Goldenseal-Mediated Inhibition of Intestinal Uptake Transporters Decreases Metformin Systemic Exposure in Mice.

Goldenseal is a perennial plant native to eastern North America. A recent clinical study reported goldenseal decreased metformin Cmax and area under the blood concentration versus time curve (AUC) by 27% and 23%, respectively, but half-life and renal clearance were unchanged. These observations suggested goldenseal altered processes involved in metformin absorption. The underlying mechanism(s) remain(s) unknown. One mechanism for the decreased metformin systemic exposure is inhibition by goldenseal of intestinal uptake transporters involved in metformin absorption. Goldenseal extract and three goldenseal alkaloids (berberine, (-)-ß-hydrastine, hydrastinine) were tested as inhibitors of organic cation transporter (OCT) 3, plasma membrane monoamine transporter (PMAT), and thiamine transporter (THTR) 2 using human embryonic kidney 293 cells overexpressing each transporter. The goldenseal extract, normalized to berberine content, was the strongest inhibitor of each transporter (IC50: 4.9, 13.1, and 5.8 µM for OCT3, PMAT, and THTR2, respectively). A pharmacokinetic study in mice compared the effects of berberine, (-)-ß-hydrastine, goldenseal extract, and imatinib (OCT inhibitor) on orally administered metformin. Goldenseal extract and imatinib significantly decreased metformin Cmax by 31% and 25%, respectively, and had no effect on half-life. Berberine and (-)-ß-hydrastine had no effect on metformin pharmacokinetics, indicating neither alkaloid alone precipitated the interaction in vivo. A follow-up murine study involving intravenous metformin and oral inhibitors examined the contributions of basolateral enteric/hepatic uptake transporters to the goldenseal-metformin interaction. Goldenseal extract and imatinib had no effect on metformin AUC and half-life, suggesting lack of inhibition of basolateral enteric/hepatic uptake transporters. Results may have implications for patients taking goldenseal with drugs that are substrates for OCT3 and THTR2. SIGNIFICANCE STATEMENT: Goldenseal is used to self-treat respiratory infections and digestive disorders. We investigated potential mechanisms for the clinical pharmacokinetic interaction observed between goldenseal and metformin, specifically inhibition by goldenseal of intestinal uptake transporters (OCT3, PMAT, THTR2) involved in metformin absorption. Goldenseal extract inhibited all three transporters in vitro and decreased metformin systemic exposure in mice. These data may have broader implications for patients co-consuming goldenseal with other drugs that are substrates for these transporters.

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.

How Low Can You Go? Selecting Intensity Thresholds for Untargeted Metabolomics Data Preprocessing.

Untargeted mass spectrometry (MS) metabolomics is an increasingly popular approach for characterizing complex mixtures. Recent studies have highlighted the impact of data preprocessing for determining the quality of metabolomics data analysis. The first step in data processing with untargeted metabolomics requires that signal thresholds be selected for which features (detected ions) are included in the dataset. Analysts face the challenge of knowing where to set these thresholds; setting them too high could mean missing relevant features, but setting them too low could result in a complex and unwieldy dataset. This study compared data interpretation for an example metabolomics dataset when intensity thresholds were set at a range of feature heights. The main observations were that low signal thresholds (1) improved the limit of detection, (2) increased the number of features detected with an associated isotope pattern and/or an MS-MS fragmentation spectrum, and (3) increased the number of in-source clusters and fragments detected for known analytes of interest. When the settings of parameters differing in intensities were applied on a set of 39 samples to discriminate the samples through principal component analyses (PCA), similar results were obtained with both low- and high-intensity thresholds. We conclude that the most information-rich datasets can be obtained by setting low-intensity thresholds. However, in the cases where only a qualitative comparison of samples with PCA is to be performed, it may be sufficient to set high thresholds and thereby reduce the complexity of the data processing and amount of computational time required.

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.

Stop the crop: Insights into the insecticidal mode of action of cinnamodial against mosquitoes.

Cinnamodial (CDIAL) is a drimane sesquiterpene dialdehyde found in the bark of Malagasy medicinal plants (Cinnamosma species; family Canellaceae). We previously demonstrated that CDIAL was insecticidal, antifeedant, and repellent against Aedes aegypti mosquitoes. The goal of the present study was to generate insights into the insecticidal mode of action for CDIAL, which is presently unknown. We evaluated the effects of CDIAL on the contractility of the ventral diverticulum (crop) isolated from adult female Ae. aegypti. The crop is a food storage organ surrounded by visceral muscle that spontaneously contracts in vitro. We found that CDIAL completely inhibited spontaneous contractions of the crop as well as those stimulated by the agonist 5-hydroxytryptamine. Several derivatives of CDIAL with known insecticidal activity also inhibited crop contractions. Morphometric analyses of crops suggested that CDIAL induced a tetanic paralysis that was dependent on extracellular Ca2+ and inhibited by Gd3+, a non-specific blocker of plasma membrane Ca2+ channels. Screening of numerous pharmacological agents revealed that a Ca2+ ionophore (A23187) was the only compound other than CDIAL to completely inhibit crop contractions via a tetanic paralysis. Taken together, our results suggest that CDIAL induces a tetanic paralysis of the crop by elevating intracellular Ca2+ through the activation of plasma membrane Ca2+ channels, which may explain the insecticidal effects of CDIAL against mosquitoes. Our pharmacological screening experiments also revealed the presence of two regulatory pathways in mosquito crop contractility not previously described: an inhibitory glutamatergic pathway and a stimulatory octopaminergic pathway. The latter pathway was also completely inhibited by CDIAL.

Clinical Pharmacokinetic Assessment of Kratom (Mitragyna speciosa), a Botanical Product with Opioid-like Effects, in Healthy Adult Participants.

Increasing use of the botanical kratom to self-manage opioid withdrawal and pain has led to increased kratom-linked overdose deaths. Despite these serious safety concerns, rigorous fundamental pharmacokinetic knowledge of kratom in humans remains lacking. We assessed the pharmacokinetics of a single low dose (2 g) of a well-characterized kratom product administered orally to six healthy participants. Median concentration-time profiles for the kratom alkaloids examined were best described by a two-compartment model with central elimination. Pronounced pharmacokinetic differences between alkaloids with the 3S configuration (mitragynine, speciogynine, paynantheine) and alkaloids with the 3R configuration (mitraciliatine, speciociliatine, isopaynantheine) were attributed to differences in apparent intercompartmental distribution clearance, volumes of distribution, and clearance. Based on noncompartmental analysis of individual concentration-time profiles, the 3S alkaloids exhibited a shorter median time to maximum concentration (1-2 vs. 2.5-4.5 h), lower area under the plasma concentration-time curve (430-490 vs. 794-5120 nM × h), longer terminal half-life (24-45 vs. ~12-18 h), and higher apparent volume of distribution during the terminal phase (960-12,700 vs. ~46-130 L) compared to the 3R alkaloids. Follow-up mechanistic in vitro studies suggested differential hepatic/intestinal metabolism, plasma protein binding, blood-to-plasma partitioning, and/or distribution coefficients may explain the pharmacokinetic differences between the two alkaloid types. This first comprehensive pharmacokinetic characterization of kratom alkaloids in humans provides the foundation for further research to establish safety and effectiveness of this emerging botanical product.

Semi-synthetic cinnamodial analogues: Structural insights into the insecticidal and antifeedant activities of drimane sesquiterpenes against the mosquito Aedes aegypti.

The Aedes aegypti mosquito serves as a major vector for viral diseases, such as dengue, chikungunya, and Zika, which are spreading across the globe and threatening public health. In addition to increased vector transmission, the prevalence of insecticide-resistant mosquitoes is also on the rise, thus solidifying the need for new, safe and effective insecticides to control mosquito populations. We recently discovered that cinnamodial, a unique drimane sesquiterpene dialdehyde of the Malagasy medicinal plant Cinnamosma fragrans, exhibited significant larval and adult toxicity to Ae. aegypti and was more efficacious than DEET-the gold standard for insect repellents-at repelling adult female Ae. aegypti from blood feeding. In this study several semi-synthetic analogues of cinnamodial were prepared to probe the structure-activity relationship (SAR) for larvicidal, adulticidal and antifeedant activity against Ae. aegypti. Initial efforts were focused on modification of the dialdehyde functionality to produce more stable active analogues and to understand the importance of the 1,4-dialdehyde and the α,ß-unsaturated carbonyl in the observed bioactivity of cinnamodial against mosquitoes. This study represents the first investigation into the SAR of cinnamodial as an insecticide and antifeedant against the medically important Ae. aegypti mosquito.

Insecticidal and Antifeedant Activities of Malagasy Medicinal Plant (Cinnamosma sp.) Extracts and Drimane-Type Sesquiterpenes against Aedes aegypti Mosquitoes.

The overuse of insecticides with limited modes of action has led to resistance in mosquito vectors. Thus, insecticides with novel modes of action are needed. Secondary metabolites in Madagascan plants of the genus Cinnamosma (Canellaceae) are commonly used in traditional remedies and known to elicit antifeedant and toxic effects in insect pests. Here we test the hypothesis that extracts of Cinnamosma sp. enriched in drimane sesquiterpenes are toxic and/or antifeedant to the yellow fever mosquito Aedes aegypti. We show that the bark and root extracts, which contain a higher abundance of drimane sesquiterpenes compared to leaves, were the most efficacious. Screening isolated compounds revealed cinnamodial to be the primary driver of adulticidal activity, whereas cinnamodial, polygodial, cinnafragrin A, and capsicodendrin contributed to the larvicidal activity. Moreover, an abundant lactone (cinnamosmolide) in the root extract synergized the larvicidal effects of cinnamodial. The antifeedant activity of the extracts was primarily contributed to cinnamodial, polygodial, and cinnamolide. Parallel experiments with warburganal isolated from Warburgia ugandensis (Canellaceae) revealed that aldehydes are critical for-and a hydroxyl modulates-insecticidal activity. Our results indicate that plant drimane sesquiterpenes provide valuable chemical platforms for developing insecticides and repellents to control mosquito vectors.