A Dimerosesquiterpene and Sesquiterpene Lactones from Artemisia argyi Inhibiting Oncogenic PI3K/AKT Signaling in Melanoma Cells.

A library of more than 2500 plant extracts was screened for activity on oncogenic signaling in melanoma cells. The ethyl acetate extract from the aerial parts of Artemisia argyi displayed pronounced inhibition of the PI3K/AKT pathway. Active compounds were tracked with the aid of HPLC-based activity profiling, and altogether 21 active compounds were isolated, including one novel dimerosequiterpenoid (1), one new disesquiterpenoid (2), three new guaianolides (3-5), 12 known sesquiterpenoids (6-17), and four known flavonoids (19-22). A new eudesmanolide derivative (13b) was isolated as an artifact formed by methanolysis. Compound 1 is the first adduct comprising a sesquiterpene lactone and a methyl jasmonate moiety. The absolute configurations of compounds 1 and 3-18 were established by comparison of their experimental and calculated ECD spectra. The absolute configuration for 2 was determined by X-ray diffraction analysis. Guaianolide 8 was the most potent sesquiterpene lactone, inhibiting the PI3K/AKT pathway with an IC50 value of 8.9 ± 0.9 µM.

Combining Activity Profiling with Advanced Annotation to Accelerate the Discovery of Natural Products Targeting Oncogenic Signaling in Melanoma.

The discovery of bioactive natural products remains a time-consuming and challenging task. The ability to link high-confidence metabolite annotations in crude extracts with activity would be highly beneficial to the drug discovery process. To address this challenge, HPLC-based activity profiling and advanced UHPLC-HRMS/MS metabolite profiling for annotation were combined to leverage the information obtained from both approaches on a crude extract scaled down to the submilligram level. This strategy was applied to a subset of an extract library screening aiming to identify natural products inhibiting oncogenic signaling in melanoma. Advanced annotation and data organization enabled the identification of compounds that were likely responsible for the activity in the extracts. These compounds belonged to two different natural product scaffolds, namely, brevipolides from a Hyptis brevipes extract and methoxylated flavonoids identified in three different extracts of Hyptis and Artemisia spp. Targeted isolation of these prioritized compounds led to five brevipolides and seven methoxylated flavonoids. Brevipolide A (1) and 6-methoxytricin (9) were the most potent compounds from each chemical class and displayed AKT activity inhibition with an IC50 of 17.6 ± 1.6 and 4.9 ± 0.2 µM, respectively.

High-Content Screening Pipeline for Natural Products Targeting Oncogenic Signaling in Melanoma.

The incidence of melanoma, the most fatal dermatological cancer, has dramatically increased over the last few decades. Modern targeted therapy with kinase inhibitors induces potent clinical responses, but drug resistance quickly develops. Combination therapy improves treatment outcomes. Therefore, novel inhibitors targeting aberrant proliferative signaling in melanoma via the MAPK/ERK and PI3K/AKT pathways are urgently needed. Biosensors were combined that report on ERK/AKT activity with image-based high-content screening and HPLC-based activity profiling. An in-house library of 2576 plant extracts was screened on two melanoma cell lines with different oncogenic mutations leading to pathological ERK/AKT activity. Out of 140 plant extract hits, 44 were selected for HPLC activity profiling. Active thymol derivatives and piperamides from Arnica montana and Piper nigrum were identified that inhibited pathological ERK and/or AKT activity. The pipeline used enabled an efficient identification of natural products targeting oncogenic signaling in melanoma.

Phytotherapy in Integrative Oncology-An Update of Promising Treatment Options.

Modern phytotherapy is part of today's conventional evidence-based medicine and the use of phytopharmaceuticals in integrative oncology is becoming increasingly popular. Approximately 40% of users of such phytopharmaceuticals are tumour patients. The present review provides an overview of the most important plants and nature-based compounds used in integrative oncology and illustrates their pharmacological potential in preclinical and clinical settings. A selection of promising anti-tumour plants and ingredients was made on the basis of scientific evidence and therapeutic practical relevance and included Boswellia, gingko, ginseng, ginger, and curcumin. In addition to these nominees, there is a large number of other interesting plants and plant ingredients that can be considered for the treatment of cancer diseases or for the treatment of tumour or tumour therapy-associated symptoms. Side effects and interactions are included in the discussion. However, with the regular and intended use of phytopharmaceuticals, the occurrence of adverse side effects is rather rare. Overall, the use of defined phytopharmaceuticals is recommended in the context of a rational integrative oncology approach.

Compounds from Toddalia asiatica: Immunosuppressant Activity and Absolute Configurations.

In a screening of an extract library from plants used in Traditional Chinese Medicine the MeOH extract of Toddalia asiatica inhibited proliferation of human primary T cells with an IC50 of 25.8 µg/mL. Activity in the extract was tracked by HPLC activity profiling, and a total of 15 compounds were characterized. Three compounds, toddalic acid (6) and both enantiomers (7a and 7b) of toddanolic acid (7), were new natural products, and two recently published compounds, (2'R)-toddalolactone 3'-O-ß-d-glucopyranoside (10) and (2'S)-toddalolactone 2'-O-ß-d-glucopyranoside (11), were described in detail for the first time. The absolute configurations of compounds 8, 9, 10, 12, 13, and 15 were determined by comparison of experimental and calculated ECD spectra. For glucosides 9 and 10, ECD data and chiral-phase HPLC of the aglycones after enzymatic hydrolysis confirmed the results. Nitidine chloride (4) inhibited proliferation of primary human T cells with an IC50 of 0.4 µM.

Antibacterial and ATP Synthesis Modulating Compounds from Salvia tingitana.

A surface extract of the aerial parts of Salvia tingitana afforded a nor-sesterterpenoid (1) and eight new sesterterpenoids (2-̵9), along with five known sesterterpenoids, five labdane and one abietane diterpenoid, one sesquiterpenoid, and four flavonoids. The structures of the new compounds were established by 1D and 2D NMR spectroscopy, HRESIMS, and VCD data and Mosher's esters analysis. The antimicrobial activity of compounds was evaluated against 30 human pathogens including 27 clinical strains and three isolates of marine origin for their possible implications on human health. The methyl ester of salvileucolide (10), salvileucolide-6,23-lactone (11), sclareol (15), and manool (17) were the most active against Gram-positive bacteria. The compounds were also tested for the inhibition of ATP production in purified mammalian rod outer segments. Terpenoids 10, 11, 15, and 17 inhibited ATP production, while only 17 inhibited also ATP hydrolysis. Molecular modeling studies confirmed the capacity of 17 to interact with mammalian ATP synthase. A significant reduction of ATP production in the presence of 17 was observed in Enterococcus faecalis and E. faecium isolates.

Sesquiterpene Lactones from Artemisia argyi: Absolute Configuration and Immunosuppressant Activity.

A library of extracts from plants used in Chinese Traditional Medicine was screened for inhibition of T lymphocyte proliferation. An ethyl acetate extract from aerial parts of Artemisia argyi showed promising activity and was submitted to HPLC-based activity profiling to track the active compounds. From the most active time window, three guaianolides (1, 2, and 5) and two seco-tanapartholides (3 and 4) were identified and, in a less active time window, five new sesquiterpene lactones (8-11, 17), along with six known sesquiterpene lactones and two known flavonoids. The absolute configurations of compounds 1, 2, 5-10, 13-15, 17, and 18 were established by comparison of experimental with calculated electronic circular dichroism (ECD) spectra. For seco-tanapartholides B (3) and A (4), ECD yielded ambiguous results, and their absolute configurations were determined by comparing experimental and calculated vibrational circular dichroism (VCD) spectra. Compounds 1-5 showed significant, noncytotoxic inhibition of T lymphocyte proliferation, with IC50 values between 1.0 and 3.7 µM.

Highly oxygenated antiplasmodial and non-hemolytic Δ7,9(11) stigmastane-type steroids from the twigs of Vernonia amygdalina Delile.

Chemical investigations of a methanolic extract of the twigs of Vernonia amygdalina Delile (Asteraceae) resulted in the isolation and identification of three previously undescribed highly oxygenated Δ7,9(11) stigmastane-type steroids namely vernonins U-W (1-3) along with six known compounds (4-9). The structural characterization of all the isolated compounds has been conducted via comprehensive 1D and 2D-NMR spectroscopy as well as HRMS. The seven steroidal derivatives 1-7 were evaluated for their antiplasmodial activity against the chloroquine-resistant strain P. falciparum Dd2 (PfDd2) and their hemolytic effect on human red blood cells (RBCs). Vernonins U (1), A (4) and stigmasterol-3-O-ß-d-glucopyranoside (7) showed the highest activity with IC50 values of (5.47 ± 0.01) µg/mL, (6.02 ± 0.13) µg/mL and (6.34 ± 0.80) µg/mL, respectively, against PfDd2, while vernonin W (3) showed moderate activity of (21.20 ± 0.40) µg/mL. None of the tested compounds displayed hemolytic effects on human RBCs up to 100 µg/mL indicating their safety. These results enrich the known chemistry of V. amygdalina and support its use in folk medicine for the treatment of malaria. This encourages further research towards new antiplasmodial drug candidates from this plant.

Biosynthesis of the bacterial antibiotic 3,7-dihydroxytropolone through enzymatic salvaging of catabolic shunt products.

The non-benzenoid aromatic tropone ring is a structural motif of numerous microbial and plant natural products with potent bioactivities. In bacteria, tropone biosynthesis involves early steps of the widespread CoA-dependent phenylacetic acid (paa) catabolon, from which a shunt product is sequestered and surprisingly further utilized as a universal precursor for structurally and functionally diverse tropone derivatives such as tropodithietic acid or (hydroxy)tropolones. Here, we elucidate the biosynthesis of the antibiotic 3,7-dihydroxytropolone in Actinobacteria by in vitro pathway reconstitution using paa catabolic enzymes as well as dedicated downstream tailoring enzymes, including a thioesterase (TrlF) and two flavoprotein monooxygenases (TrlCD and TrlE). We furthermore mechanistically and structurally characterize the multifunctional key enzyme TrlE, which mediates an unanticipated ipso-substitution involving a hydroxylation and subsequent decarboxylation of the CoA-freed side chain, followed by ring oxidation to afford tropolone. This study showcases a remarkably efficient strategy for 3,7-dihydroxytropolone biosynthesis and illuminates the functions of the involved biosynthetic enzymes.

Phytochemical Comparison of Medicinal Cannabis Extracts and Study of Their CYP-Mediated Interactions with Coumarinic Oral Anticoagulants.

Introduction: Treatment with cannabis extracts for a variety of diseases has gained popularity. However, differences in herb-drug interaction potential of extracts from different plant sources are poorly understood. In this study, we provide a characterization of cannabis extracts prepared from four cannabis chemotypes and an in vitro assessment of their Cytochrome P450 (CYP)-mediated herb-drug interaction profiles. Methods: Plant extracts were either commercially obtained or prepared using ethanol as solvent, followed by overnight decarboxylation in a reflux condenser system. The extracts were characterized for their cannabinoid content using NMR and HPLC-PDA-ELSD-ESIMS. CYP inhibition studies with the cannabis extracts and pure cannabinoids (tetrahydrocannabinol [THC] and cannabidiol [CBD]) were performed using pooled, mixed gender human liver microsomes. Tolbutamide and testosterone were used as specific substrates to assess the inhibitory potential of the extracts on CYP2C9 and CYP3A4, and the coumarinic oral anticoagulants warfarin, phenprocoumon, and acenocoumarol were studied as model compounds since in vivo herb-drug interactions have previously been reported for this compound class. Results: In accordance with the plant chemotypes, two extracts were rich in THC and CBD (at different proportions); one extract contained mostly CBD and the other mostly cannabigerol (CBG). Residual amounts of the corresponding acids were found in all extracts. The extracts with a single major cannabinoid (CBD or CBG) inhibited CYP2C9- and CYP3A4-mediated metabolism stronger than the extracts containing both major cannabinoids (THC and CBD). The inhibition of CYP3A4 and CYP2C9 by the extract containing mostly CBD was comparable to their inhibition by pure CBD. In contrast, the inhibitory potency of extracts containing both THC and CBD did not correspond to the combined inhibitory potency of pure THC and CBD. Although being structural analogs, the three coumarin derivatives displayed major differences in their herb-drug interaction profiles with the cannabis extracts and the pure cannabinoids. Conclusion: Despite the fact that cannabinoids are the major components in ethanolic, decarboxylated cannabis extracts, it is difficult to foresee their herb-drug interaction profiles. Our in vitro data and the literature-based evidence on in vivo interactions indicate that cannabis extracts should be used cautiously when co-administered with drugs exhibiting a narrow therapeutic window, such as coumarinic anticoagulants, regardless of the cannabis chemotype used for extract preparation.

Flavonoids from Ericameria nauseosa inhibiting PI3K/AKT pathway in human melanoma cells.

The PI3K/AKT and MAPK/ERK pathways are frequently mutated in metastatic melanoma. In a screen of over 2500 plant extracts, the dichloromethane extract of Ericameria nauseosa significantly inhibited oncogenic activity of AKT in MM121224 human melanoma cells. This extract was analyzed by analytical HPLC, and the column effluent was fractionated and tested for activity to generate the so-called HPLC-based activity profile. Compounds eluting within active time-windows of the chromatogram were subsequently isolated in a larger scale to afford 11 flavones (1-11), four flavanones (12-15), two diterpenes (16, 17), and a seco-caryophyllene (18). All isolated compounds were tested for activity, whereby only flavonoids were found active. Of these, flavones were shown to be more active than the flavanones. The most potent flavone was compound 9, that was displaying an IC50 of 14.7 ± 1.4 µM on AKT activity in MM121224 cells. The terpenoids (16-18) were found to be inactive in the assay. Both diterpenes, a grindelic acid derivative (16) and an ent-neo-clerodane (17) were identified as new natural products. Their absolute configuration was established by ECD. Compound 17 is the first description of a clerodane type diterpene in the genus Ericameria.

Immunosuppressant flavonoids from Scutellaria baicalensis.

Some plants used in Traditional Chinese Medicine serve as treatment for disease states where a suppression of the cellular immune response is desired. However, the compounds responsible for the immunosuppressant effects of these plants are not necessarily known. The immunosuppressant compounds in the roots of Scutellaria baicalensis, one of the most promising plants identified in a previous screening, were tracked by HPLC activity profiling and concomitant on-line spectroscopic analysis. Compounds were then isolated by preparative chromatography, and structures elucidated by spectroscopic methods. Twelve flavonoids (5-16) were identified from the active time windows, and structurally related flavones 2, 4, and 17, and flavanones 1 and 3 were isolated from adjacent fractions. All flavonoids possessed an unusual substitution pattern on the B-ring, with an absence of substituents at C-3 and C-4. Compounds 11, 13, 14, and 16 inhibited T-cell proliferation (IC50 values at 12.1-39 µM) at non-cytotoxic concentrations. The findings may support the use of S. baicalensis in disorders where a modulation of the cellular immune response is desirable.

European medicinal mushrooms: Do they have potential for modern medicine? - An update.

BACKGROUND: The application of mushrooms for health purposes has a long tradition and is very common in Asian countries. This trend is also becoming increasingly popular in the western hemisphere. However, mushrooms from European tradition are being treated in a restrained manner despite having significant potential as drugs or as sources of pure bioactive substances. AIM: The present review provides an overview of the most important mushrooms used in European ethnomedical traditions and explores their pharmacological potential and the challenges for the development of new drugs from these sources of natural products. METHOD: Mushroom species were selected based on information in old herbal books and dispensaries, uninterrupted use and scientific literature in the PubMed database up to June 2019. RESULTS: Traditional experiences and modern studies have demonstrated that medical mushrooms used in European traditions have promising distinct pharmacological potential mediated through defined mechanisms (anti-tumour, anti-inflammatory, anti-oxidative and anti-bacterial). However, the number of modern chemical, biological and pharmacological studies remains relatively small, and some mushroom species have not been studied at all. Unfortunately, no valid clinical studies can be found. Unlike the case with herbal and fungal drugs from traditional Chinese medicine, we are far from comprehensively exploring this potential. CONCLUSIONS: Mushrooms from traditional European medicine have the potential to be used in modern medicine. Considerable research, interdisciplinary collaboration, involvement of the pharmaceutical industry, time and money are necessary to explore this potential not only in the form of dietary supplements but also in the form of approved drugs.

Boswellia carteri extract and 3-O-acetyl-alpha-boswellic acid suppress T cell function.

Resins from various Boswellia species have a long track record in different cultures as a treatment for inflammatory diseases. This study was designed to provide evidence for the anti-inflammatory capacity and medicinal use of Boswellia carteri (Burseraceae). A dichloromethane (DCM) extract of B. carteri gum resin and isolated compounds thereof were immunologically characterized. Flow cytometric-based analysis was performed to investigate the impact of B. carteri extract on proliferation, viability, and function of anti-CD3 and anti-CD28 activated human primary T cells. The secretion level of IL-2 and IFN-γ was determined by a bead array-based flow cytometric technique. HPLC-based activity profiling of the B. carteri extract identified active compounds. The impact of B. carteri extract and isolated compounds on the IL-2 transcription factor activity was addressed using specially designed Jurkat reporter cells. The extract of B. carteri suppressed the proliferation of human primary T lymphocytes in vitro in a concentration-dependent manner, without inducing cytotoxicity. Thereby, the B. carteri extract further reduced the degranulation capacity and cytokine secretion of stimulated human T cells. Transcription factor analysis showed that the immunosuppressive effects of the extract are based on specific NFAT-conditioned suppression within T cell signaling. Through HPLC-based activity profiling of the extract, 3-O-acetyl-alpha-boswellic acid was identified as the compound responsible for the NFAT-based mechanism. The recent study presents a scientific base for the immunosuppressive effects of B. carteri gum resin extract including a mode-of-action via the NFAT-conditioned suppression of T lymphocyte proliferation. The immunosuppressive effects of 3-O-acetyl-alpha-boswellic acid are depicted for the first time.

Immunosuppressive Activity of Artemisia argyi Extract and Isolated Compounds.

The need for novel drugs for the treatment of autoimmune diseases is high, since available pharmaceuticals often have substantial side effects and limited efficacy. Natural products are a good starting point in the development of immunosuppressive leads. Since enhanced T cell proliferation is a common feature of autoimmune diseases, we investigated the T cell proliferation inhibitory potential of an extract library of plants used in traditional Chinese medicine. Using a newly established cell-based screening platform, an ethyl acetate extract of Artemisia argyi H.Lév. & Vaniot (Asteraceae, A. argyi) was found to suppress the proliferation of human primary T lymphocytes in vitro in an IL-2-dependent manner. Flow cytometry- and ELISA-based techniques further demonstrated that the A. argyi extract reduced the activation and function of T cells. Transcription factor analysis and flow cytometric calcium influx investigations indicated that the immunomodulatory effect was based on specific modification of T cell signaling in a non-cytotoxic manner which is mediated via the NFAT pathway and a non-sequestrant inhibition of the calcium influx. A series of guaianolide and seco-guaianolide sesquiterpene lactones, as well as a flavonoid, were identified in a previous study as the bioactive compounds in the A. argyi extract. The effects of these bioactive compounds were compared to those of the crude extract. The tested sesquiterpene lactones act via the transcription factor NFAT and NF-κB, thereby exhibiting their immunosuppressive potential, but have an overall effect on T cell biology on a more-downstream level than the crude A. argyi extract.