Charlotte Simmler

Universal Quantitative NMR Analysis of Complex Natural Samples

Nuclear Magnetic Resonance (NMR) is a universal and quantitative analytical technique. Being a unique structural tool, NMR also competes with metrological techniques for purity determination and reference material analysis. In pharmaceutical research, applications of quantitative NMR (qNMR) cover mostly the identification and quantification of drug and biological metabolites. Offering an unbiased view of the sample composition, and the possibility to simultaneously quantify multiple compounds, qNMR has become the method of choice for metabolomic studies and quality control of complex natural samples such as foods, plants or herbal remedies, and biofluids. In this regard, NMR-based metabolomic studies, dedicated to both the characterization of herbal remedies and clinical diagnosis, have increased considerably.

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.

Phytochemistry and Biological Properties of Glabridin

Glabridin, a prenylated isoflavonoid of G. glabra L. roots (European licorice, Fabaceae), has been associated with a wide range of biological properties such as antioxidant, anti-inflammatory, anti-atherogenic, regulation of energy metabolism, estrogenic, neuroprotective, anti-osteoporotic, and skin-whitening. While glabridin is one of the most studied licorice flavonoids, a comprehensive literature survey linked to its numerous bioactivities is unavailable. The present review provides a comprehensive description of glabridin as a key chemical and biological marker of G. glabra, by covering both its phytochemical characterization and reported biological activities. Both glabridin and standardized licorice extracts have significant impact on food, dietary supplements (DSs) and cosmetic markets, as evidenced by the amount of available patents and scientific articles since 1976, when glabridin was first described. Nevertheless, a thorough literature survey also reveals that information about the isolation and chemical characterization of this important marker is scattered and less detailed than expected. Accordingly, the first part of this review gathers and provides all analytical and spectroscopic data required for the comprehensive phytochemical characterization of glabridin. The four most frequently described and most relevant bioactivities of glabridin are its anti-inflammatory, anti-atherogenic, estrogenic-like effects, and its capacity to regulate energy metabolism. While all bioactivities reported for glabridin belong to a wide array of targets, its principal biological properties are likely interconnected. To this end, the current state of the literature suggests that the biological activity of glabridin mainly results from its capacity to down-regulate intracellular reactive oxygen species, bind to antioxidant effectors, and act on estrogen receptors, potentially as a plant-based Selective Estrogen Receptor Modulator (phytoSERM).

Evaluation of Estrogenic Activity of Licorice Species in Comparison with Hops Used in Botanicals for Menopausal Symptoms

The increased cancer risk associated with hormone therapies has encouraged many women to seek non-hormonal alternatives including botanical supplements such as hops (Humulus lupulus) and licorice (Glycyrrhiza spec.) to manage menopausal symptoms. Previous studies have shown estrogenic properties for hops, likely due to the presence of 8-prenylnarigenin, and chemopreventive effects mainly attributed to xanthohumol. Similarly, a combination of estrogenic and chemopreventive properties has been reported for various Glycyrrhiza species. The major goal of the current study was to evaluate the potential estrogenic effects of three licorice species (Glycyrrhiza glabra, G. uralensis, and G. inflata) in comparison with hops. Extracts of Glycyrrhiza species and spent hops induced estrogen responsive alkaline phosphatase activity in endometrial cancer cells, estrogen responsive element (ERE)-luciferase in MCF-7 cells, and Tff1 mRNA in T47D cells. The estrogenic activity decreased in the order H. lupulus > G. uralensis > G. inflata > G. glabra. Liquiritigenin was found to be the principle phytoestrogen of the licorice extracts; however, it exhibited lower estrogenic effects compared to 8-prenylnaringenin in functional assays. Isoliquiritigenin, the precursor chalcone of liquiritigenin, demonstrated significant estrogenic activities while xanthohumol, a metabolic precursor of 8-prenylnaringenin, was not estrogenic. Liquiritigenin showed ERβ selectivity in competitive binding assay and isoliquiritigenin was equipotent for ER subtypes. The estrogenic activity of isoliquiritigenin could be the result of its cyclization to liquiritigenin under physiological conditions. 8-Prenylnaringenin had nanomolar estrogenic potency without ER selectivity while xanthohumol did not bind ERs. These data demonstrated that Glycyrrhiza species with different contents of liquiritigenin have various levels of estrogenic activities, suggesting the importance of precise labeling of botanical supplements. Although hops shows strong estrogenic properties via ERα, licorice might have different estrogenic activities due to its ERβ selectivity, partial estrogen agonist activity, and non-enzymatic conversion of isoliquiritigenin to liquiritigenin.

Antioxidant Biomarkers from Vanda coerulea Stems Reduce Irradiated HaCaT PGE-2 Production as a Result of COX-2 Inhibition

Background In our investigations towards the isolation of potentially biologically active constituents from Orchidaceae, we carried out phytochemical and biological analyses of Vanda species. A preliminary biological screening revealed that Vanda coerulea (Griff. ex. Lindl) crude hydro-alcoholic stem extract displayed the best DPPH /•OH radical scavenging activity and in vitro inhibition of type 2 prostaglandin (PGE-2) release from UVB (60 mJ/cm2) irradiated HaCaT keratinocytes. Principal Findings Bio-guided fractionation and phytochemical analysis led to the isolation of five stilbenoids: imbricatin (1) methoxycoelonin (2) gigantol (3) flavidin (4) and coelonin (5). Stilbenoids (1–3) were the most concentrated in crude hydro-alcoholic stem extract and were considered as Vanda coerulea stem biomarkers. Dihydro-phenanthropyran (1) and dihydro-phenanthrene (2) displayed the best DPPH/•OH radical scavenging activities as well as HaCaT intracellular antioxidant properties (using DCFH-DA probe: IC50 8.8 µM and 9.4 µM, respectively) compared to bibenzyle (3) (IC50 20.6 µM). In turn, the latter showed a constant inhibition of PGE-2 production, stronger than stilbenoids (1) and (2) (IC50 12.2 µM and 19.3 µM, respectively). Western blot analysis revealed that stilbenoids (1–3) inhibited COX-2 expression at 23 µM. Interestingly, stilbenoids (1) and (2) but not (3) were able to inhibit human recombinant COX-2 activity. Conclusions Major antioxidant stilbenoids (1–3) from Vanda coerulea stems displayed an inhibition of UVB-induced COX-2 expression. Imbricatin (1) and methoxycoelonin (2) were also able to inhibit COX-2 activity in a concentration-dependent manner thereby reducing PGE-2 production from irradiated HaCaT cells. Our studies suggest that stilbenoids (1–3) could be potentially used for skin protection against the damage caused by UVB exposure.

Diarylheptanoids from Dioscorea villosa (Wild Yam)

A fractionation methodology aimed at the metabolomic mining of new phytoconstituents for the widely used botanical, wild yam (Dioscorea villosa), makes use of 1D qHNMR and 2D NMR profiles along the preparative fractionation pathway. This quantifiable and structural guidance led to the isolation of 14 diarylheptanoids (1-14), including five new compounds (1-5) with a tetrahydropyrano core skeleton. The structures, including the absolute configurations of both new and previously known diarylheptanoids, were assigned by a combination of HRESIMS, 1D and 2D NMR, (1)H iterative full spin analysis (HiFSA), and Moshers ester method. The isolation yields were consistent with yields predicted by qHNMR, which confirms the (semi)quantifiable capabilities of NMR-based preparative metabolomic mining. The qHNMR-aided approach enabled the identification of new and potentially significant chemical entities from a small fraction of the plant extract and, thereby, facilitated the characterization of the residual complexity of the D. villosa secondary metabolome. LC-MS profiling of different D. villosa accessions further confirmed that the diarylheptanoids represent genuine secondary metabolites, which can serve as a new class of markers for botanical integrity analysis of D. villosa.

Metabolite Profiling and Classification of DNA-Authenticated Licorice Botanicals

Raw licorice roots represent heterogeneous materials obtained from mainly three Glycyrrhiza species. G. glabra, G. uralensis, and G. inflata exhibit marked metabolite differences in terms of flavanones (Fs), chalcones (Cs), and other phenolic constituents. The principal objective of this work was to develop complementary chemometric models for the metabolite profiling, classification, and quality control of authenticated licorice. A total of 51 commercial and macroscopically verified samples were DNA authenticated. Principal component analysis and canonical discriminant analysis were performed on (1)H NMR spectra and area under the curve values obtained from UHPLC-UV chromatograms, respectively. The developed chemometric models enable the identification and classification of Glycyrrhiza species according to their composition in major Fs, Cs, and species specific phenolic compounds. Further key outcomes demonstrated that DNA authentication combined with chemometric analyses enabled the characterization of mixtures, hybrids, and species outliers. This study provides a new foundation for the botanical and chemical authentication, classification, and metabolomic characterization of crude licorice botanicals and derived materials. Collectively, the proposed methods offer a comprehensive approach for the quality control of licorice as one of the most widely used botanical dietary supplements.

Differential Effects of Glycyrrhiza Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 μM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for womens health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.

Glucosyloxybenzyl Eucomate Derivatives from Vanda teres Stimulate HaCaT Cytochrome c Oxidase.

Eucomic acid [(2R)-2-(p-hydroxybenzyl)malic acid)] (1) and three new glucopyranosyloxybenzyl eucomate derivatives, vandaterosides I (2), II (3), and III (4), were isolated and identified from the stems of Vanda teres. Their cellular antiaging properties were evaluated in a human immortalized keratinocyte cell line (HaCaT) by monitoring their effect on cytochrome c oxidase activity, implicated in mitochondrial respiratory function and cellular energy production. Eucomic acid (1) and vandateroside II (3) increased cytochrome c oxidase activity and/or expression, without enhancing cellular mitochondrial content. These two V. teres biomarkers apparently contributed to stimulate respiratory functions in keratinocytes. Since aging and its pathologies may be ascribed to a decline in mitochondrial functions, these biomarkers have the potential to become new natural ingredients for antiaging preparations to remedy age-related disorders such as skin aging.

Biochemical characterization and anti-inflammatory properties of an isothiocyanate-enriched moringa (Moringa oleifera) seed extract

Moringa oleifera Lam. is a tropical plant, used for centuries as food and traditional medicine. The aim of this study was to develop, validate and biochemically characterize an isothiocyanate-enriched moringa seed extract (MSE), and to compare the anti-inflammatory effects of MSE-containing moringa isothiocyanate-1 (MIC-1) with a curcuminoid-enriched turmeric extract (CTE), and a material further enriched in its primary phytochemical, curcumin (curcumin-enriched material; CEM). MSE was prepared by incubating ground moringa seeds with water to allow myrosinase-catalyzed enzymatic formation of bioactive MIC-1, the predominant isothiocyanate in moringa seeds. Optimization of the extraction process yielded an extract of 38.9% MIC-1. Phytochemical analysis of MSE revealed the presence of acetylated isothiocyanates, phenolic glycosides unique to moringa, flavonoids, fats and fatty acids, proteins and carbohydrates. MSE showed a reduction in the carrageenan-induced rat paw edema (33% at 500 mg/kg MIC-1) comparable to aspirin (27% at 300 mg/kg), whereas CTE did not have any significant effect. In vitro, MIC-1 at 1 μM significantly reduced the production of nitric oxide (NO) and at 5 μM, the gene expression of LPS-inducible nitric oxide synthase (iNOS) and interleukins 1β and 6 (IL-1β and IL-6), whereas CEM did not show any significant activity at all concentrations tested. MIC-1 (10μM) was also more effective at upregulating the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) target genes NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase pi 1 (GSTP1), and heme oxygenase 1 (HO1) than the CEM. Thus, in contrast to CTE and CEM, MSE and its major isothiocyanate MIC-1 displayed strong anti-inflammatory and antioxidant properties in vivo and in vitro, making them promising botanical leads for the mitigation of inflammatory-mediated chronic disorders.