DESIGNER fraction concept unmasks minor bioactive constituents in red clover (Trifolium pratense L.).

In botanical extracts, highly abundant constituents can mask or dilute the effects of other, and often, more relevant biologically active compounds. To facilitate the rational chemical and biological assessment of these natural products with wide usage in human health, we introduced the DESIGNER approach of Depleting and Enriching Selective Ingredients to Generate Normalized Extract Resources. The present study applied this concept to clinical Red Clover Extract (RCE) and combined phytochemical and biological methodology to help rationalize the utility of RCE supplements for symptom management in postmenopausal women. Previous work has demonstrated that RCE reduces estrogen detoxification pathways in breast cancer cells (MCF-7) and, thus, may serve to negatively affect estrogen metabolism-induced chemical carcinogenesis. Clinical RCE contains ca. 30% of biochanin A and formononetin, which potentially mask activities of less abundant compounds. These two isoflavonoids are aryl hydrocarbon receptor (AhR) agonists that activate P450 1A1, responsible for estrogen detoxification, and P450 1B1, producing genotoxic estrogen metabolites in female breast cells. Clinical RCE also contains the potent phytoestrogen, genistein, that downregulates P450 1A1, thereby reducing estrogen detoxification. To identify less abundant bioactive constituents, countercurrent separation (CCS) of a clinical RCE yielded selective lipophilic to hydrophilic metabolites in six enriched DESIGNER fractions (DFs 01-06). Unlike solid-phase chromatography, CCS prevented any potential loss of minor constituents or residual complexity (RC) and enabled the polarity-based enrichment of certain constituents. Systematic analysis of estrogen detoxification pathways (ERα-degradation, AhR activation, CYP1A1/CYP1B1 induction and activity) of the DFs uncovered masked bioactivity of minor/less abundant constituents including irilone. These data will allow the optimization of RCE with respect to estrogen detoxification properties. The DFs revealed distinct biological activities between less abundant bioactives. The present results can inspire future carefully designed extracts with phytochemical profiles that are optimized to increase in estrogen detoxification pathways and, thereby, promote resilience in women with high-risk for breast cancer. The DESIGNER approach helps to establish links between complex chemical makeup, botanical safety and possible efficacy parameters, yields candidate DFs for (pre)clinical studies, and reveals the contribution of minor phytoconstituents to the overall safety and bioactivity of botanicals, such as resilience promoting activities relevant to women's health.

Geraniol-Derived Monoterpenoid Glucosides from Rhodiola rosea: Resolving Structures by QM-HifSA Methodology.

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 1 and 2 exhibited 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. rosea extract. Moreover, the three previously undescribed geranioloids 3-5 were 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.

Development of Centrifugal Partition Chromatography for the Purification of Antibody-Drug Conjugates.

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.

Pharmaceutical analysis by NMR can accommodate strict impurity thresholds: The case of choline.

The ICH guidelines recommend reporting thresholds for regular impurities in drug substances at the level of 0.05% or 0.03% (w/w) depending on the maximum daily intake. Therefore, any instrumental method of analysis applicable to the impurity analysis should be able to detect and quantify the analytes at those levels. This investigation was designed to verify the suitability of 1H NMR spectroscopy for the detection of impurities, as a first step in the process before attempting quantification. In order to minimize demand on equipment, this study employed a 400 MHz instrument for structural confirmation and signal assignments of choline (1) and O-(2-hydroxyethyl)choline (2), a known impurity. The limit of detection (LOD) of 2 in 10 mg of 1 was established as 0.01% on a 400 MHz instrument and 2% on a 60 MHz (benchtop) NMR spectrometer. Thus, impurities for which quantification is required are readily detected at 400 MHz or above. These results are in contrast to the widespread belief that 1H NMR sensitivity is insufficient for pharmaceutical impurity analysis. The choice of solvent was recognized as a critical parameter for 1H NMR LOD analysis. Furthermore, publicly available NMR raw data (HMDB) proved to be valuable for unveiling the otherwise cryptic information hidden in complex signal patterns via 1H NMR iterative Full Spin Analysis. Finally, the study uncovered the less noticed, yet characteristic, 14N-1H coupling in the -N+(CH3)3 groups, adding strong arguments for the Raw NMR Data Initiative. Collectively, the data prove that the analytical capabilities of high-field NMR easily fulfill the ICH requirements for detection of impurity in the presence of an actual substance of interest which makes it a step closer to achieving regulatory standards.

Selective Preparation and High Dynamic-Range Analysis of Cannabinoids in "CBD Oil" and Other Cannabis sativa Preparations.

Much confusion exists about the chemical composition of widely sold Cannabis sativa products 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.

Investigation of red clover (Trifolium pratense) isoflavonoid residual complexity by off-line CCS-qHNMR.

The importance of Trifolium pratense L. as a dietary supplement and its use in traditional medicine prompted the preparation of a thorough metabolite profile. This included the identification and quantitation of principal constituents as well as low abundant metabolites that constitute the residual complexity (RC) of T. pratense bioactives. The purity and RC of isoflavonoid fractions from standardized red clover extract (RCE) was determined using an off-line combination of countercurrent separation (CCS) and two orthogonal analytical methodologies: quantitative 1H NMR spectroscopy with external calibration (EC-qHNMR) and LC-MS. A single-step hydrostatic CCS methodology (Centrifugal Partition Chromatography [CPC]) was developed that fractionated the isoflavonoids with a hexanes-ethyl acetate-methanol-water (HEMWat) 5.5/4.5/5/5, v/v solvent system (SS) into 75 fractions containing 3 flavonolignans, 2 isoflavonoid glycosides, as well as 17 isoflavonoids and related compounds. All metabolites were identified and quantified by qHNMR spectroscopy. The data led to the creation of a complete isoflavonoid profile to complement the biological evaluation. For example, fraction 69 afforded 90.5% w/w biochanin A (17), with 0.33% w/w of prunetin (16), and 0.76% w/w of maackiain (15) as residual components. Fraction 27 with 89.4% w/w formononetin (13) as the major component had, in addition, a residual complexity consisting of 3.37%, 0.73%, 0.68% w/w of pseudobaptigenin (11), kaempferol (10) and pratensein (8), respectively. Despite the relatively high resolving power of CPC, and not unexpectedly, the chromatographic fractions retained varying degrees of the original metabolomic diversity. Collectively, the extent of metabolomic diversity should be recognized and used to guide the development of isolation strategies, especially when generating samples for bioactivity evaluation. The simultaneous structural and quantitative characterization enabled by qNMR, supported by LC-MS measurements, enables the evaluation of a relatively large number of individual fractions and, thereby, advances both the chemical and biological evaluation of active principles in complex natural products.

Do Certain Flavonoid IMPS Have a Vital Function?

Flavonoids are a vast group of metabolites that are essential for vascular plant physiology and, thus, occur ubiquitously in plant-based/-derived foods. The solitary designation of thousands of known flavonoids hides the fact that their metabolomes are structurally highly diverse, consist of disparate subgroups, yet undergo a certain degree of metabolic interconversion. Unsurprisingly, flavonoids have been an important theme in nutrition research. Already in the 1930s, it was discovered that the ability of synthetic Vitamin C to treat scurvy was inferior to that of plant extracts containing Vitamin C. Subsequent experimental evidence led to the proposal of Vitamin P (permeability) as an essential phytochemical nutrient. However, attempts to isolate and characterize Vitamin P gave confusing and sometimes irreproducible results, which today can be interpreted as rooted in the unrecognized (residual) complexity of the intervention materials. Over the years, primarily flavonoids (and some coumarins) were known as having Vitamin P-like activity. More recently, in a NAPRALERT meta-analysis, essentially all of these Vitamin P candidates were identified as IMPs (Invalid/Improbable/Interfering Metabolic Panaceas). While the historic inability to define a single compound and specific mode of action led to general skepticism about the Vitamin P proposition for "bioflavonoids," the more logical conclusion is that several abundant and metabolically labile plant constituents fill this essential role in human nutrition at the interface of vitamins, cofactors, and micronutrients. Reviewing 100+ years of the multilingual Vitamin P and C literature provides the rationales for this conclusion and new perspectives for future research.

Tandem of Countercurrent Separation and qHNMR Enables Gravimetric Analyses: Absolute Quantitation of the Rhodiola rosea Metabolome.

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 rosea crude 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 Rhodiola marker 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.

Silica Gel-mediated Oxidation of Prenyl Motifs Generates Natural Product-Like Artifacts.

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 of 1H 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.

Auto-hydrolysis of red clover as "green" approach to (iso)flavonoid enriched products.

Optimal parameters for the auto-hydrolysis of (iso)flavone glycosides to aglycones in ground Trifolium pratense L. plant material were established as a "green" method for the production of a reproducible red clover extract (RCE). The process utilized 72-h fermentation in DI water at 25 and 37 °C. The aglycones obtained at 25 °C, as determined by UHPLC-UV and quantitative 1H NMR (qHNMR), increased significantly in the auto-hydrolyzed (ARCE) (6.2-6.7% w/w biochanin A 1, 6.1-9.9% formononetin 2) vs a control ethanol (ERCE) extract (0.24% 1, 0.26% 2). After macerating ARCE with 1:1 (v/v) diethyl ether/hexanes (ARCE-d/h), 1 and 2 increased to 13.1-16.7% and 14.9-18.4% w, respectively, through depletion of fatty components. The final extracts showed chemical profiles similar to that of a previous clinical RCE. Biological standardization revealed that the enriched ARCE-d/h extracts produced the strongest estrogenic activity in ERα positive endometrial cells (Ishikawa cells), followed by the precursor ARCE. The glycoside-rich ERCE showed no estrogenic activity. The estrogenicity of ARCE-d/h was similar to that of the clinical RCE. The lower potency of the ARCE compared to the prior clinical RCE indicated that substantial amounts of fatty acids/matter likely reduce the estrogenicity of crude hydrolyzed preparations. The in vitro dynamic residual complexity of the conversion of biochanin A to genistein was evaluated by LC-MS-MS. The outcomes help advance translational research with red clover and other (iso)flavone-rich botanicals by inspiring the preparation of (iso)flavone aglycone-enriched extracts for the exploration of new in vitro and ex vivo bioactivities that are unachievable with genuine, glycoside-containing extracts.

Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response.

The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.

The Untargeted Capability of NMR Helps Recognizing Nefarious Adulteration in Natural Products.

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.

Isolation and Pharmacological Characterization of Six Opioidergic Picralima nitida Alkaloids.

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 4 to 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. nitida alkaloids 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.

Quantum mechanical NMR full spin analysis in pharmaceutical identity testing and quality control.

Issues related to pharmaceutical quality are arising at an alarming rate. Pharmaceutical quality concerns both the Active Pharmaceutical Ingredients (APIs) and the Finished Drug Product/ Formulation. Recently, there has been a significant increase in the number of reports of harmful impurities in marketed drug formulations. Impurities range from solvents, reactants, adulterants, and catalysts to synthetic byproducts. Quality concerns in commercial preparations may also arise due to shelf life stability. Furthermore, a number of falsified and substandard drug cases have been reported. Most of the techniques which are currently in place can, at best, detect the impurities, but cannot identify them unless they are already known and can be compared to a standard. On the other hand, 1H NMR spectroscopy detects all the hydrogen containing species, typically provides information to elucidate structures partially or even completely, and through its absolute quantitative capabilities even can detect the presence hydrogen-free species indirectly. The structural properties that produce 1H NMR signals as characteristic representations of a given molecule are the chemical shifts (δ in ppm) and coupling constants (J in Hz). Along with the line widths (ω1/2 in Hz), these parameters are bound to both the molecule and the NMR experimental conditions by quantum mechanical (QM) principles. This means that the 1H NMR spectra of APIs can be precisely calculated and compared to the experimental data. This review explains how 1H NMR spectroscopy coupled with Full Spin Analysis can contribute towards the quality control of pharmaceuticals by improving structural dereplication and achieving simultaneous quantification of both APIs and their contaminants.

The Essential Medicinal Chemistry of Cannabidiol (CBD).

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.

Selective Chlorophyll Removal Method to "Degreen" Botanical Extracts.

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.

Quantum Mechanics-Based Structure Analysis of Cyclic Monoterpene Glycosides from Rhodiola rosea.

NMR- and MS-guided metabolomic mining for new phytoconstituents from a widely used dietary supplement, Rhodiola rosea, yielded two new (+)-myrtenol glycosides, 1 and 2, and two new cuminol glycosides, 3 and 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 1 and 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. rosea as a resilience botanical. Application of a new NMR data analysis software package, CT, for QM-based iteration of NMR spectra is also discussed.

Linear regression analysis of silychristin A, silybin A and silybin B contents in Silybum marianum.

Quantitative correlations between the contents of the flavonolignans silychristin A and silybins A/B provide biosynthetic clues that support a pathway in which one mesomeric form of a taxifolin radical is undergoing an oxidative coupling with a coniferyl alcohol radical. The flavonolignan content and patterns reported in the literature for 53 samples, representing populations of the Silybum marianum plant growing in different parts of the world, were subject to a meta-analysis. Linear regression analyses were carried out on these data sets, and a mathematical model was derived that predicts the content of silychristin A relative to the metabolomic pattern of its congeners. The validity of the model was verified by applying it to test samples. This approach could potentially become a tool to enhance the understanding of both the relative composition of the silymarin complex and the biosynthetic pathways that underlie its formation.

Studying Mass Balance and the Stability of (Z)-Ligustilide from Angelica sinensis Helps to Bridge a Botanical Instability-Bioactivity Chasm.

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 1 being an unstable, rapidly degrading compound, ultimately leading to dynamic residual complexity. While Angelica sinensis is 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 1 obtained from A. sinensis to investigate the factors that influence the chemical transformation of 1, the products formed, and the rationale for monitoring 1 in natural product preparations. Countercurrent separation was used to purify 1 from a supercritical fluid extract of A. sinensis, achieving 93.4% purity in a single step. Subsequent purification by preparative HPLC afforded 1 with a 98.0% purity. Providing a mass balance setting, we monitored chemical changes occurring to highly purified 1 under 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 1 is 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.

Preparation of DESIGNER extracts of red clover (Trifolium pratense L.) by centrifugal partition chromatography.

Starting with an isoflavone-rich red clover extract (RCE), this study expands on the DESIGNER approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources using countercurrent separation (CCS) methodology. A hydrostatic CCS (also known as centrifugal partition chromatography, CPC) technique was used to enrich and deplete selected bioactive isoflavones of RCE extracts. In order to efficiently prepare large enough DESIGNER extracts from RCE for biological testing including in vivo assays, it was necessary to choose a balance between resolution and a loading capacity of at least 1 g per separation for the selected solvent system (SS). Adding 3 mL of DMSO to the sample containing equal amounts of upper and lower phases of hexanes-ethyl acetate-methanol-water (HEMWat 5.5/4.5/5/5, v/v) allowed 1 g of RCE to be dissolved in the sample without disrupting the chromatographic resolution of the target isoflavones. CPC experiments using other solubility modifiers, acetone and acetonitrile indicated that these modifiers increase solubility significantly, even better than DMSO, but the separation of target compounds was sufficiently disturbed to be unacceptable for producing the desired DESIGNER extracts. The preparation of DESIGNER extracts was achieved with two sequential CPC separations. The first produced a biochanin A enriched fraction (93.60% w/w) with only small amounts of other isoflavones: 2.30% w/w prunetin, 1.17% w/w formononetin, and 0.12% w/w irilone. Gravimetric investigations of this step demonstrated the high efficiency of CCS technology for full and unbiased sample recovery, confirmed experimentally to be 99.80%. A formononetin enriched fraction from this first separation was re-chromatographed on a more polar HEMWat (4/6/4/6, v/v) SS to produce a formononetin enriched DESIGNER fraction of 94.70% w/w purity. The presence of the minor (iso)flavonoids: 3.16% w/w pseudobaptigenin, 0.39% w/w kaempferol, and 0.31% w/w genistein was also monitored in these fractions. Chromatographic fractions, combined fractions, and DESIGNER extracts were analyzed with quantitative 1H NMR (qHNMR) spectroscopy which provided purity information, quantitation, and structural identification of the components.