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Monoterpenoids are integral to the chemical composition of the widely used adaptogenic dietary supplement Rhodiola rosea. The present study expands the chemical space and stereochemical information about these taxon-specific constituents from the isolation and characterization of five geraniol-derived glucosides, 1–5. While 1and 2exhibited almost identical NMR spectra and shared the same 2D structure ascribed to the 4-hydroxygeraniolglucoside previously described as rosiridin, the NMR-based Mosher ester method revealed the enantiomeric nature of their aglycone moieties. This marks the first report of enantiomeric aglycones among geraniol derivatives. These findings also resolve the long-standing dispute regarding the absolute configuration of rosiridin and congeneric C-4 hydroxylated geraniols and may help explain incongruent bioactivity reports of R. roseaextract. Moreover, the three previously undescribed geranioloids 3–5were fully characterized by extensive spectroscopic analysis. Quantum mechanics-driven 1H iterative functionalized spin analysis (QM-HifSA) was performed for all isolates and provides detailed NMR spin parameters, with adequate decimal place precision, which enable the distinction of such close congeners exhibiting near identical NMR spectra with high specificity. The outcomes also reinforce the importance of reporting chemical shifts and coupling constants with adequate decimal place precision as a means of achieving specificity and reproducibility in structural analysis.

Prenyl moieties are commonly encountered in the natural products of terpenoid and mixed biosynthetic origin. The reactivity of unsaturated prenyl motifs is less recognized and shown here to affect the acyclic Rhodiola rosea monoterpene glycoside, kenposide A (8), which oxidizes readily on silica gel when exposed to air. The major degradation product mediated under these conditions was a new aldehyde, 9. Exhibiting a shortened carbon skeleton formed through the breakdown of the terminal isopropenyl group, 9 is prone to acetalization in protic solvents. Further investigation of minor degradation products of both 8 and 8-prenylapigenin (8-PA, 12), a flavonoid with an ortho -prenyl substituent, revealed that the aldehyde formation was likely realized through epoxidation and subsequent cleavage at the prenyl olefinic bond. Employment of1 H NMR full spin analysis (HiFSA) achieved the assignment of all chemical shifts and coupling constants of the investigated terpenoids and facilitated the structural validation of the degradation product, 9. This study indicates that prenylated compounds are generally susceptible to oxidative degradation, particularly in the presence of catalytic mediators, but also under physiological conditions. Such oxidative artifact/metabolite formation leads to a series of compounds with prenyl-derived (cyclic) partial structures that are analogous to species formed during Phase I metabolism in vivo. Phytochemical and pharmacological studies should take precautions or at least consider the impact of (unavoidable) exposure of prenyl-containing compounds to catalytic and/or oxidative conditions. [ABSTRACT FROM AUTHOR]

Prenyl moieties are commonly encountered in the natural products of terpenoid and mixed biosynthetic origin. The reactivity of unsaturated prenyl motifs is less recognized and shown here to affect the acyclic Rhodiola rosea monoterpene glycoside, kenposide A (8), which oxidizes readily on silica gel when exposed to air. The major degradation product mediated under these conditions was a new aldehyde, 9. Exhibiting a shortened carbon skeleton formed through the breakdown of the terminal isopropenyl group, 9 is prone to acetalization in protic solvents. Further investigation of minor degradation products of both 8 and 8-prenylapigenin (8-PA, 12), a flavonoid with an ortho -prenyl substituent, revealed that the aldehyde formation was likely realized through epoxidation and subsequent cleavage at the prenyl olefinic bond. Employment of1 H NMR full spin analysis (HiFSA) achieved the assignment of all chemical shifts and coupling constants of the investigated terpenoids and facilitated the structural validation of the degradation product, 9. This study indicates that prenylated compounds are generally susceptible to oxidative degradation, particularly in the presence of catalytic mediators, but also under physiological conditions. Such oxidative artifact/metabolite formation leads to a series of compounds with prenyl-derived (cyclic) partial structures that are analogous to species formed during Phase I metabolism in vivo. Phytochemical and pharmacological studies should take precautions or at least consider the impact of (unavoidable) exposure of prenyl-containing compounds to catalytic and/or oxidative conditions. [ABSTRACT FROM AUTHOR]

NMR- and MS-guided metabolomic mining for new phytoconstituents from a widely used dietary supplement, Rhodiola rosea, yielded two new (+)-myrtenol glycosides, 1and 2, and two new cuminol glycosides, 3and 4, along with three known analogues, 5–7. The structures of the new compounds were determined by extensive spectroscopic data analysis. Quantum mechanics-driven 1H iterative full spin analysis (QM-HiFSA) decoded the spatial arrangement of the methyl groups in 1and 2, as well as other features not recognizable by conventional methods, including higher order spin-coupling effects. Expanding applied HiFSA methodology to monoterpene glycosides advances the toolbox for stereochemical assignments, facilitates their structural dereplication, and provides a more definitive reference point for future phytochemical and biological studies of R. roseaas a resilience botanical. Application of a new NMR data analysis software package, CT, for QM-based iteration of NMR spectra is also discussed.

Occurring in hops (Humulus lupulus) and beer as a racemic mixture, (2R,2S)-8-prenylnaringenin (8-PN) is a potent phytoestrogen in hop dietary supplements used by women as alternatives to conventional hormone therapy. With a half-life exceeding 20 h, 8-PN is excreted primarily as 8-PN-7-O-glucuronide or 8-PN-4′-O-glucuronide. Human liver microsomes and 11 recombinant human UDP-glucuronosyltransferases (UGTs) were used to catalyze the formation of the two oxygen-linked glucuronides of purified (2R)-8-PN and (2S)-8-PN, which were subsequently identified using mass spectrometry and nuclear magnetic resonance spectroscopy. Formation of (2R)- and (2S)-8-PN-7-O-glucuronides predominated over the 8-PN-4′-O-glucuronides except for intestinal UGT1A10, which formed more (2S)-8-PN-4′-O-glucuronide. (2R)-8-PN was a better substrate for all 11 UGTs except for UGT1A1, which formed more of both (2S)-8-PN glucuronides than (2R)-8-PN glucuronides. Although several UGTs conjugated both enantiomers of 8-PN, some conjugated just one enantiomer, suggesting that human phenotypic variation might affect the routes of metabolism of this chiral estrogenic constituent of hops.

Botanical dietary supplements contain multiple bioactive compounds that target numerous biological pathways. The lack of uniform standardization requirements is one reason that inconsistent clinical effects are reported frequently. The multifaceted biological interactions of active principles can be disentangled by a coupled pharmacological/phytochemical approach using specialized (“knock-out”) extracts. This is demonstrated for hops, a botanical for menopausal symptom management. Employing targeted, adsorbent-free countercurrent separation, Humulus lupulusextracts were designed for pre- and postmenopausal women by containing various amounts of the phytoestrogen 8-prenylnaringenin (8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis of their estrogenic (alkaline phosphatase), chemopreventive (NAD(P)H-quinone oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that the estrogenicity of hops is a function of 8-PN, whereas their NQO1 induction and cytotoxic properties depend on XH levels. Antagonization of the estrogenicity of 8-PN by elevated XH concentrations provided evidence for the interdependence of the biological effects. A designed postmenopausal hop extract was prepared to balance 8-PN and XH levels for both estrogenic and chemopreventive properties. An extract designed for premenopausal women contains reduced 8-PN levels and high XH concentrations to minimize estrogenic while retaining chemopreventive properties. This study demonstrates the feasibility of modulating the concentrations of bioactive compounds in botanical extracts for potentially improved efficacy and safety.

Investigating the phytochemical equivalence of the aerial parts of Actaea racemosa(syn. Cimicifuga racemosa) relative to the widely used roots/rhizomes, this study provides a perspective for the potential use of renewable (“green”) plant parts as a source of black cohosh botanical preparations. In addition to the characterization of Nω-methylserotonin as one representative marker of the Actaeaalkaloids, nine cycloartane triterpenes were isolated and characterized, including the two new triterpene glycosides (1S,15R)-1,15,25-trihydroxy-3-O-β-d-xylopyranosyl-acta-(16S,23R,24R)-16,23;16,24-binoxoside (1) and 3-O-α-l-arabinopyranosyl-(1S,24R)-1,24,25-trihydroxy-15-oxo-acta-(16R,23R)-16,23-monoxoside (2). Their structures were elucidated by spectroscopic data interpretation. The relative configuration of 1was deduced by 1H iterative full-spin analysis (HiFSA), making it the first example of the complete analysis of the complex 1H NMR spectrum of a triterpene glycoside. In addition to the new compounds 1and 2, the aerial plant parts were shown to contain the previously known binoxosides 3, 4, 6, and 7, the monoxoside 8, and the binoxols 5and 9. Overall, the metabolome of the aerial plant parts consists of a variety of Actaeatriterpenes, similar to those found in roots/rhizomes, a tendency toward C-1 and C-7 hydroxylation of the cycloartanol skeleton, a greater abundance of aglycones, and the presence of comparable amounts of Nω-methylserotonin.

This study introduces a flexible and compound targeted approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources, generating DESIGNER extracts, by means of chemical subtraction or augmentation of metabolites. Targeting metabolites based on their liquid–liquid partition coefficients (Kvalues), Ktargeting uses countercurrent separation methodology to remove single or multiple compounds from a chemically complex mixture, according to the following equation: DESIGNER extract = total extract ± target compound(s). Expanding the scope of the recently reported depletion of extracts by immunoaffinity or solid phase liquid chromatography, the present approach allows a more flexible, single- or multi-targeted removal of constituents from complex extracts such as botanicals. Chemical subtraction enables both chemical and biological characterization, including detection of synergism/antagonism by both the subtracted targets and the remaining metabolite mixture, as well as definition of the residual complexity of all fractions. The feasibility of the DESIGNER concept is shown by K-targeted subtraction of four bioactive prenylated phenols, isoxanthohumol (1), 8-prenylnaringenin (2), 6-prenylnaringenin (3), and xanthohumol (4), from a standardized hops (Humulus lupulusL.) extract using specific solvent systems. Conversely, adding K-targeted isolates allows enrichment of the original extract and hence provides an augmented DESIGNER material. Multiple countercurrent separation steps were used to purify each of the four compounds, and four DESIGNER extracts with varying depletions were prepared. The DESIGNER approach innovates the characterization of chemically complex extracts through integration of enabling technologies such as countercurrent separation, K-by-bioactivity, the residual complexity concepts, as well as quantitative analysis by 1H NMR, LC-MS, and HiFSA-based NMR fingerprinting.

Licorice botanicals are produced from the roots of Glycyrrhizaspecies (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 Glycyrrhiza uralensis. 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, p-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 G. uralensisextract. 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.

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, 1H iterative full spin analysis (HiFSA), and Mosher’s 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. villosasecondary metabolome. LC-MS profiling of different D. villosaaccessions 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.

Off-line combination of countercurrent separation (CCS) and quantitative 1H NMR (qHNMR) methodologies enabled the systematic dissection and gravimetric quantification of a chemically complex Rhodiola roseacrude extract (RCE). The loss-free nature and high selectivity of CCS achieved the quantitative discrimination of fatty acids (FAs), sugars, and proanthocyanidins (PACs) from ten other metabolite classes: phenylpropanoids, phenylethanoids, acyclic monoterpenoid glycosides, pinene derived glycosides, benzyl alcohol glycosides, cyanogenic glycosides, flavonoids, gallic acids, methylparabens, and cuminol glycosides. The ability of CCS to remove (“knockout”) PACs completely resolved challenges with baselines that plague NMR and UHPLC analyses and produce inaccurate integral and AUC quantitation, respectively. NMR analysis of the non-PAC fractions enabled unambiguous identification of metabolites and their characteristic resonances for subsequent multitarget absolute quantification by qHNMR using a single, nonidentical internal calibrant (IC). An orthogonal LC–MS/MS method validated the gravimetric nature of the CCS-qHNMR analytical tandem. Underlying this LC-based cross-validation, comprehensive phytochemical isolation and characterization established 19 single-compound reference standards that represented all ten metabolite classes. Finally, quantum mechanical 1H iterative Full Spin Analysis (HiFSA) of each standard provided a blueprint for future structural dereplication, identification, and quantification of Rhodiolamarker constituents. The combination of two gravimetric analytical methods, loss-free CCS and IC-qHNMR, realizes the first chemical standardization of a botanical material that comprehensively captures a metabolome and permits absolute quantification.

Prenyl moieties are commonly encountered in the natural products of terpenoid and mixed biosynthetic origin. The reactivity of unsaturated prenyl motifs is less recognized and shown here to affect the acyclic Rhodiola rosea monoterpene glycoside, kenposide A (8), which oxidizes readily on silica gel when exposed to air. The major degradation product mediated under these conditions was a new aldehyde, 9. Exhibiting a shortened carbon skeleton formed through the breakdown of the terminal isopropenyl group, 9 is prone to acetalization in protic solvents. Further investigation of minor degradation products of both 8 and 8-prenylapigenin (8-PA, 12), a flavonoid with an ortho -prenyl substituent, revealed that the aldehyde formation was likely realized through epoxidation and subsequent cleavage at the prenyl olefinic bond. Employment of1 H NMR full spin analysis (HiFSA) achieved the assignment of all chemical shifts and coupling constants of the investigated terpenoids and facilitated the structural validation of the degradation product, 9. This study indicates that prenylated compounds are generally susceptible to oxidative degradation, particularly in the presence of catalytic mediators, but also under physiological conditions. Such oxidative artifact/metabolite formation leads to a series of compounds with prenyl-derived (cyclic) partial structures that are analogous to species formed during Phase I metabolism in vivo. Phytochemical and pharmacological studies should take precautions or at least consider the impact of (unavoidable) exposure of prenyl-containing compounds to catalytic and/or oxidative conditions. [ABSTRACT FROM AUTHOR]

Prenyl moieties are commonly encountered in the natural products of terpenoid and mixed biosynthetic origin. The reactivity of unsaturated prenyl motifs is less recognized and shown here to affect the acyclic Rhodiola rosea monoterpene glycoside, kenposide A (8), which oxidizes readily on silica gel when exposed to air. The major degradation product mediated under these conditions was a new aldehyde, 9. Exhibiting a shortened carbon skeleton formed through the breakdown of the terminal isopropenyl group, 9 is prone to acetalization in protic solvents. Further investigation of minor degradation products of both 8 and 8-prenylapigenin (8-PA, 12), a flavonoid with an ortho -prenyl substituent, revealed that the aldehyde formation was likely realized through epoxidation and subsequent cleavage at the prenyl olefinic bond. Employment of1 H NMR full spin analysis (HiFSA) achieved the assignment of all chemical shifts and coupling constants of the investigated terpenoids and facilitated the structural validation of the degradation product, 9. This study indicates that prenylated compounds are generally susceptible to oxidative degradation, particularly in the presence of catalytic mediators, but also under physiological conditions. Such oxidative artifact/metabolite formation leads to a series of compounds with prenyl-derived (cyclic) partial structures that are analogous to species formed during Phase I metabolism in vivo. Phytochemical and pharmacological studies should take precautions or at least consider the impact of (unavoidable) exposure of prenyl-containing compounds to catalytic and/or oxidative conditions. [ABSTRACT FROM AUTHOR]

Given the importance of Thymus glabrescensas a useful antibacterial remedy, we have evaluated the antibacterial and streptomycin-modifying activity of Thymus glabrescensessential oil, geraniol, geranyl acetate and thymol. It was shown that all substance-streptomycin combinations produced predominantly antagonistic interactions. Furthermore, combinations between geraniol and thymol showed dominant additive effect. Chemoinformatics results, combined with experimental data, suggest that antagonistic interactions with streptomycin were not a consequence of the antimicrobial action at the same target, but an outcome of the membrane impairment, followed by the membrane potential/proton motive force dissipation, which decreased the streptomycin uptake. Furthermore, the membrane toxicity of geraniol and thymol was confirmed by their additive antibacterial interactions and parameters of their penetration and accumulation throughout a cell membrane. This study should greatly help in an intelligent and a controlled pharmacomodulation of antibiotics.

The composition and antimicrobial activity of the essential oil of Heracleum sibiricumL. (Apiaceae) was studied. The aerial part of plant was hydro-distilled and chemical composition of the essential oil was analyzed by GC and GC-MS. Forty-six compounds, corresponding to 95.12% of the total oil, were identified. Esters represented the major chemical class (69.55%) while the main constituents were octyl butanoate (36.82%), hexyl butanoate (16.08%), 1-octanol (13.62%) and octyl hexanoate (8.10%). Antibacterial activity of the essential oil and reference antibiotics against nine bacterial strains was tested by the broth microdilution method. The results of the bioassays showed that essential oil had slight antimicrobial activities against all tested microorganisms (MIC and MBC values were in the range of 2431.2 to 9724.8 μg/mL). Reference antibiotics were active in concentrations between 0.5 and 16.0 μg/mL. The results confirm that Gram-positive bacteria were more susceptible to the essential oil of H. sibiricum, in comparison with Gram-negative bacteria.