Bryophyllum pinnatum Inhibits Oxytocin and Vasopressin Signaling in Myometrial Cells.

The medicinal plant Bryophyllum pinnatum was previously shown to block oxytocin (OT)-induced signals in myometrial cells, consistent with its tocolytic effect observed in patients. OT activates not only OT receptors but also V1A receptors, two receptors with high receptor homology that are both expressed in the myometrium and play a crucial role in myometrial contraction signaling. We aimed to study the molecular pharmacology of B. pinnatum herbal preparations using specific receptor ligands, the human myometrial cell line hTERT-C3, and cell lines expressing recombinant human OT and V1A receptors.We found that press juice from B. pinnatum (BPJ) inhibits both OT- and vasopressin (AVP)-induced intracellular calcium increases in hTERT-C3 myometrial cells. In additional assays performed with cells expressing recombinant receptors, BPJ also inhibited OT and V1A receptor-mediated signals with a similar potency (IC50 about 0.5 mg/mL). We further studied endogenous OT- and AVP-sensitive receptors in hTERT-C3 cells and found that OT and AVP stimulated those receptors with similar potency (EC50 of ~ 1 nM), suggesting expression of both receptor subtypes. This interpretation was corroborated by the antagonist potencies of atosiban and relcovaptan that we found. However, using qPCR, we almost exclusively found expression of OT receptors suggesting a pharmacological difference between recombinant OT receptors and native receptors expressed in hTERT-C3 cells.In conclusion, we show that B. pinnatum inhibits both OT and AVP signaling, which may point beyond its tocolytic effects to other indications involving a disbalance in the vasopressinergic system.

Potential benefits of Malva sylvestris in dry-eye disease pathology in vitro based on antioxidant, wound-healing and anti-inflammatory properties.

Dry eye disease (DED) is a common chronic ocular surface disease. Available therapies are effective but often associated with side effects. This study investigates the potential of a Malva sylvestris L. flower extract and two defined preparations, a mucilage and a polyphenol rich fraction, on cells that are essential for the DED pathology. Furthermore, single compounds were isolated and characterised out of the polyphenol fraction. The M. sylvestris extract and its two fractions reduced reactive oxygen species (ROS) in an ultraviolet-induced model and promoted wound healing capacity of HCE-T cells, but only the polyphenol fraction and the flower extract exhibited significant radical scavenging activity. The flower extract and the polyphenol fraction inhibited cytokine secretion (IL-6, TNF-α, IL-8) from HCE-T cells and THP-1 cells. In contrast, the mucilage fraction led to an increase in mediator secretion. The NF-κB activity and calcium influx in THP-1 and Jurkat cells, respectively was decreased by treatment with the flower extract and the polyphenol fraction, whereas the mucilage fraction had no influence on these parameters. Moreover, the flower extract and the mucilage fraction at low concentration could stimulate meibomian gland cells' lipid accumulation. The isolated single compounds showed no effect on analysed parameters, except a coumarin derivative and malvin which showed ROS inhibition effects.

St. John's Wort Formulations Induce Rat CYP3A23-3A1 Independent of Their Hyperforin Content.

The pregnane X receptor (PXR) is a ligand-activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant was used as it contains a high hyperforin content and Rebalance because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant and Rebalance transactivated the CYP3A reporter 3.8-fold and 2.8-fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared with control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant, we observed 1.8 times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount, and a 1.6-fold increase in activity compared with controls. For Rebalance we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared with control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. SIGNIFICANCE STATEMENT: Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the pregnane X receptor-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro. Importantly, CYP3A induction mimics findings in humans, but our results suggest that another so far unknown constituent of SJW is responsible for the expression- and function-modifying effects in rat liver.

Alkaloids in commercial preparations of California poppy - Quantification, intestinal permeability and microbiota interactions.

California poppy products are commonly used for the treatment of nervousness, anxiety and sleeping disorders. Pharmacologically relevant constituents include the main alkaloids californidine, escholtzine and protopine. However, only limited information is available about the alkaloid content in commercial preparations and their intestinal absorption. Moreover, a possible metabolization of these alkaloids by the gut microbiota, and their impact on microbial activity and viability have not been investigated. Californidine, escholtzine and protopine were quantified by UHPLC-MS/MS in eight commercial California poppy products. The intestinal permeability of alkaloids was studied in Caco-2 cell as a model for absorption in the small intestine. The gut microbial biotransformation was explored in artificial gut microbiota from the in vitro PolyFermS model. In addition, the impact of these alkaloids and a California poppy extract on the microbial production of short-chain fatty acids (SCFAs) and the viability of microbiota was investigated. Contents of californidine, escholtzine and protopine in California poppy products were in the ranges of 0.13-2.55, 0.05-0.63 and 0.008-0.200 mg/g, respectively. In the Caco-2 cell model, californidine was low-to-moderately permeable while escholtzine and protopine were highly permeable. An active transport process was potentially involved in the transfer of the three alkaloids. The three compounds were not metabolized by the artificial gut microbiota over 24 h. Neither the California poppy extract nor the alkaloids markedly impacted microbial SCFA production and bacterial viability.

Qualitative and Quantitative Secondary Metabolite Profiles in a Large Set of Sumatra Benzoin Samples.

Sumatra benzoin, a resin produced by Styrax benzoin and Styrax paralleloneurum, is used as an aromatic agent and may have the potential to be developed as a new agricultural fungicide. In this context, we performed a comprehensive metabolite profiling of a commercial grade A resin by high-performance liquid chromatography coupled with photodiode array detection, evaporative light scattering detection, and mass spectrometry (HPLC-PDA-ELSD-MS) analysis in combination with 1H NMR. Thirteen compounds including a new cinnamic acid ester containing two p-coumaroyl residues were identified after preparative isolation. These compounds accounted for an estimated 90% of the crude resin according to 1H NMR analysis. The two major constituents, p-coumaryl cinnamate (5) and sumaresinolic acid (11), were quantified by HPLC analysis. In a next step, the chemical profiles and the content in p-coumaryl cinnamate were compared in a large set of resin samples of different quality grades that were obtained from various commercial suppliers in Sumatra. The qualitative profiles of the samples were very similar, but significant quantitative differences were observed between different quality grades and origins of the samples for the relative contents.

Immunological evaluation of herbal extracts commonly used for treatment of mental diseases during pregnancy.

Nonpsychotic mental diseases (NMDs) affect approximately 15% of pregnant women in the US. Herbal preparations are perceived a safe alternative to placenta-crossing antidepressants or benzodiazepines in the treatment of nonpsychotic mental diseases. But are these drugs really safe for mother and foetus? This question is of great relevance to physicians and patients. Therefore, this study investigates the influence of St. John's wort, valerian, hops, lavender, and California poppy and their compounds hyperforin and hypericin, protopine, valerenic acid, and valtrate, as well as linalool, on immune modulating effects in vitro. For this purpose a variety of methods was applied to assess the effects on viability and function of human primary lymphocytes. Viability was assessed via spectrometric assessment, flow cytometric detection of cell death markers and comet assay for possible genotoxicity. Functional assessment was conducted via flow cytometric assessment of proliferation, cell cycle and immunophenotyping. For California poppy, lavender, hops, and the compounds protopine and linalool, and valerenic acid, no effect was found on the viability, proliferation, and function of primary human lymphocytes. However, St. John's wort and valerian inhibited the proliferation of primary human lymphocytes. Hyperforin, hypericin, and valtrate inhibited viability, induced apoptosis, and inhibited cell division. Calculated maximum concentration of compounds in the body fluid, as well as calculated concentrations based on pharmacokinetic data from the literature, were low and supported that the observed effects in vitro would probably have no relevance on patients. In-silico analyses comparing the structure of studied substances with the structure of relevant control substances and known immunosuppressants revealed structural similarities of hyperforin and valerenic acid to the glucocorticoids. Valtrate showed structural similarities to the T cells signaling modulating drugs.

Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 Evodia preparations.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines, In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study.

Transplacental passage of hyperforin, hypericin, and valerenic acid.

Safe medications for mild mental diseases in pregnancy are needed. Phytomedicines from St. John's wort and valerian are valid candidates, but safety data in pregnancy are lacking. The transplacental transport of hyperforin and hypericin (from St. John's wort), and valerenic acid (from valerian) was evaluated using the ex vivo cotyledon perfusion model (4 h perfusions, term placentae) and, in part, the in vitro Transwell assay with BeWo b30 cells. Antipyrine was used for comparison in both models. U(H)PLC-MS/MS bioanalytical methods were developed to quantify the compounds. Perfusion data obtained with term placentae showed that only minor amounts of hyperforin passed into the fetal circuit, while hypericin did not cross the placental barrier and valerenic acid equilibrated between the maternal and fetal compartments. None of the investigated compounds affected metabolic, functional, and histopathological parameters of the placenta during the perfusion experiments. Data from the Transwell model suggested that valerenic acid does not cross the placental cell layer. Taken together, our data suggest that throughout the pregnancy the potential fetal exposure to hypericin and hyperforin - but not to valerenic acid - is likely to be minimal.

Intestinal permeability and gut microbiota interactions of pharmacologically active compounds in valerian and St. John's wort.

Phytomedicines such as valerian and St. John's wort are widely used for the treatment of sleeping disorders, anxiety and mild depression. They are perceived as safe alternatives to synthetic drugs, but limited information is available on the intestinal absorption and interaction with human intestinal microbiota of pharmacologically relevant constituents valerenic acid in valerian, and hyperforin and hypericin in St. John's wort. The intestinal permeability of these compounds and the antidepressant and anxiolytic drugs citalopram and diazepam was investigated in the Caco-2 cell model with bidirectional transport experiments. In addition, interaction of compounds and herbal extracts with intestinal microbiota was evaluated in artificial human gut microbiota. Microbiota-mediated metabolisation of compounds was assessed, and bacterial viability and short-chain fatty acids (SCFA) production were measured in the presence of compounds or herbal extracts. Valerenic acid and hyperforin were highly permeable in Caco-2 cell monolayers. Hypericin showed low-to-moderate permeability. An active transport process was potentially involved in the transfer of valerenic acid. Hyperforin and hypericin were mainly transported through passive transcellular diffusion. All compounds were not metabolized over 24 h in the artificial gut microbiota. Microbial SCFA production and bacterial viability was not substantially impaired nor promoted by exposure to the compounds or herbal extracts.

Corrigendum: Bryophyllum pinnatum compounds inhibit oxytocin-induced signaling pathways in human myometrial cells.

[This corrects the article DOI: 10.3389/fphar.2021.632986.].

Aryltetralin lignans from Hyptis brachiata inhibiting T lymphocyte proliferation.

Increased activation and proliferation of T lymphocytes plays an essential role in the development of chronic inflammation and autoimmune diseases. Currently used immunosuppressive drugs often do not provide long-lasting relief of symptoms and show a gradual loss of efficacy over time, and are accompanied by various side effects. Therefore, novel immunosuppressive lead substances are needed. For this purpose, an in-house library consisting of 600 extracts of plants from Panama was screened for inhibition of human T lymphocyte proliferation. As one of the hits, an ethyl acetate extract from the aerial parts of Hyptis brachiata (Lamiaceae) exhibited strong inhibitory effects. Subsequent investigation resulted in the isolation of seven aryltetralin lignans, five arylnaphthalene lignans, two flavonoids, three triterpenes, and cinnamyl cinnamate. Aryltetralin lignans inhibited T lymphocyte proliferation in a concentration-dependent manner without induction of apoptosis. No relevant inhibition was observed for the arylnaphthalene lignans, flavonoids, and triterpenes. Additional cell cycle arrest investigations revealed that isolated aryltetralin lignans potently inhibited cell division in G2/M phase similarly to podophyllotoxin. Multifluorescence panel analyses of the extract also showed weak suppressive effects on the production of IL-2 and TNF-α. Therefore, preparations made out of H. brachiata could be further explored as an interesting herbal alternative in the treatment of autoimmune diseases.

Placental Passage of Protopine in an Ex Vivo Human Perfusion System.

The placental passage of protopine was investigated with a human ex vivo placental perfusion model. The model was first validated with diazepam and citalopram, 2 compounds known to cross the placental barrier, and antipyrine as a positive control. All compounds were quantified by partially validated U(H)PLC-MS/MS bioanalytical methods. Protopine was transferred from the maternal to the fetal circuit, with a steady-state reached after 90 min. The study compound did not affect placental viability or functionality, as glucose consumption, lactate production, and beta-human chorionic gonadotropin, and leptin release remained constant. Histopathological evaluation of all placental specimens showed unremarkable, age-appropriate parenchymal maturation with no pathologic findings.

Polyacetylenic caffeoyl amides from Ammodaucus leucotrichus.

Six undescribed polyacetylenic caffeoyl amides, five known flavones and three known lignans were obtained from the fruits of the North African traditional medicinal plant Ammodaucus leucotrichus Coss. & Durieu (Apiaceae). Isolation was achieved by a combination of chromatographic methods, and structures were established by extensive 1D and 2D NMR spectroscopy, mass spectrometry, electronic circular dichroism, and by GC-MS analysis of sugar derivatives. Polyacetylenic caffeoyl amides are reported for the first time as specialized metabolites.

Best Practice in the chemical characterisation of extracts used in pharmacological and toxicological research-The ConPhyMP-Guidelines.

Background: Research on medicinal plants and extracts derived from them differs from studies performed with single compounds. Extracts obtained from plants, algae, fungi, lichens or animals pose some unique challenges: they are multicomponent mixtures of active, partially active and inactive substances, and the activity is often not exerted on a single target. Their composition varies depending on the method of preparation and the plant materials used. This complexity and variability impact the reproducibility and interpretation of pharmacological, toxicological and clinical research. Objectives: This project develops best practice guidelines to ensure reproducibility and accurate interpretations of studies using medicinal plant extracts. The focus is on herbal extracts used in pharmacological, toxicological, and clinical/intervention research. Specifically, the consensus-based statement focuses on defining requirements for: 1) Describing the plant material/herbal substances, herbal extracts and herbal medicinal products used in these studies, and 2) Conducting and reporting the phytochemical analysis of the plant extracts used in these studies in a reproducible and transparent way. The process and methods: We developed the guidelines through the following process: 1) The distinction between the three main types of extracts (extract types A, B, and C), initially conceptualised by the lead author (MH), led the development of the project as such; 2) A survey among researchers of medicinal plants to gather global perspectives, opportunities, and overarching challenges faced in characterising medicinal plant extracts under different laboratory infrastructures. The survey responses were central to developing the guidelines and were reviewed by the core group; 3) A core group of 9 experts met monthly to develop the guidelines through a Delphi process; and. 4) The final draft guidelines, endorsed by the core group, were also distributed for feedback and approval to an extended advisory group of 20 experts, including many journal editors. Outcome: The primary outcome is the "Consensus statement on the Phytochemical Characterisation of Medicinal Plant extracts" (ConPhyMP) which defines the best practice for reporting the starting plant materials and the chemical methods recommended for defining the chemical compositions of the plant extracts used in such studies. The checklist is intended to be an orientation for authors in medicinal plant research as well as peer reviewers and editors assessing such research for publication.

Ingadosides A-C, acacic acid-type saponins from Inga sapindoides with potent inhibitory activity against downy mildew.

As part of a project aiming at the discovery of environmentally friendly alternatives to copper in organic agriculture, a 96% ethanolic extract from the leaves of Inga sapindoides showed potent inhibitory activity against grapevine downy mildew (Plasmopara viticola) in vitro (MIC100 25 µg/mL). Separation of the n-BuOH soluble fraction by silica gel column chromatography followed by a combination of RP18 and HILIC HPLC resulted in the isolation of a series of bidesmosidic saponins characterized by the presence of a monoterpenoid unit attached to a triterpenoid aglycone, a p-methoxycinnamoyl residue, and rare sugar residues such as N-acetyl-d-glucosamine, d-quinovose, and d-fucose. The isolated compounds inhibited the formation or activity of P. viticola zoospores with MIC100 values of 3 or 6 µg/mL, respectively. I. sapindoides, a tree which is often cultivated for shading coffee plantations in Central America, may represent a sustainable source of fungicidal products to be used in the replacement of copper.

Pheophorbide a identified in an Eupatorium perfoliatum extract is a novel lymphatic vascular activator.

The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies.

Constituents of Passiflora incarnata, but Not of Valeriana officinalis, Interact with the Organic Anion Transporting Polypeptides (OATP)2B1 and OATP1A2.

Herbal medication used in the treatment of sleep disorders and anxiety often contain extracts of Valeriana officinalis or Passiflora incarnata. Valerenic acid in V. officinalis and apigenin, orientin, and vitexin in P. incarnata are thought to contribute to their therapeutic effect. It was the aim of this study to test whether these constituents of herbal extracts are interacting with the uptake of estrone 3-sulfate, pregnenolone sulfate, and dehydroepiandrosterone sulfate mediated by the uptake transporters organic anion transporting polypeptide 2B1 (OATP2B1) or organic anion transporting polypeptide 1A2 (OATP1A2). Madin-Darby canine kidney cells overexpressing OATP2B1 or OATP1A2 were used to determine the influence of the constituents on the cellular accumulation of the sulfated steroids. Subsequently, competitive counterflow experiments were applied to test whether identified inhibitors are also substrates of the transporters. Valerenic acid only interacted with OATP2B1, whereas apigenin, orientin, and vitexin interacted with OATP2B1 and OATP1A2. Competitive counterflow revealed that orientin is a substrate of both transporters, while apigenin was transported by OATP1A2 and vitexin by OATP2B1. In a next step, commercially available P. incarnata preparations were assessed for their influence on the transporters, revealing inhibition of transporter-mediated estrone 3-sulfate uptake. HPLC-UV-MS analysis confirmed the presence of orientin and vitexin in these preparations, thereby suggesting that these constituents are involved in the interaction. Our data indicate that constituents of P. incarnata may alter the function of OATP2B1 and OATP1A2, which could affect the uptake of other compounds relying on uptake mediated by the transporters.

Medicinal Plants for the Treatment of Mental Diseases in Pregnancy: An In Vitro Safety Assessment.

Pregnancy is a critical period for medical care, during which the well-being of woman and fetus must be considered. This is particularly relevant in managing non-psychotic mental disorders since treatment with central nervous system-active drugs and untreated NMDs may have negative effects. Some well-known herbal preparations (phytopharmaceuticals), including St. John's wort, California poppy, valerian, lavender, and hops, possess antidepressant, sedative, anxiolytic, or antidepressant properties and could be used to treat mental diseases such as depression, restlessness, and anxiety in pregnancy. Our goal was to assess their safety in vitro, focusing on cytotoxicity, induction of apoptosis, genotoxicity, and effects on metabolic properties and differentiation in cells widely used as a placental cell model (BeWo b30 placenta choriocarcinoma cells). The lavender essential oil was inconspicuous in all experiments and showed no detrimental effects. At low-to-high concentrations, no extract markedly affected the chosen safety parameters. At an artificially high concentration of 100 µg/mL, extracts from St. John's wort, California poppy, valerian, and hops had minimal cytotoxic effects. None of the extracts resulted in genotoxic effects or altered glucose consumption or lactate production, nor did they induce or inhibit BeWo b30 cell differentiation. This study suggests that all tested preparations from St. John's wort, California poppy, valerian, lavender, and hops, in concentrations up to 30 µg/mL, do not possess any cytotoxic or genotoxic potential and do not compromise placental cell viability, metabolic activity, and differentiation. Empirical and clinical studies during pregnancy are needed to support these in vitro data.