Modulation of in Vitro SARS-CoV-2 Infection by Stephania tetrandra and Its Alkaloid Constituents.

Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography-mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.

Metabolomics-Guided Comparison of Pollen and Microalgae-Based Artificial Diets in Honey Bees.

Managed honey bee colonies used for crop pollination are fed artificial diets to offset nutritional deficiencies related to land-use intensification and climate change. In this study, we formulated novel microalgae diets using Chlorella vulgaris and Arthrospira platensis (spirulina) biomass and fed them to young adult honey bee workers. Diet-induced changes in bee metabolite profiles were studied relative to a natural pollen diet using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) metabolomics. Untargeted analyses of pollen- and microalgae-fed bees revealed significant overlap, with 248 shared features determined by LC-MS and 87 shared features determined by GC-MS. Further metabolomic commonalities were evident upon subtraction of unique diet features. Twenty-five identified metabolites were influenced by diet, which included complex lipids, essential fatty acids, vitamins, and phytochemicals. The metabolomics results are useful to understand mechanisms underlying favorable growth performance as well as increased antioxidant and heat shock protein gene expression in bees fed the microalgae diets. We conclude that the tested microalgae have potential as sustainable feed additives and as a source of bee health-modulating natural products. Metabolomics-guided diet development could eventually help tailor feed interventions to achieve precision nutrition in honey bees and other livestock animals.

Kratom (Mitragyna speciosa) Validation: Quantitative Analysis of Indole and Oxindole Alkaloids Reveals Chemotypes of Plants and Products.

Many consumers are turning to kratom (Mitragyna speciosa) to self-manage pain and opioid addiction. In the United States, an array of capsules, powders, and loose-leaf kratom products are readily available. Additionally, several online sites supply live kratom plants. A prerequisite to establishing quality control and quality assurance standards for the kratom industry, or understanding how alkaloid levels effect clinical outcomes, is the identification and quantitation of major and minor alkaloid constituents within available products and preparations. To this end, an ultra-high performance liquid chromatography-high resolution mass spectrometry method was developed for the analysis of 8 indole alkaloids (7-hydroxymitragynine, ajmalicine, paynantheine, mitragynine, speciogynine, isopaynantheine, speciociliatine, and mitraciliatine) and 6 oxindole alkaloids (isomitraphylline, isospeciofoleine, speciofoline, corynoxine A, corynoxeine, and rhynchophylline) in US-grown kratom plants and commercial products. These commercial products shared a qualitatively similar alkaloid profile, with 12 - 13 detected alkaloids and high levels of the indole alkaloid mitragynine (13.9 ± 1.1 - 270 ± 24 mg/g). The levels of the other major alkaloids (paynantheine, speciociliatine, speciogynine, mitraciliatine, and isopaynantheine) and the minor alkaloids varied in concentration from product to product. The alkaloid profile of US-grown M. speciosa "Rifat" showed high levels of the indole alkaloid speciogynine (7.94 ± 0.83 - 11.55 ± 0.18 mg/g) and quantifiable levels of isomitraphylline (0.943 ± 0.033 - 1.47 ± 0.18 mg/g). Notably, the alkaloid profile of a US-grown M. speciosa seedling was comparable to the commercial products with a high level of mitragynine (15.01 ± 0.20 mg/g). This work suggests that there are several M. speciosa chemotypes.

More Than Just a Weed: An Exploration of the Antimicrobial Activity of Rumex crispus using a Multivariate Data Analysis Approach.

Plants have a long history of use for their medicinal properties. The complexity of botanical extracts presents unique challenges and necessitates the application of innovative approaches to correctly identify and quantify bioactive compounds. For this study, we used untargeted metabolomics to explore the antimicrobial activity of Rumex crispus (yellow dock), a member of the Polygonaceae family used as an herbal remedy for bacterial infections. Ultra-performance liquid chromatography coupled with high resolution mass-spectrometry (UPLC-MS) was used to identify and quantify the known antimicrobial compound emodin. In addition, we used biochemometric approaches to integrate data measuring antimicrobial activity from R. crispus root starting material and fractions against methicillin-resistant Staphylococcus aureus (MRSA) with UPLC-MS data. Our results support the hypothesis that multiple constituents, including the anthraquinone emodin, contribute to the antimicrobial activity of R. crispus against MRSA.

Interlaboratory Comparison of Untargeted Mass Spectrometry Data Uncovers Underlying Causes for Variability.

Despite the value of mass spectrometry in modern natural products discovery workflows, it remains very difficult to compare data sets between laboratories. In this study we compared mass spectrometry data for the same sample set from two different laboratories (quadrupole time-of-flight and quadrupole-Orbitrap) and evaluated the similarity between these two data sets in terms of both mass spectrometry features and their ability to describe the chemical composition of the sample set. Somewhat surprisingly, the two data sets, collected with appropriate controls and replication, had very low feature overlap (25.7% of Laboratory A features overlapping 21.8% of Laboratory B features). Our data clearly demonstrate that differences in fragmentation, charge state, and adduct formation in the ionization source are a major underlying cause for these differences. Consistent with other recent literature, these findings challenge the conventional wisdom that electrospray ionization mass spectrometry (ESI-MS) yields a simple one-to-one correspondence between analytes in solution and features in the data set. Importantly, despite low overlap in feature lists, principal component analysis (PCA) generated qualitatively similar PCA plots. Overall, our findings demonstrate that comparing untargeted metabolomics data between laboratories is challenging, but that data sets with low feature overlap can yield the same qualitative description of a sample set using PCA.

Bacterial efflux inhibitors are widely distributed in land plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Secondary metabolites play a critical role in plant defense against disease and are of great importance to ethnomedicine. Bacterial efflux pumps are active transport proteins that bacterial cells use to protect themselves against multiple toxic compounds, including many antimicrobials. Efflux pump inhibitors from plants can block these efflux pumps, increasing the potency of antimicrobial compounds. This study demonstrates that efflux pump inhibition against the Gram-positive bacterial pathogen Staphylococcus aureus is widespread in extracts prepared from individual species throughout the land plant lineage. It therefore suggests a general mechanism by which plants used by indigenous species may be effective as a topical treatment for some bacterial infections. AIM OF THE STUDY: The goal of this research was to evaluate the distribution of efflux pump inhibitors in nine plant extracts with an ethnobotanical use suggestive of an antimicrobial function for the presence of efflux pump inhibitory activity against Staphylococcus aureus. MATERIALS AND METHODS: Plants were collected, dried, extracted, and vouchers submitted to the Herbarium of the University of North Carolina Chapel Hill (NCU). The extracts were analyzed by quantitative mass spectrometry (UPLC-MS) to determine the presence and concentration of flavonoids with known efflux pump inhibitory activity. A mass spectrometry-based assay was employed to measure efflux pump inhibition for all extracts against Staphylococcus aureus. The assay relies on UPLC-MS measurement of changes in ethidium concentration in the spent culture broth when extracts are incubated with bacteria. RESULTS: Eight of these nine plant extracts inhibited toxic compound efflux at concentrations below the MIC (minimum inhibitory concentration) value for the same extract. The most active extracts were those prepared from Osmunda claytoniana L. and Pinus strobes L., which both demonstrated IC50 values for efflux inhibition of 19 ppm. CONCLUSIONS: Our findings indicate that efflux pump inhibitors active against Staphylococcus aureus are common in land plants. By extension, this activity is likely to be important in many plant-derived antimicrobial extracts, including those used in traditional medicine, and evaluation of efflux pump inhibition may often be valuable when studying natural product efficacy.

Identification of adulteration in botanical samples with untargeted metabolomics.

Adulteration remains an issue in the dietary supplement industry, including botanical supplements. While it is common to employ a targeted analysis to detect known adulterants, this is difficult when little is known about the sample set. With this study, untargeted metabolomics using liquid chromatography coupled to ultraviolet-visible spectroscopy (LC-UV) or high-resolution mass spectrometry (LC-MS) was employed to detect adulteration in botanical dietary supplements. A training set was prepared by combining Hydrastis canadensis L. with a known adulterant, Coptis chinensis Franch., in ratios ranging from 5 to 95% adulteration. The metabolomics datasets were analyzed using both unsupervised (principal component analysis and composite score) and supervised (SIMCA) techniques. Palmatine, a known H. canadensis metabolite, was quantified as a targeted analysis comparison. While the targeted analysis was the most sensitive method tested in detecting adulteration, statistical analyses of the untargeted metabolomics datasets detected adulteration of the goldenseal samples, with SIMCA providing the greatest discriminating potential. Graphical abstract.

Apicidin Attenuates MRSA Virulence through Quorum-Sensing Inhibition and Enhanced Host Defense.

Recurrent epidemics of drug-resistant Staphylococcus aureus illustrate the rapid lapse of antibiotic efficacy following clinical implementation. Over the last decade, community-associated methicillin-resistant S. aureus (MRSA) has emerged as a dominant cause of infections, and this problem is amplified by the hyper-virulent nature of these isolates. Herein, we report the discovery of a fungal metabolite, apicidin, as an innovative means to counter both resistance and virulence. Owing to its breadth and specificity as a quorum-sensing inhibitor, apicidin antagonizes all MRSA agr systems in a non-biocidal manner. In skin challenge experiments, the apicidin-mediated abatement of MRSA pathogenesis corresponds with quorum-sensing inhibition at in vivo sites of infection. Additionally, we show that apicidin attenuates MRSA-induced disease by potentiating innate effector responses, particularly through enhanced neutrophil accumulation and function at cutaneous challenge sites. Together, these results indicate that apicidin treatment represents a strategy to limit MRSA virulence and promote host defense.

Reinforcement of polymeric nanoassemblies for ultra-high drug loadings, modulation of stiffness and release kinetics, and sustained therapeutic efficacy.

The optimization of current polymeric nanoparticle therapies is restricted by low drug loadings and limited tunability of core properties. To overcome these shortcomings, a novel self-association approach is utilized to fabricate a dual-loaded poly(1,2-glycerol carbonate)-graft-succinic acid-paclitaxel (PGC-PTX) conjugate nanoparticle (NP) in which the physical entrapment of free paclitaxel (PTX) affords unprecedented ultra-high drug loadings >100 wt%, modulation of mechanical stiffness, and tunable release kinetics. Despite high incorporation of free PTX (up to 50 wt%), the dual-loaded PGC-PTX nanocarriers (i.e., PGC-PTX + PTX NPs) exhibit controlled and sustained drug release over 15 days, without burst release effects. Importantly, optimization of drug/material efficiency concomitantly affords improved in vitro efficacy. In vivo, PGC-PTX + PTX NPs are safely administered at doses exceeding the median lethal dose of standard PTX, while a single high dose significantly extends survival relative to weekly PTX administrations in a murine model of peritoneal carcinomatosis.

Mast cell degranulation and calcium influx are inhibited by an Echinacea purpurea extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide.

ETHNOPHARMACOLOGICAL RELEVANCE: Native Americans used plants from the genus Echinacea to treat a variety of different inflammatory conditions including swollen gums, sore throats, skin inflammation, and gastrointestinal disorders. Today, various Echinacea spp. preparations are used primarily to treat upper respiratory infections. AIM OF THE STUDY: The goal of this study was to evaluate the effects of an ethanolic E. purpurea (L) Moench root extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide (A15) on mast cells, which are important mediators of allergic and inflammatory responses. Inhibition of mast cell activation may help explain the traditional use of Echinacea. MATERIALS AND METHODS: A15 was evaluated for its effects on degranulation, calcium influx, cytokine and lipid mediator production using bone marrow derived mast cells (BMMCs) and the transformed rat basophilic leukemia mast cell line RBL-2H3. Methods included enzymatic assays, fluorimetry, ELISAs, and microscopy. A root extract of E. purpurea, and low and high alkylamide-containing fractions prepared from this extract, were also tested for effects on mast cell function. Finally, we tested A15 for effects on calcium responses in RAW 264.7 macrophage and Jurkat T cell lines. RESULTS: A15 inhibited ß-hexosaminidase release from BMMCs and RBL-2H3 cells after treatment with the calcium ionophore A23187 by 83.5% and 48.4% at 100µM, respectively. Inhibition also occurred following stimulation with IgE anti-DNP/DNP-HSA. In addition, A15 inhibited 47% of histamine release from A23187-treated RBL-2H3 cells. A15 prevented the rapid rise in intracellular calcium following FcεRI crosslinking and A23187 treatment suggesting it acts on the signals controlling granule release. An E. purpurea root extract and a fraction with high alkylamide content derived from this extract also displayed these activities while fractions with little to no detectable amounts of alkylamide did not. A15 mediated inhibition of calcium influx was not limited to mast cells as A23187-stimulated calcium influx was blocked in both RAW 264.7 and Jurkat cell lines with 60.2% and 43.6% inhibition at 1min post-stimulation, respectively. A15 also inhibited the release of TNF-α, and PGE2 to a lesser degree, following A23187 stimulation indicating its broad activity on mast cell mediator production. CONCLUSIONS: These findings suggest that Echinacea extracts and alkylamides may be useful for treating allergic and inflammatory responses mediated by mast cells. More broadly, since calcium is a critical second messenger, the inhibitory effects of alkylamides on calcium uptake would be predicted to dampen a variety of pathological responses, suggesting new uses for this plant and its constituents.

Secondary Metabolites from the Leaves of the Medicinal Plant Goldenseal (Hydrastis canadensis).

The study presented herein constitutes an extensive investigation of constituents in Hydrastis canadensis L. (Ranunculaceae) leaves. It describes the isolation and identification of two previously unknown compounds, 3,4-dimethoxy-2-(methoxycarbonyl)benzoic acid (1) and 3,5,3'-trihydroxy-7,4'-dimethoxy-6,8-C-dimethyl-flavone (2), along with the known compounds (±)-chilenine (3), (2R)-5,4'-dihydroxy-6-C-methyl-7-methoxy-flavanone (4), 5,4'-dihydroxy-6,8-di-C-methyl-7-methoxy-flavanone (5), noroxyhydrastinine (6), oxyhydrastinine (7) and 4',5'-dimethoxy-4-methyl-3'-oxo-(1,2,5,6-tetrahydro-4H-1,3-dioxolo-[4',5':4,5]-benzo[1,2-e]-1,2-oxazocin)-2-spiro-1'-phtalan (8). Compounds 3-8 have been reported from other sources, but this is the first report of their presence in H. canadensis extracts. A mass spectrometry based assay was employed to demonstrate bacterial efflux pump inhibitory activity against Staphylococcus aureus for 2, with an IC50 value of 180 ± 6 µM. This activity in addition to that of other bioactive compounds such as flavonoids and alkaloids, may explain the purported efficacy of H. canadensis for treatment of bacterial infections. Finally, this report includes high mass accuracy fragmentation spectra for all compounds investigated herein which were uploaded into the Global Natural Products Social molecular networking library and can be used to facilitate their future identification in H. canadensis or other botanicals.

Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine-Suppressive and Cytokine-Inducing Compounds, Including Some That Originate from Endophytic Bacteria.

Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than $100 million annually. In an attempt to shed light on Echinacea's mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.