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This exploratory study was designed to identify factors implicating microbial influence on medicinal plant metabolomes. Utilizing a whole-microbiome approach, amplicon sequencing was used to identify the makeup of fungal and bacterial assemblages from endophytic (interior) and epiphytic (external) environments in two different sets of congeneric host-plant pairs, with collection of multiple samples of two medicinal plant species (Actaea racemosa, Rhodiola rosea) and two generic analogs (Actaea rubra, Rhodiola integrifolia). Diversity analysis of microbial assemblages revealed the influence of three primary factors driving variance in microbial community composition: host-plant taxonomy, the compartmentalization of microbial communities within discrete plant parts, and the scale of distance (microhabitat heterogeneity) between sampling locations. These three factors accounted for ~ 60% of variance within and between investigated microbiomes. Across all our collections, bacterial populations were more diverse than fungi (per compartment), and microbial density in epiphytic compartments (aerial parts, rhizosphere) were higher than those of endophytes (leaf and root). These comparative data point to key loci associated with variation between congeneric pairs and plant genera, providing insight into the complex and contrasting relationships found within this multi-kingdom coevolutionary relationship. Although reflective of only a limited set of botanical source materials, these data document the richness of a relatively unexplored component of the plant world and highlight the relevance of a whole-microbiome ecology-driven approach to botanical research and directed natural product investigations. [ABSTRACT FROM AUTHOR]

Quantum mechanics (QM)-driven 1Hiterative functionalized spin analysis produces HifSA profiles, which encode the complete 1H spin parameters (“nuclear genotype”) of analytes of interest. HifSA profiles enable the establishment of digital reference standards (dRS) that are portable, FAIR (findable - accessible - interoperable - reusable), and fit for the purpose of quantitative 1H NMR (qHNMR) analysis at any magnetic field. This approach enhances the sustainability of analytical standards. Moreover, the analyte-specific complete chemical shift and J-coupling information in HifSA-based dRS enable computational quantitation of substances in mixtures via QM-total-line-shape fitting (QM-qHNMR). We present the proof of concept for HifSA-based dRS by resolving the highly overlapping NMR resonances in the experimental spectra (“nuclear phenotypes”) of the diastereomeric mixture of (2RS, 4RS)- and (2RS, 4SR)-difenoconazole (DFZ), a widely used antifouling food additive. The underlying 1H spin parameters are highly conserved in various solvents, are robust against variation in measurement temperature, and work across a wide range of magnetic fields. QM-qHNMR analysis of DFZ samples at 80, 400, 600, and 800 MHz showed high congruence with metrological reference values. Furthermore, this study introduces QM-qHNMR combined with chiral shift reagents for the analysis of all four DFZ stereoisomers: (2R, 4R)-, (2S, 4S)-, (2R, 4S)-, and (2S, 4R)-DFZ to perform chiral qHNMR measurements.

In nature, proanthocyanidins (PACs) with A-type linkages are relatively rare, likely due to biosynthetic constraints in the formation of additional ether bonds to be introduced into the more common B-type precursors. However, A-type linkages confer greater structural rigidity on PACs than do B-type linkages. Prior investigations into the structure–activity relationships (SAR) describing how plant-derived PACs with B- and complex AB-type linkages affect their capacity for dentin biomodification indicate that a higher ratio of double linkages leads to a greater interaction with dentin type I collagen. Thus, A-type PACs emerge as particularly intriguing candidates for interventional functional biomaterials. This study employed a free-radical-mediated oxidation using DPPH to transform trimeric and tetrameric B-type PACs, 2and 4, respectively, into their exclusively A-type linked analogues, 3and 5, respectively. The structures and absolute configurations of the semisynthetic products, including the new all-A-type tetramer 5, were determined by comprehensive spectroscopic analysis. Additionally, molecular modeling investigated the conformational characteristics of all trimers and tetramers, 1–5. Our findings suggest that the specific interflavan linkages significantly impact the flexibility and low-energy conformations of the connected monomeric units, which conversely can affect the bioactive conformations relevant for dentin biomodification.

Systematic assessments of species extinction risk at regular intervals are necessary for informing conservation action1,2. Ongoing developments in taxonomy, threatening processes and research further underscore the need for reassessment3,4. Here we report the findings of the second Global Amphibian Assessment, evaluating 8,011 species for the International Union for Conservation of Nature Red List of Threatened Species. We find that amphibians are the most threatened vertebrate class (40.7% of species are globally threatened). The updated Red List Index shows that the status of amphibians is deteriorating globally, particularly for salamanders and in the Neotropics. Disease and habitat loss drove 91% of status deteriorations between 1980 and 2004. Ongoing and projected climate change effects are now of increasing concern, driving 39% of status deteriorations since 2004, followed by habitat loss (37%). Although signs of species recoveries incentivize immediate conservation action, scaled-up investment is urgently needed to reverse the current trends.

Given that the essence of Science is a search for the truth, one might expect that those identifying as scientists would be conscientious and observant of the demands this places on them. However, that expectation is not fulfilled universally as, not too surprisingly, egregious examples of unethical behavior appear and are driven by money, personal ambition, performance pressure, and other incentives. The reproducibility-, fact-, and truth-oriented modus operandiof Science has come to face a variety of challenges. Organized into 11 cases, this article outlines examples of compromised integrity from borderline to blatant unethical behavior that disgrace our profession unnecessarily. Considering technological developments in neural networks/artificial intelligence, a host of factors are identified as impacting Good Ethical Practices. The goal is manifold: to raise awareness and offer perspectives for refocusing on Science and true scientific evidence; to trigger discussion and developments that strengthen ethical behavior; to foster the recognition of the beauty, simplicity, and rewarding nature of scientific integrity; and to highlight the originality of intelligence.

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.

Monoclonal antibody–drug conjugates (ADCs) are an expanding therapeutic class of biomolecules for which relatively few analytical and preparative separation options exist. Purification of ADCs with a specific drug antibody ratio is even more challenging. We report the first application of countercurrent separation (CCS) to this problem. An ADC mimic was successfully chromatographed using an aqueous two-phase system (ATPS) consisting of PEG 1000/sodium citrate pH 7.5/water, 17.75/17.75/64.50 (w/w/w). Notably, different partition coefficients (K) in this ATPS for the ADC mimic (0.09 < K< 0.16) and its monoclonal antibody backbone, IgG (0.16 < K< 0.27), were observed using CCS. Differential elution behavior of such high-molecular-weight biomolecules, 146,441 vs. ∼150,000 Da, using CCS has no precedent. The results provide a proof of concept for further exploration of the application of ATPSs and CCS to the separation of ADCs.

Licorice, the roots and rhizomes of Glycyrrhiza glabraL., has been used as a medicinal herb, herbal adjuvant, and flavoring agent since ancient times. Recently, licorice extracts have become popular as dietary supplements used by females to alleviate menopausal symptoms. Exposure to licorice products containing high levels of glycyrrhizic acid can cause hypokalemia, but independent from this effect, preclinical data indicate that licorice can inhibit certain cytochrome P450 (P450) enzymes. To evaluate whether clinically relevant pharmacokinetic interactions of licorice with P450 enzymes exist, a phase 1 clinical investigation was carried out using a licorice extract depleted in glycyrrhizic acid (content <1%) and a cocktail containing caffeine, tolbutamide, alprazolam, and dextromethorphan, which are probe substrates for the enzymes CYP1A2, CYP2C9, CYP3A4/5, and CYP2D6, respectively. The botanically authenticated and chemically standardized extract of roots from G. glabrawas consumed by 14 healthy menopausal and postmenopausal female participants twice daily for 2 weeks. The pharmacokinetics of each probe drug were evaluated immediately before and after supplementation with the licorice extract. Comparison of the average areas under the time-concentration curves (AUCs) for each probe substrate in serum showed no significant changes from licorice consumption, whereas time to reach peak concentration for caffeine and elimination half-life for tolbutamide showed small changes. According to the US Food and Drug Administration guidance, which is based on changes in the AUC of each probe substrate drug, the investigated licorice extract should not cause any clinically relevant pharmacokinetic interactions with respect to CYP3A4/5, CYP2C9, CYP2D6, or CYP1A2.SIGNIFICANCE STATEMENTDespite generally-recognized-as-safe status, the licorice species Glycyrrhiza glabrahas been associated with some toxicity. Preclinical studies suggest that G. glabramight cause pharmacokinetic drug interactions by inhibiting several cytochrome P450 enzymes. This phase 1 clinical study addressed these concerns by evaluating clinically relevant effects with respect to CYP3A4/5, CYP2C9, CYP2D6, and CYP1A2. These results showed that a standardized G. glabraextract did not cause any clinically relevant pharmacokinetic drug interactions with four major cytochrome P450 enzymes.

Investigation of a pine bark extract for bioactive proanthocyanidin oligomers resulted in the isolation of structurally related dimeric secoB-type procyanidin derivatives, 1–5. This includes scalemic mixtures of gambiriin A1 (1a) and A2 (2a) and their newly described optical antipodes, ent-gambiriin A1 (1b) and ent-gambiriin A2 (2b), respectively, as well as a racemic mixture of the newly described (ent-)gambiriin A5 (3a/3b). Furthermore, the study now fully characterizes the previously reported optically pure dimers gambiriin B1 (4) and gambirflavan D1 (5), and characterized the novel secoB-type procyanidin trimer, 6(gambirifuran C1). Thermal conversion of catechin in aqueous solution provided further evidence for the structures of 1–6and led to the purification of semisynthetic 1aand 2aas well as additional dimers 7–10. Elucidating the structures of the natural dimers, 1–5, from comprehensive NMR and ECD data and synthetic evidence provided crucial reference points for establishing the structure of the secoB-type procyanidin trimer, 6. Serving as assigned building blocks, data from the dimers supported the 3D structural assignment of 6based on NMR substituent chemical shift differences (s.c.s., syn. ΔδC) and component-based empirical ECD calculations. Within the newly characterized series of PAC-related molecules, 5exhibited high dentin biomodification potential. In addition, considering the nomenclature issues and plausible biosynthetic pathways of this group of compounds led to a consolidated nomenclature of all currently known secoB-type procyanidins. These findings, thereby, expand the chemical space of bioactive catechin oligomers, which have promise as agents for the natural enhancement of dental biomaterials. Finally, the current knowledge of the chemical space of secoB-type procyanidin derivatives was compiled to the level of absolute configuration.

Four new rufomycins, compounds 1–4, named rufomycins 56, 57, 58, and 61, respectively, exhibiting new skeletal features, were obtained from Streptomyces atratusstrain MJM3502 and were fully characterized. Compounds 1and 2possess a 4-imidazolidinone ring not previously encountered in this family of cyclopeptides, thereby resulting in a [5,17] bicyclic framework. The in vitro anti-Mycobacterium tuberculosispotency of compounds 3and 4is remarkable, with minimum inhibitory concentration values of 8.5 and 130 nM, respectively.

Objective: To compare the clinical effects of needling interventions eliciting local twitch responses (LTRs) versus needling without eliciting LTRs when applied to muscle trigger points (TrPs) associated with spinal pain of musculoskeletal origin.Databases and data treatment: Electronic databases were searched for randomized or non-randomized clinical trials where one group received needling intervention where LTRs were elicited and was compared with another group receiving the same intervention without elicitation of LTRs in spinal pain disorders associated with TrPs. Outcomes included pain intensity, pain-related disability, and pressure pain thresholds. The risk of bias (RoB) was assessed using the Cochrane risk of bias tool or ROBINS-I tool, methodological quality was assessed with the PEDro score, and quality of evidence was evaluated using the GRADE approach.Results: Six trials were included. The application of a needling intervention eliciting LTRs was associated with a significant reduction in pain intensity immediately after treatment (mean difference (MD): −2.03 points, 95% confidence interval (CI): −3.77 to −0.29; standardized MD (SMD): −1.35, 95% CI: −2.32 to −0.38, p= 0.02) when compared to the same needling intervention without elicitation of LTRs. No effect at short-term follow-up (MD: −0.20 points, 95% CI: −1.46 to 1.06, p= 0.75) was observed. No significant differences based on elicitation or non-elicitation of LTRs were found in related disability (SMD: −0.05, 95% CI: −0.41 to 0.30, p= 0.77) or pressure pain thresholds (MD: 23.39 kPa, 95% CI: −13.68 to 60.47, p= 0.22).Discussion: Low-level evidence suggests an immediate effect of obtaining LTRs during needling interventions on pain intensity, with no significant effects on related disability or pressure pain sensitivity in spinal pain disorders associated with muscle TrPs.Registration number: OSF Registry—https://doi.org/10.17605/OSF.IO/5ZX9N

The University of Illinois at Chicago/National Institutes of Health Center for Botanical Dietary Supplements Research began in 1999 with an emphasis on botanical dietary supplements for women's health. We have concentrated on plants that may improve women's health, especially to reduce hot flashes in menopausal women, alleviate the symptoms of premenstrual syndrome, and reduce persistent urinary tract infections. The primary focus of this article is to describe the operation of our center, from acquiring and identifying botanicals to isolating and identifying active constituents, to elucidating their mechanisms of action, and to conducting phase I and phase II clinical studies. Black cohosh (Actaea racemosa; syn Cimicifuga racemosa) has been used as a model to illustrate the steps involved in taking this plant from the field to clinical trials. Bioassays are described that were necessary to elucidate the pertinent biological studies of plant extracts and their mechanisms of action. We conclude that this type of research can only be successful with the use of a multidisciplinary approach. KEY WORDS Black cohosh, botanical dietary supplements, menopause, multidisciplinary research

Much confusion exists about the chemical composition of widely sold Cannabis sativaproducts that utilize the cannabidiol (CBD) acronym and related terms such as “CBD oil”, “CBD plus hemp oil”, “full spectrum CBD”, “broad spectrum CBD”, and “cannabinoids”. Their rational chemical and subsequent biological assessment requires both knowledge of the chemical complexity and the characterization of significant individual constituents. Applicable to hemp preparations in general, this study demonstrates how the combination of liquid–liquid-based separation techniques, NMR analysis, and quantum mechanical-based NMR interpretation facilitates the process of natural product composition analysis by allowing specific structural characterization and absolute quantitation of cannabinoids present in such products with a large dynamic range. Countercurrent separation of a commercial “CBD oil” yielded high-purity CBD plus a more polar cannabinoid fraction containing cannabigerol and cannabidivarin, as well as a less polar cannabinoid fraction containing cannabichromene, trans-Δ9-tetrahydrocannabinol, cis-Δ9-tetrahydrocannabinol, and cannabinol. Representatives of six cannabinoid classes were identified within a narrow range of polarity, which underscores the relevance of residual complexity in biomedical research on cannabinoids. Characterization of the individual components and their quantitation in mixed fractions were undertaken by TLC, HPLC, 1H (q)NMR spectroscopy, 1H iterative full spin analysis (HiFSA), 13C NMR, and 2D NMR. The developed workflow and resulting analytical data enhance the reproducible evaluation of “CBD et al.” products, which inevitably represent complex mixtures of varying molecular populations, structures, abundances, and polarity features.

To enable the further exploration of structure–activity relationships (SARs) of proanthocyanidins (PACs) with dentin biomodification abilities, Cinnamomum verumwas selected for scaled-up purification of mixed A-/B-type, medium-size PAC oligomers. Sequential purification by centrifugal partition chromatography (CPC), Sephadex LH-20, and semiprep HPLC chromatography yielded four underivatized tetrameric (5–8) and two pentameric (9–10) PACs. Their unambiguous structural characterization involved extensive spectral and chemical degradation approaches to show that epicatechin units are connected by plant-specific combinations of doubly linked A- and singly linked B-type interflavanyl bonds. The biomechanical properties (via dynamic mechanical analysis) and physicochemical structure (via infrared spectroscopy) were assessed to evaluate the biomodification potency of PAC-treated collagen in a preclinical dentin model. This study revealed that (4→8) versus (4→6) bonds in PAC interflavan linkages have limited influence on biomechanical outcomes of dentin. By exhibiting a 25-fold increase in the complex modulus of treated dentin compared to control, aesculitannin E (5) was found to be the most potent PAC known to date for enhancing the mechanical properties of dentin in this preclinical model.

Rufomycin and ilamycin are synonymous for the same class of cyclopeptides, currently encompassing 33 structurally characterized isolates and 9 semisynthetic derivatives. Elucidation of new structures prioritized the consolidation of the names and established the structures of four diastereoisomeric rufomycins with a 2-piperidinone, named rufomycins 4–7, including full 1H/13C NMR assignments. The characteristic HSQC cross-peak for the CH-5, the hemiaminal carbon in amino acid #5, allows assignment of the stereocenters C-4 and C-5 within this ring. Semisynthetic derivatives (rufomycinSS 1, 2, and 3) were prepared from a rufomycins 4 and 6 mixture to validate the structural assignments. Based on the X-ray crystal structures of rufomycins 2 and 4, considering the NMR differences of rufomycins 7 vs 4–6 compared to rufomycinSS 1 vs 2 and 3, and taking into account that two major conformers, A and B, occur in both rufomycinSS 2 and 3, structural modeling was pursued. Collectively, this paper discusses the NMR spectroscopic differences of the stereoisomers and their possible 3D conformers and correlates these with the anti-Mycobacterium tuberculosisactivity. In addition, a look at the history prioritizes names and numbering schemes for this group of antibiotics and leads to consolidated nomenclature for all currently known members, natural and semisynthetic derivatives, and serves to accommodate future discoveries.

Objective: The aim of this study was to evaluate the effect of acupuncture/electroacupuncture, alone or combined with other interventions, on pain intensity, pain-related disability, and strength in lateral epicondylalgia (LE) of musculoskeletal origin.Databases and data treatment: Electronic databases were searched for randomized clinical trials, where at least one group received acupuncture or electroacupuncture for LE of musculoskeletal origin. To be eligible, trials had to include humans and collect outcomes on pain intensity or pain-related disability in LE. Data were extracted by two reviewers. The risk of bias (RoB) of the trials was assessed using the Cochrane RoB tool, methodological quality was assessed with the Physiotherapy Evidence Database (PEDro) score, and the level of evidence was summarized using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE). Standardized mean differences (SMDs) using random effects were calculated.Results: A total of 14 trials (10 acupuncture) were included. The meta-analysis found a moderate effect size of acupuncture (SMD = −0.66, 95% confidence interval (CI) = −1.22 to −0.10), but not electroacupuncture (SMD = −0.08, 95% CI = −0.99 to 0.83), in the reduction of elbow pain as compared to a comparative group. Acupuncture exhibited a significant moderate effect size (SMD = −0.51, 95% CI = −0.91 to −0.11) in the improvement of related-disability. Acupuncture (SMD = 0.36, 95% CI = 0.16 to 0.57), but not electroacupuncture (SMD = 0.34, 95% CI = −0.29 to 0.98), exhibited a significant but small effect size on strength. Most significant effects were in the short term. The RoB was low but the heterogeneity of trial results led to a downgrading of the GRADE evidence level.Conclusion: Low-level evidence suggests positive effects of acupuncture, but not electroacupuncture, for pain, related-disability, and strength, in LE of musculoskeletal origin, in the short term.

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]

The goal of the qNMR Summit is to take stock of the status quo and the recent developments in qNMR research and applications in a timely and accurate manner. It provides a platform for both advanced and novice qNMR practitioners to receive a well-rounded update and discuss potential qNMR-related applications and collaborations. For over a decade, scientists from academia, industry, nonprofit institutions, and governmental bodies have focused on the standardization of qNMR methodology, as well as its metrological and pharmacopeial utility. This paper reviews key content of qNMR Summits 1.0 to 4.0 and puts into perspective the outcomes and available transcripts of the October 2019 Summit 5.0, with attendees from the United States, Canada, Japan, Korea, and several European countries. Summit presentations focused on qNMR methodology in the pharmaceutical industry, advanced quantitation algorithms, and promising developments.

Two new diprenylated coumaric acid isomers (1aand 1b) and two known congeners, capillartemisin A (2) and B (3), were isolated from Artemisia scopariaas bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR methods and LC-MS, with their purity assessed by 1D 1H pure shift qNMR spectroscopic analysis. The bioactivity of compounds was evaluated by enhanced accumulation of lipids, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the plant’s 80% EtOH extract, these purified compounds showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This suggests that additional bioactive substances are responsible for the multiple metabolically favorable effects on adipocytes observed with Artemisia scopariaextract.

Dentin endogenous proteases are involved in the progression of dental caries. This study investigated the proteolytic activity of endogenous enzymes of carious and sound dentin of primary teeth and the potential selective inhibitory effect of plant-derived proanthocyanidins (PACs).

Curcuma longa(turmeric) has an extensive history of ethnomedical use for common ailments, and “curcumin”-containing dietary supplements (CDS) are a highly visible portion of today’s self-medication market. Owing to raw material cost pressure, CDS products are affected by economically motivated, nefarious adulteration with synthetic curcumin (“syncumin”), possibly leading to unexpected toxicological issues due to “residual” impurities. Using a combination of targeted and untargeted (phyto)chemical analysis, this study investigated the botanical integrity of two commercial “turmeric” CDS with vitamin and other additives that were associated with reported clinical cases of hepatotoxicity. Analyzing multisolvent extracts of the CDS by 100% quantitative 1H NMR (qHNMR), alone and in combination with countercurrent separation (CCS), provided chemical fingerprints that allowed both the targeted identification and quantification of declared components and the untargeted recognition of adulteration. While confirming the presence of curcumin as a major constituent, the universal detection capability of NMR spectroscopy identification of significant residual impurities, including potentially toxic components. While the loss-free nature of CCS captured a wide polarity range of declared and unwanted chemical components, and also increased the dynamic range of the analysis, (q)HNMR determined their mass proportions and chemical constitutions. The results demonstrate that NMR spectroscopy can recognize undeclared constituents even if they represent only a fraction of the mass balance of a dietary supplement product. The chemical information associated with the missing 4.8% and 7.4% (m/m) in the two commercial samples, exhibiting an otherwise adequate curcumin content of 95.2% and 92.6%, respectively, pointed to a product integrity issue and adulteration with undeclared synthetic curcumin. Impurities from synthesis are most plausibly the cause of the observed adverse clinical effects. The study exemplifies how the simultaneously targeted and untargeted analytical principle of the 100% qHNMR method, performed with entry-level high-field instrumentation (400 MHz), can enhance the safety of dietary supplements by identifying adulterated, non-natural “natural” products.

Quantitative 1H nuclear magnetic resonance (qHNMR) is a highly regarded analytical methodology for purity determination as it balances metrological rigor, practicality, and versatility well. While ideal for intrinsically mass-limited samples, external calibration (EC) qHNMR is overshadowed by the prevalence of internal calibration and perceived rather than real practical limitations. To overcome this hurdle, this study applied the principle of reciprocity, certified reference materials (caffeine as analyte, dimethyl sulfone as calibrant), and a systematic evaluation of data acquisition workflows to extract key factors for the achievement of accuracy and precision in EC-qHNMR. Automatic calibration of the 90° pulse width (90 PW) formed the foundation for the principle of reciprocity and used optimized nutation experiments, showing good agreement with values derived from manual high-precision measurement of 360 PW. Employing the automatic 90 PW calibration, EC-qHNMR with automatic vs manual tuning and matching (T&M) yielded the certified purity value within 1% error. The timing of T&M (before vs after shimming) turned out to be critically important: sufficient time is required to achieve full-temperature equilibrium relative to thermal gradients in the air inside the probe and the sample. Achievable accuracy across different NMR solvents varies with differences in thermal conductivity and leads to 2% or greater errors. With matching solvents, the demonstrated accuracy of ∼1.0% underscores the feasibility of EC-qHNMR as a highly practical research tool.

The seeds of the akuamma tree (Picralima nitida) have been used as a traditional treatment for pain and fever. Previous studies have attributed these effects to a series of indole alkaloids found within the seed extracts; however, these pharmacological studies were significantly limited in scope. Herein, an isolation protocol employing pH-zone-refining countercurrent chromatography was developed to provide six of the akuamma alkaloids in high purity and quantities sufficient for more extensive biological evaluation. Five of these alkaloids, akuammine (1), pseudo-akuammigine (3), akuammicine (4), akuammiline (5), and picraline (6), were evaluated against a panel of >40 central nervous system receptors to identify that their primary targets are the opioid receptors. Detailed in vitro investigations revealed 4to be a potent kappa opioid receptor agonist, and three alkaloids (1–3) were shown to have micromolar activity at the mu opioid receptor. The mu opioid receptor agonists were further evaluated for analgesic properties but demonstrated limited efficacy in assays of thermal nociception. These findings contradict previous reports of the antinociceptive properties of the P. nitidaalkaloids and the traditional use of akuamma seeds as analgesics. Nevertheless, their opioid-preferring activity does suggest the akuamma alkaloids provide distinct scaffolds from which novel opioids with unique pharmacologic properties and therapeutic utility can be developed.

Proanthocyanidins (PACs) are near-ubiquitous and chemically complex metabolites, prototypical of higher plants. Their roles in food/feed/nutrition and ethnomedicine are widely recognized but poorly understood. With the analysis of evidence that underlies this challenge, this perspective identifies shortcomings in capturing and delineating PAC structures as key factors. While several groups have forwarded new representations, a consensus method that captures PAC structures concisely and offers high integrity for electronic storage is required to reduce confusion in this expansive field. The PAC block arrays (PACBAR) system fills this gap by providing precise and human- and machine-readable structural descriptors that capture PAC metabolomic structural diversity. PACBAR enables communication of PAC structures for the development of precise structure–activity relationships and will assist in advancing PAC research to the next level.

The present study elucidated the structures of three A-type tri- and tetrameric proanthocyanidins (PACs) isolated from Cinnamomum verumbark to the level of absolute configuration and determined their dental bioactivity using two therapeutically relevant bioassays. After selecting a PAC oligomer fraction via a biologically diverse bioassay-guided process, in tandem with centrifugal partition chromatography, phytochemical studies led to the isolation of PAC oligomers that represent the main bioactive principles of C. verum: two A-type tetrameric PACs, epicatechin-(2β→O→7,4β→8)-epicatechin-(4β→6)-epicatechin-(2β→O→7,4β→8)-catechin (1) and parameritannin A1 (2), together with a trimer, cinnamtannin B1 (3). Structure determination of the underivatized proanthocyanidins utilized a combination of HRESIMS, ECD, 1D/2D NMR, and 1H iterative full spin analysis data and led to NMR-based evidence for the deduction of absolute configuration in constituent catechin and epicatechin monomeric units.

Extracts of red clover (Trifolium pratenseL.), containing estrogenic isoflavones like genistein and daidzein and the proestrogenic isoflavones formononetin and biochanin A, are used by women as dietary supplements for the management of menopausal symptoms. Although marketed as a safer alternative to hormone therapy, red clover isoflavones have been reported to inhibit some cytochrome P450 (CYP) enzymes involved in drug metabolism. To evaluate the potential for clinically relevant drug–red clover interactions, we tested a standardized red clover dietary supplement (120 mg isoflavones per day) for interactions with the pharmacokinetics of four FDA-approved drugs (caffeine, tolbutamide, dextromethorphan, and alprazolam) as probe substrates for the enzymes CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5, respectively. Fifteen peri- and postmenopausal women completed pharmacokinetic studies at baseline and 2 weeks after consuming red clover. The averaged pharmacokinetic profiles of probe substrates in serum showed no significant alterations and no changes in the areas under the curve (AUC) over 96 h. Subgroup analysis based on the demographic characteristics (BMI, menopausal status, race, and age) also showed no differences in AUC for each probe substrate. Analysis of red clover isoflavones in serum showed primarily conjugated metabolites that explain, at least in part, the red clover pharmacokinetic safety profile.

This Perspective of the published essential medicinal chemistry of cannabidiol (CBD) provides evidence that the popularization of CBD-fortified or CBD-labeled health products and CBD-associated health claims lacks a rigorous scientific foundation. CBD’s reputation as a cure-all puts it in the same class as other “natural” panaceas, where valid ethnobotanicals are reduced to single, purportedly active ingredients. Such reductionist approaches oversimplify useful, chemically complex mixtures in an attempt to rationalize the commercial utility of natural compounds and exploit the “natural” label. Literature evidence associates CBD with certain semiubiquitous, broadly screened, primarily plant-based substances of undocumented purity that interfere with bioassays and have a low likelihood of becoming therapeutic agents. Widespread health challenges and pandemic crises such as SARS-CoV-2 create circumstances under which scientists must be particularly vigilant about healing claims that lack solid foundational data. Herein, we offer a critical review of the published medicinal chemistry properties of CBD, as well as precise definitions of CBD-containing substances and products, distilled to reveal the essential factors that impact its development as a therapeutic agent.

The use of microorganisms in industrial fermentations requires robust strains tolerant to stresses that challenge its performance during the bioprocess. One approach to obtain such a strain, adaptive evolution methodology, is carried out in this work with an emphasis on the biochemistry of stress tolerance. This work evaluated the robustness and cellulosic ethanol efficiency of an evolutionary adapted strain ofScheffersomyces stipitis NRRL Y-7124 (HAJ) obtained after successive batch cultures with increasing concentrations of acid hydrolysate lignocellulosic jojoba residue. Strain robustness was associated with its ability to tolerate stresses present along an industrial cellulosic bioethanol production process (i.e., thermal, oxidative or osmotic stress; high concentration of ethanol or phenolic compounds). Under such conditions, HAJ exhibited 4-fold higher viability and 8-fold higher vitality (metabolic performance) values than the parental strain. Whereas all stresses assayed produced a significant increase in reactive oxygen species (ROS) concentrations in Y-7124 (up to 15-fold higher than controls), in HÁJ only ethanol induced a significant rise in ROS levels, associated to variations in superoxide dismutase (SOD) and catalase (CAT) enzymatic activities. The highest increase in SOD activity was associated with ethanol stress, the most oxidative stress assayed, being 3.5-fold higher in HAJ versus Y-7124. Intracellular concentrations of cell protectants trehalose and glycogen increased significantly after stresses related to hydric deficiencies (sorbitol and ethanol), with HAJ showing a higher increase than the parental strain. Ethanol production efficiency on a non-detoxified, non-supplemented acid-hydrolyzed lignocellulosic medium was 40% higher for HAJ versus Y-7124. Our results propose that stress cross-tolerance of this yeast is associated to its oxidative stress tolerance, and that high levels of molecules like trehalose should be a goal for obtaining a robust strain that can be used industrially.

Many botanicals used for women’s health contain estrogenic (iso)flavonoids. The literature suggests that estrogen receptor beta (ERβ) activity can counterbalance estrogen receptor alpha (ERα)-mediated proliferation, thus providing a better safety profile. A structure–activity relationship study of (iso)flavonoids was conducted to identify ERβ-preferential structures, overall estrogenic activity, and ER subtype estrogenic activity of botanicals containing these (iso)flavonoids. Results showed that flavonoids with prenylation on C8 position increased estrogenic activity. C8-prenylated flavonoids with C2–C3 unsaturation resulted in increased ERβ potency and selectivity [e.g., 8-prenylapigenin (8-PA), EC50(ERβ): 0.0035 ± 0.00040 μM], whereas 4′-methoxy or C3 hydroxy groups reduced activity [e.g., icaritin, EC50(ERβ): 1.7 ± 0.70 μM]. However, nonprenylated and C2–C3 unsaturated isoflavonoids showed increased ERβ estrogenic activity [e.g., genistein, EC50(ERβ): 0.0022 ± 0.0004 μM]. Licorice (Glycyrrhiza inflata, [EC50(ERα): 1.1 ± 0.20; (ERβ): 0.60 ± 0.20 μg/mL], containing 8-PA, and red clover [EC50(ERα): 1.8 ± 0.20; (ERβ): 0.45 ± 0.10 μg/mL], with genistein, showed ERβ-preferential activity as opposed to hops [EC50(ERα): 0.030 ± 0.010; (ERβ): 0.50 ± 0.050 μg/mL] and Epimedium sagittatum[EC50(ERα): 3.2 ± 0.20; (ERβ): 2.5 ± 0.090 μg/mL], containing 8-prenylnaringenin and icaritin, respectively. Botanicals with ERβ-preferential flavonoids could plausibly contribute to ERβ-protective benefits in menopausal women.

An investigation of the dental bioactive proanthocyanidin (PAC) oligomer fractions led to three structurally distinct new PACs (1–3) from pine bark. Pinutwindoublin (1) is the first reported trimer with double A-type interflavanyl linkages (2α→O→5,4α→6 and 2α→O→7,4α→8). Pinuspirotetrin (2) represents the first reported PAC tetramer with a heterodimeric framework consisting of one spiro-type and one A-type dimer. Pinumassohexin (3) was elucidated as a mixed A + B-type hexamer that consists of a peanut-derived tetramer, peanut procyanidin E, and an A-type dimer (5). Compound 3increased the modulus of elasticity of dentin by an impressive 4.3 times at a concentration of 0.65%.

Guided by dentin biomechanical bioactivity, this phytochemical study led to the elucidation of an extended set of structurally demanding proanthocyanidins (PACs). Unambiguous structure determination involved detailed spectroscopic and chemical characterization of four A-type dimers (2and 4–6), seven trimers (10–16), and six tetramers (17–22). New outcomes confirm the feasibility of determining the absolute configuration of the catechol monomers in oligomeric PACs by one-dimensional (1D) and two-dimensional (2D) NMR. Electronic circular dichroism as well as phloroglucinolysis followed by mass spectrometry and chiral phase high-performance liquid chromatography (HPLC) analysis generated the necessary chiral reference data. In the context of previously reported dentin-bioactive PACs, accurately and precisely assigned 13C NMR resonances enabled absolute stereochemical assignments of PAC monomers via (i) inclusion of the 13C NMR γ-gauche effect and (ii) determination of differential 13C chemical shift values (ΔδC) in comparison with those of the terminal monomer (unit II) in the dimers 2and 4–6. Among the 13 fully elucidated PACs, eight were identified as new, and one structure (11) was revised based on new knowledge gained regarding the subtle, stereospecific spectroscopic properties of PACs.

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.

Chlorophylls are present in all extracts from the aerial parts of green plant materials. Chlorophylls may act as in vitro bioassay nuisance compounds, possibly preventing the reproducibility and accurate measurement of readouts due to their UV/vis absorbance, fluorescence properties, and tendency to precipitate in aqueous media. Despite the diversity of methods used traditionally to remove chlorophylls, details about their mode of operation, specificity, and reproducibility are scarce. Herein, we report a selective and efficient 45 min liquid–liquid/countercurrent chlorophyll cleanup method using Centrifugal Partition Chromatography (CPC) with a solvent system composed of hexanes–EtOAc–MeOH–water (5:5:5:5, v/v) in elution-extrusion mode. The broader utility of the method was assessed with four different extracts prepared from three well-characterized plant materials: Epimedium sagittatum(leaves), Senna alexandrina(leaves), and Trifolium pratense(aerial parts). The reproducibility of the method, the selectivity of the chlorophyll removal, as well as the preservation of the phytochemical integrity of the resulting chlorophyll-free (“degreened”) extracts were evaluated using HPTLC, UHPLC-UV, 1H NMR spectroscopy, and LC-MS as orthogonal phytochemical methods. The cleanup process adequately preserves the metabolomic diversity as well as the integrity of the original extracts. This method was found to be sufficiently rapid for the “degreening” of botanical extracts in higher-throughput sample preparation for further biological screening.

Botanical dietary supplements produced from hops (Humulus lupulus) containing the chemopreventive compound xanthohumol and phytoestrogen 8-prenylnaringenin are used by women to manage menopausal symptoms. Because of the long half-lives of prenylated hop phenols and reports that they inhibit certain cytochrome P450 enzymes, a botanically authenticated and chemically standardized hop extract was tested for Phase I pharmacokinetic drug interactions. Sixteen peri- and postmenopausal women consumed the hop extract twice daily for 2 weeks, and the pharmacokinetics of tolbutamide, caffeine, dextromethorphan, and alprazolam were evaluated before and after supplementation as probe substrates for the enzymes CYP2C9, CYP1A2, CYP2D6, and CYP3A4/5, respectively. The observed area under the time–concentration curves were unaffected, except for alprazolam which decreased 7.6% (564.6 ± 46.1 h·μg/L pre-hop and 521.9 ± 36.1 h·μg/L post-hop; p-value 0.047), suggesting minor induction of CYP3A4/5. No enzyme inhibition was detected. According to FDA guidelines, this hop dietary supplement caused no clinically relevant pharmacokinetic interactions with respect to CYP2C9, CYP1A2, CYP2D6, or CYP3A4/5. The serum obtained after consumption of the hop extract was analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry to confirm compliance. Abundant Phase II conjugates of the hop prenylated phenols were observed including monoglucuronides and monosulfates as well as previously unreported diglucuronides and sulfate-glucuronic acid diconjugates.

Utilizing the distinct HMBC cross-peak patterns of lower-field range (LFR; 11.80–14.20 ppm) hydroxyl singlets, presented NMR methodology characterizes flavonoid metabolomes both qualitatively and quantitatively. It enables simultaneous classification of the structural types of 5-OH flavonoids and biogenetically related 2′-OH chalcones, as well as quantification of individual metabolites from 1H NMR spectra, even in complex mixtures. Initially, metabolite-specific LFR 1D 1H and 2D HMBC patterns were established via literature mining and experimental data interpretation, demonstrating that LFR HMBC patterns encode the different structural types of 5-OH flavonoids/2′-OH chalcones. Taking advantage of the simplistic multiplicity of the H,H-uncoupled LFR 5-/2′-OH singlets, individual metabolites could subsequently be quantified by peak fitting quantitative 1H NMR (PF-qHNMR). Metabolomic analysis of enriched fractions from three medicinal licorice (Glycyrrhiza) species established proof-of-concept for distinguishing three major structural types and eight subtypes in biomedical applications. The method identified 15 G. uralensis(GU) phenols from the six possible subtypes of 5,7-diOH (iso)flav(an)ones with 6-, 8-, and nonprenyl substitution, including the new 6-prenyl-licoisoflavanone (1) and two previously unknown compounds (4and 7). Relative (100%) qNMR established quantitative metabolome patterns suitable for species discrimination and plant metabolite studies. Absolute qNMR with combined external and internal (solvent) calibration (ECIC) identified and quantified 158 GU metabolites. HMBC-supported qHNMR analysis of flavonoid metabolomes (“flavonomics”) empowers the exploration of structure–abundance–activity relationships of designated bioactivity. Its ability to identify and quantify numerous metabolites simultaneously and without identical reference materials opens new avenues for natural product discovery and botanical quality control and can be adopted to other flavonoid- and chalcone-containing taxa.

This study represents a systematic chemical and biological study of the rufomycin (RUF) class of cyclic heptapeptides, which our anti-TB drug discovery efforts have identified as potentially promising anti-TB agents that newly target the caseinolytic protein C1, ClpC1. Eight new RUF analogues, rufomycins NBZ1–NBZ8 (1–8), as well as five known peptides (9–13) were isolated and characterized from the Streptomyces atratusstrain MJM3502. Advanced Marfey’s and X-ray crystallographic analysis led to the assignment of the absolute configuration of the RUFs. Several isolates exhibited potent activity against both pathogens M. tuberculosisH37Rv and M. abscessus, paired with favorable selectivity (selectivity index >60), which collectively underscores the promise of the rufomycins as potential anti-TB drug leads.

Distal renal tubular acidosis is a rare renal tubular disorder characterized by hyperchloremic metabolic acidosis and impaired urinary acidification. Mutations in three genes (ATP6V0A4, ATP6V1B1and SLC4A1)constitute a monogenic causation in 58-70% of familial cases of distal renal tubular acidosis. Recently, mutations in FOXI1have been identified as an additional cause. Therefore, we hypothesized that further monogenic causes of distal renal tubular acidosis remain to be discovered. Panel sequencing and/or whole exome sequencing was performed in a cohort of 17 families with 19 affected individuals with pediatric onset distal renal tubular acidosis. A causative mutation was detected in one of the three “classical” known distal renal tubular acidosis genes in 10 of 17 families. The seven unsolved families were then subjected to candidate whole exome sequencing analysis. Potential disease causing mutations in three genes were detected: ATP6V1C2, which encodes another kidney specific subunit of the V-type proton ATPase (1 family); WDR72(2 families), previously implicated in V-ATPase trafficking in cells; and SLC4A2(1 family),a paralog of the known distal renal tubular acidosis gene SLC4A1.Two of these mutations were assessed for deleteriousness through functional studies. Yeast growth assays for ATP6V1C2revealed loss-of-function for the patient mutation, strongly supporting ATP6V1C2as a novel distal renal tubular acidosis gene. Thus, we provided a molecular diagnosis in a known distal renal tubular acidosis gene in 10 of 17 families (59%) with this disease, identified mutations in ATP6V1C2as a novel human candidate gene, and provided further evidence for phenotypic expansion in WDR72mutations from amelogenesis imperfecta to distal renal tubular acidosis.

Botanical dietary supplements (BDS) containing hops are sold as women’s health supplements due to the potent hop phytoestrogen, 8-prenylnaringenin (8-PN), and the cytoprotective chalcone, xanthohumol. Previous studies have shown a standardized hop extract to beneficially influence chemical estrogen carcinogenesis in vitroby fostering detoxified 2-hydroxylation over genotoxic 4-hydroxylation estrogen metabolism. In this study, hop extract and its bioactive compounds were investigated for its mechanism of action within the chemical estrogen carcinogenesis pathway, which is mainly mediated through the 4-hydroxylation pathway catalyzed by CYP1B1that can form gentoxic quinones. Aryl hydrocarbon receptor (AhR) agonists induce CYP1A1and CYP1B1, while estrogen receptor alpha (ERα) inhibits transcription of CYP1A1, the enzyme responsible for 2-hydroxylated estrogens and the estrogen detoxification pathway. An In-Cell Western MCF-7 cell assay revealed hop extract and 6-prenylnaringenin (6-PN) degraded ERα via an AhR-dependent mechanism. Reverse transcription PCR and xenobiotic response element luciferase assays showed hop extract and 6-PN-mediated activation of AhR and induction of CYP1A1. A reduction in estrogen-mediated DNA (cytosine-5)-methyltransferase 1 (DNMT1) downregulation of CYP1A1accompanied this activity in a chromatin immunoprecipitation assay. Ultimately, hop extract and 6-PN induced preferential metabolism of estrogens to their detoxified form in vitro. These results suggest that the standardized hop extract and 6-PN activate AhR to attenuate epigenetic inhibition of CYP1A1through degradation of ERα, ultimately increasing 2-hydroxylated estrogens. A new mechanism of action rationalizes the positive influence of hop BDS and 6-PN on oxidative estrogen metabolism in vitroand, thus, potentially on chemical estrogen carcinogenesis. The findings underscore the importance of elucidating various biological mechanisms of action and standardizing BDS to multiple phytoconstituents for optimal resilience promoting properties.

Natural products (NPs) continue to inform the discovery and development of a diversity of drugs, both marketed and investigational. Pain, one of the most common of human experiences and profound challenges in medicine and biology, has emerged at the core of an urgent societal problem, in the United States and globally. The present study employs a retrospective analysis of an extensive set of published literature curated in the NAPRALERT database to identify NPs with experimental evidence of bioactivity supporting the selection and prioritization of NP leads with promise in pain management. The NAPRALERT pain data set currently documents >38,000 pain-relevant experiments reported in >1,750 distinct journals. The evidence presented here was annotated from >10,000 distinct scientific publications identifying NP extracts and isolates with experimental biological data indicating positive mitigation of pain, inflammation, and/or modulation of nociceptive signaling targets. Correlation of ethnomedical uses with experimental data represents a value-added approach to the selection and prioritization of leads. Dissemination of this unique NP/pain data set, with experimental data and information applicable to basic, translational, and clinical science stakeholders alike, furnishes practical evidence in support of a rational selection of NPs for directed pain research. A large portion of the NAPRALERT pain-relevant data set, along with a set of query tools designed to assist user-directed selection and prioritization of leads, are presented as Supporting Information in order to mitigate the limitations inherent in presenting such a large data set in (print) format. To support user efforts, this report involves explication of NAPRALERT data organization and the articulation of rational approaches to user-guided selection of evidence-based NP leads.

The dichloromethane extract of the roots of Jatropha dioicaafforded riolozatrione (1) and a C-6 epimer of riolozatrione, 6-epi-riolozatrione (2), as a new structure and only the second reported riolozane diterpenoid. The two known diterpenoids jatrophatrione (3) and citlalitrione (4) were also isolated and characterized. Both epimers 1and 2are genuine plant constituents, with 2likely being the biosynthesis precursor of 1due to the tendency for the quantitative transformation of 2into 1under base catalysis. The structural characterization and distinction of the stereoisomers utilized 1H iterative full-spin analysis, yielding complete J-correlation maps that were represented as quantum interaction and linkage tables. The absolute configuration of compounds 1–4was established by means of vibrational circular dichroism and via X-ray diffraction analysis for 1, 2, and 4. Additionally, the cytotoxic and antiherpetic in vitro activities of the isolates were evaluated.

The characteristic signals observed in NMR spectra encode essential information on the structure of small molecules. However, extracting all of this information from complex signal patterns is not trivial. This report demonstrates how computer-aided spectral analysis enables the complete interpretation of 1D 1H NMR data. The effectiveness of this approach is illustrated with a set of organic molecules, for which replicas of their 1H NMR spectra were generated. The potential impact of this methodology on organic chemistry research is discussed.

Complementing classical drug discovery, phytochemicals act on multiple pharmacological targets, especially in botanical extracts, where they form complex bioactive mixtures. The reductionist approach used in bioactivity-guided fractionation to identify single bioactive phytochemicals is inadequate for capturing the full therapeutic potential of the (bio)chemical interactions present in such complex mixtures. This study used a DESIGNER (Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources) approach to selectively remove the known bioactives, 4′-O-methyldavidigenin (1; 4,2′-dihydroxy-4′-methoxydihydrochalcone, syn. DMC-1) and its isomer 4-O-methyldavidigenin (2; syn. DMC-2), from the mixture of phytochemicals in an ethanol extract from Artemisia dracunculusto determine to what degree the more abundant 2accounts for the established antidiabetic effect of the A. dracunculusextract. Using an otherwise chemically intact “knock-out extract” depleted in 2and its regioisomer, 1, in vitro and in vivo outcomes confirmed that 2(and likely 1) acts as major bioactive(s) that enhance(s) insulin signaling in skeletal muscle, but also revealed that 2does not account for the breadth of detectable biological activity of the extract. This is the first report of generating, at a sufficiently large preparative scale, a “knock-out extract” used as a pharmacological tool for in vitro and in vivo studies to dissect the biological impact of a designated bioactive in a complex phytochemical mixture.

Direct analyte-probed nanoextraction (DAPNe) is a technique that allows extraction of drug and endogenous compounds from a discrete location on a tissue sample using a nano capillary filled with solvent. Samples can be extracted from spot diameters as low as 6 μm. Studies previously undertaken by our group have shown that the technique can provide good precision (5%) for analyzing drug molecules in 150 μm diameter areas of homogenized tissue, provided an internal standard is sprayed on to the tissue prior to analysis. However, without an isotopically labeled standard, the repeatability is poor, even after normalization to the spot area or matrix compounds. By application to tissue homogenates spiked with drug compounds, we can demonstrate that it is possible to significantly improve the repeatability of the technique by incorporating a liquid chromatography separation step. Liquid chromatography is a technique for separating compounds prior to mass spectrometry (LC-MS) which enables separation of isomeric compounds that cannot be discriminated using mass spectrometry alone, as well as reducing matrix interferences. Conventionally, LC-MS is carried out on bulk or homogenized samples, which means analysis is essentially an average of the sample and does not take into account discrete areas. This work opens a new opportunity for spatially resolved liquid chromatography mass spectrometry with precision better than 20%.

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.

Numerous reports assigning (Z)-ligustilide (1) the role of a major bioactive principle in Apiaceae botanicals are called into question by the recurrent demonstrations of 1being an unstable, rapidly degrading compound, ultimately leading to dynamic residual complexity. While Angelica sinensisis recognized for its therapeutic value in (peri-)menopausal symptom management, its purported active principle, 1, represents a typical example of the instability–bioactivity chasm of botanicals. To help bridge the gap, this study used both the essential oil and purified 1obtained from A. sinensisto investigate the factors that influence the chemical transformation of 1, the products formed, and the rationale for monitoring 1in natural product preparations. Countercurrent separation was used to purify 1from a supercritical fluid extract of A. sinensis, achieving 93.4% purity in a single step. Subsequent purification by preparative HPLC afforded 1with a 98.0% purity. Providing a mass balance setting, we monitored chemical changes occurring to highly purified 1under various conditions and at different time points, in sealed NMR tubes by quantitative 1H NMR (qHNMR). The nondestructive nature of NMR enabled a comprehensive assessment of degradation products. Moreover, in being a mole-based determination, the total intensity (integral) of all NMR signals intrinsically represents the theoretical mass balance within the sample solution. The results demonstrated that 1is most stable while within the original plant material. Exposure to light had a profound impact on the chemical transformation of 1, leading to the formation of ligustilide dimers and trimers, as verified by both NMR and LC-HRMS studies. Moreover, the results shown for 1, augmented by other recent outcomes, have serious implications for the meaningful biological evaluation of NPs that exhibit instability/reactivity, while having a plethora of “promising” bioactivities reported in the literature and being frequently associated with unsubstantiated health claims.

Aimed at exploring the dentin biomodification potential of proanthocyanidins (PACs) for the development of dental biomaterials, this study reports the phytochemical and dental evaluation of nine B-type PACs from grape seed extract (GSE). Out of seven isolated dimers (1–7), four new compounds (2, 3, 5, and 6) involved relatively rare ent-catechin or ent-epicatechin monomeric flavan-3-ol units. Low-temperature NMR analyses conducted along with phloroglucinolysis and electronic circular dichroism enabled unequivocal structural characterization and stereochemical assignment. Additionally, one known (8) and one new (9) B-type trimer were characterized. Differential 13C NMR chemical shifts (Δδ) were used to determine the absolute configuration of 9, relative to the dimers 1and 2as the possible constituent subunits. Compared to the dimers, the trimers showed superior dentin biomodification properties. The dimers, 1–7, exhibited pronounced differences in their collagenase inhibitory activity, while enhancing dentin stiffness comparably. This suggests that PAC structural features such as the degree of polymerization, relative and absolute configuration have a differentialinfluence on enhancement of dentin biomechanical and biostability. As mechanical enhancement to dentin and resistance to proteolytic biodegradation are both essential properties functional and stable dentin substrate, the structurally closely related PACs suggest a new metric, the dentin biomodification potential (DBMP) that may rationalize both properties.

The suadimins A–C (1–3) from Melodinus suaveolensare the first example of monoterpenoid quinoline alkaloid dimers featuring an unprecedented carbon skeleton. Their structures and absolute configurations were established on the basis of extensive spectroscopic analyses, electric circular dichroism (ECD), and X-ray crystallography. Suadimin A showed significant antimycobacterial activity in vitro with an MIC90of 6.76 μM against the H37Rv strain of M. tuberculosis.