Advancing botanical safety: A strategy for selecting, sourcing, and characterizing botanicals for developing toxicological tools.

Increases in botanical use, encompassing herbal medicines and dietary supplements, have underlined a critical need for an advancement in safety assessment methodologies. However, botanicals present unique challenges for safety assessment due to their complex and variable composition arising from diverse growing conditions, processing methods, and plant varieties. Historically, botanicals have been largely evaluated based on their history of use information, based primarily on traditional use or dietary history. However, this presumption lacks comprehensive toxicological evaluation, demanding innovative and consistent assessment strategies. To address these challenges, the Botanical Safety Consortium (BSC) was formed as an international, cross-sector forum of experts to identify fit-for purpose assays that can be used to evaluate botanical safety. This global effort aims to assess botanical safety assessment methodologies, merging traditional knowledge with modern in vitro and in silico assays. The ultimate goal is to champion the development of toxicity tools for botanicals. This manuscript highlights: 1) BSC's strategy for botanical selection, sourcing, and preparation of extracts to be used in in vitro assays, and 2) the approach utilized to characterize botanical extracts, using green tea and Asian ginseng as examples, to build confidence for use in biological assays.

Tree mode of death and mortality risk factors across Amazon forests.

The carbon sink capacity of tropical forests is substantially affected by tree mortality. However, the main drivers of tropical tree death remain largely unknown. Here we present a pan-Amazonian assessment of how and why trees die, analysing over 120,000 trees representing > 3800 species from 189 long-term RAINFOR forest plots. While tree mortality rates vary greatly Amazon-wide, on average trees are as likely to die standing as they are broken or uprooted-modes of death with different ecological consequences. Species-level growth rate is the single most important predictor of tree death in Amazonia, with faster-growing species being at higher risk. Within species, however, the slowest-growing trees are at greatest risk while the effect of tree size varies across the basin. In the driest Amazonian region species-level bioclimatic distributional patterns also predict the risk of death, suggesting that these forests are experiencing climatic conditions beyond their adaptative limits. These results provide not only a holistic pan-Amazonian picture of tree death but large-scale evidence for the overarching importance of the growth-survival trade-off in driving tropical tree mortality.

A multi-detector chromatographic approach for characterization and quantitation of botanical constituents to enable in silico safety assessments.

An approach has been developed to characterize the individual chemical constituents of botanicals. The challenge was to identify and quantitate the significant analytes in these complex mixtures, largely in the absence of authentic standards. The data-rich information content generated by this three-detector configuration was specifically intended to be used to conduct safety and/or quality evaluations for complex botanical mixtures, on a chemical constituent basis. The approach utilized a broad gradient UHPLC chromatographic separation. Following the chromatographic separation and UV detection, the eluent was split and sent into a charged aerosol detector (CAD), for quantitation, and a quadrupole/time-of-flight high-resolution mass spectrometer for component identification. The known bias of the otherwise universal CAD response, for organic solvent composition of the mobile phase, was compensated by the addition of an inverse gradient make-up stream. This approach and the orthogonal information content from the chromatography and three different detectors was specifically designed to enable in-silico safety assessments. These guide, minimize, or even eliminate the need for in vivo and in vitro safety assessments. The methodology was developed and demonstrated using standardized extracts of Ginkgo biloba. Results from the development of this novel approach and the characterization example reported here demonstrate the suitability of this instrumental configuration for enabling in-silico safety assessments and proving general quality assessments of botanicals.

In silico approach to safety of botanical dietary supplement ingredients utilizing constituent-level characterization.

Botanicals used in dietary supplements industry can have toxicology concerns related to endpoint gaps that cannot be fully resolved by a history of use, or existence of conflicting safety data. However, traditional toxicological studies on botanicals are scientifically and pragmatically challenging due to testing of complex mixtures of constituents, cost, time, and animal usage. Alternatively, we developed an in silico decision-tree approach to address data gaps and inform need for further studies by toxicologically evaluating the chemical composition of botanicals. Following advanced multi-detector analytical characterization of a botanical, each chemical constituent is: (a.) quantitatively benchmarked against similar constituents in commonly consumed foods or botanicals with well-established safety profiles, (b.) systematically evaluated for toxicity data utilizing structure-activity relationships, and, (c.) compared to established thresholds of toxicological concern in absence of safety data or structural analogs. Finally, where safety endpoint gaps are identified which cannot be resolved without additional in vitro or in vivo studies, the botanical compositional data are critical to inform on study design. Results with three herbal preparations demonstrate the utility of this novel approach to identify potential hazards and establish safe human use levels for botanicals in a cost efficient and informative manner that minimizes animal use.

Safety assessment of mushrooms in dietary supplements by combining analytical data with in silico toxicology evaluation.

Despite growing popularity in dietary supplements, many medicinal mushrooms have not been evaluated for their safe human consumption using modern techniques. The multifaceted approach described here relies on five key principles to evaluate the safety of non-culinary fungi for human use: (1) identification by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region (commonly referred to as ITS barcoding), (2) screening an extract of each fungal raw material against a database of known fungal metabolites, (3) comparison of these extracts to those prepared from grocery store-bought culinary mushrooms using UHPLCPDA-ELS-HRMS, (4) review of the toxicological and chemical literature for each fungus, and (5) evaluation of data establishing presence in-market. This weight-of-evidence approach was used to evaluate seven fungal raw materials and determine safe human use for each. Such an approach may provide an effective alternative to conventional toxicological animal studies (or more efficiently identifies when studies are necessary) for the safety assessment of fungal dietary ingredients.

Variations in Amazon forest productivity correlated with foliar nutrients and modelled rates of photosynthetic carbon supply.

The rate of above-ground woody biomass production, W(P), in some western Amazon forests exceeds those in the east by a factor of 2 or more. Underlying causes may include climate, soil nutrient limitations and species composition. In this modelling paper, we explore the implications of allowing key nutrients such as N and P to constrain the photosynthesis of Amazon forests, and also we examine the relationship between modelled rates of photosynthesis and the observed gradients in W(P). We use a model with current understanding of the underpinning biochemical processes as affected by nutrient availability to assess: (i) the degree to which observed spatial variations in foliar [N] and [P] across Amazonia affect stand-level photosynthesis; and (ii) how these variations in forest photosynthetic carbon acquisition relate to the observed geographical patterns of stem growth across the Amazon Basin. We find nutrient availability to exert a strong effect on photosynthetic carbon gain across the Basin and to be a likely important contributor to the observed gradient in W(P). Phosphorus emerges as more important than nitrogen in accounting for the observed variations in productivity. Implications of these findings are discussed in the context of future tropical forests under a changing climate.