolites that generate or participate in NADES formation highlights the growing insight that biosynthetically primordial metabolites (PRIMs) are as essential to the biological function and bioactivity of unrefined natural products as the biosynthetically more highly evolutionary metabolites (HEVOs) that can be isolated from crude mixtures

Can Invalid Bioactives Undermine Natural Product-Based Drug Discovery?

High-throughput biology has contributed a wealth of data on chemicals, including natural products (NPs). Recently, attention was drawn to certain, predominantly synthetic, compounds that are responsible for disproportionate percentages of hits but are false actives. Spurious bioassay interference led to their designation as <u>p</u>an-<u>a</u>ssay <u>in</u>terference compound<u>s</u> (PAINS). NPs lack comparable scrutiny, which this study aims to rectify. Systematic mining of 80+ years of the phytochemistry and biology literature, using the NAPRALERT database, revealed that only 39 compounds represent the NPs most reported by occurrence, activity, and distinct activity. Over 50% are not explained by phenomena known for synthetic libraries, and all had manifold ascribed bioactivities, designating them as <u>i</u>nvalid <u>m</u>etabolic <u>p</u>anaceas (IMPs). Cumulative distributions of ∼200,000 NPs uncovered that NP research follows power-law characteristics typical for behavioral phenomena. Projection into occurrence–bioactivity–effort space produces the hyperbolic black hole of NPs, where IMPs populate the high-effort base

Can Invalid Bioactives Undermine Natural Product-Based Drug Discovery?

High-throughput biology has contributed a wealth of data on chemicals, including natural products (NPs). Recently, attention was drawn to certain, predominantly synthetic, compounds that are responsible for disproportionate percentages of hits but are false actives. Spurious bioassay interference led to their designation as <u>p</u>an-<u>a</u>ssay <u>in</u>terference compound<u>s</u> (PAINS). NPs lack comparable scrutiny, which this study aims to rectify. Systematic mining of 80+ years of the phytochemistry and biology literature, using the NAPRALERT database, revealed that only 39 compounds represent the NPs most reported by occurrence, activity, and distinct activity. Over 50% are not explained by phenomena known for synthetic libraries, and all had manifold ascribed bioactivities, designating them as <u>i</u>nvalid <u>m</u>etabolic <u>p</u>anaceas (IMPs). Cumulative distributions of ∼200,000 NPs uncovered that NP research follows power-law characteristics typical for behavioral phenomena. Projection into occurrence–bioactivity–effort space produces the hyperbolic black hole of NPs, where IMPs populate the high-effort base

Quantification of a Botanical Negative Marker without an Identical Standard: Ginkgotoxin in <i>Ginkgo biloba</i>

A new strategy for the analysis of natural products uses a combination of quantitative ¹H NMR (qHNMR) and adsorbent-free countercurrent separation (CS) methodology to establish a quantification method for ginkgotoxin (4′-<i>O</i>-methylpyridoxine) in <i>Ginkgo biloba</i> preparations. The target analyte was concentrated in a one-step CS process using the ChMWat +2 solvent system (CHCl₃–MeOH–H₂O, 10:5:5) and subsequently assayed by qHNMR. While commercial <i>G. biloba</i> seeds contained 59 μg of ginkgotoxin per seed, the compound was below the limit of detection (9 ppm) in a typical leaf extract. Due to the enrichment potential and loss-free operation of CS, the combination of CS and qHNMR is a generally suitable approach for threshold assays aimed at quantifying target compounds such as botanical negative markers at the low ppm level. As the proof of principle is demonstrated for relatively small CS capacities (20 mL, 1:40 loading) and modest NMR sensitivity (<i>n</i> = 16, 400 MHz, 5 mm RT probe), the approach can be adapted to quantification at the ppb level. The procedure enables the quantification of a botanical negative marker in the absence of identical reference material, which otherwise is a prerequisite for LC-based assays

Correction to Importance of Purity Evaluation and the Potential of Quantitative ¹H NMR as a Purity Assay

Correction to Importance of Purity Evaluation and the Potential of Quantitative ¹H NMR as a Purity Assa

Importance of Purity Evaluation and the Potential of Quantitative ¹H NMR as a Purity Assay

In any biomedical and chemical context, a truthful description of chemical constitution requires coverage of both structure and purity. This qualification affects all drug molecules, regardless of development stage (early discovery to approved drug) and source (natural product or synthetic). Purity assessment is particularly critical in discovery programs and whenever chemistry is linked with biological and/or therapeutic outcome. Compared with chromatography and elemental analysis, quantitative NMR (qNMR) uses nearly universal detection and provides a versatile and orthogonal means of purity evaluation. Absolute qNMR with flexible calibration captures analytes that frequently escape detection (water, sorbents). Widely accepted structural NMR workflows require minimal or no adjustments to become practical ¹H qNMR (qHNMR) procedures with simultaneous qualitative and (absolute) quantitative capability. This study reviews underlying concepts, provides a framework for standard qHNMR purity assays, and shows how adequate accuracy and precision are achieved for the intended use of the material

Real-Time Volumetric Phase Monitoring: Advancing Chemical Analysis by Countercurrent Separation

Countercurrent separation (CCS) utilizes the differential partitioning behavior of analytes between two immiscible liquid phases. We introduce the first platform (“CherryOne”) capable of real-time monitoring, metering, and control of the dynamic liquid–liquid CCS process. Automated phase monitoring and volumetrics are made possible with an array of sensors, including the new permittivity-based phase metering apparatus (PMA). Volumetric data for each liquid phase are converted into a dynamic real-time display of stationary phase retention (Sf) and eluent partition coefficients (<i>K</i>), which represent critical parameters of CCS reproducibility. When coupled with the elution–extrusion operational mode (EECCC), automated Sf and <i>K</i> determination empowers untargeted and targeted applications ranging from metabolomic analysis to preparative purifications

Orthogonal Analysis Underscores the Relevance of Primary and Secondary Metabolites in Licorice

Licorice botanicals are produced from the roots of <i>Glycyrrhiza</i> species (Fabaceae), encompassing metabolites of both plant and rhizobial origin. The composition in both primary and secondary metabolites (1°/2°Ms) reflects the physiologic state of the plant at harvest. Interestingly, the relative abundance of 1°Ms vs 2°Ms in licorice extracts remains undetermined. A centrifugal partition chromatography (CPC) method was developed to purify liquiritin derivatives that represent major bioactive 2°Ms and to concentrate the polar 1°Ms from the crude extract of <i>Glycyrrhiza uralensis</i>. One objective was to determine the purity of the generated reference materials by orthogonal UHPLC-UV/LC-MS and qHNMR analyses. The other objectives were to evaluate the presence of 1°Ms in purified 2°Ms and define their mass balance in a crude botanical extract. Whereas most impurities could be assigned to well-known 1°Ms, <i>p</i>-hydroxybenzylmalonic acid, a new natural tyrosine analogue, was also identified. Additionally, in the most polar fraction, sucrose and proline represented 93% (w/w) of all qHNMR-quantified 1°Ms. Compared to the 2°Ms, accounting for 11.9% by UHPLC-UV, 1°Ms quantified by qHNMR defined an additional 74.8% of <i>G. uralensis</i> extract. The combined orthogonal methods enable the mass balance characterization of licorice extracts and highlight the relevance of 1°Ms, and accompanying metabolites, for botanical quality control