In Vitro and In Silico Studies of Neolignans from Magnolia grandiflora L. Seeds against Human Cannabinoids and Opioid Receptors.

Magnolia grandiflora L. (Magnoliaceae) is a plant of considerable medicinal significance; its flowers and seeds have been used in various traditional remedies. Radioligand binding assays of n-hexane seeds extract showed displacement of radioligand for cannabinoid (CB1 and CB2) and opioid δ (delta), κ (kappa), and µ (mu) receptors. Bioactivity-guided fractionation afforded 4-O-methylhonokiol (1), magnolol (2), and honokiol (3), which showed higher binding to cannabinoid rather than opioid receptors in radioligand binding assays. Compounds 1-3, together with the dihydro analog of 2 (4), displayed selective affinity towards CB2R (Ki values of 0.29, 1.4, 1.94, and 0.99 µM, respectively), compared to CB1R (Ki 3.85, 17.82, 14.55, and 19.08 µM, respectively). An equal mixture of 2 and 3 (1:1 ratio) showed additive displacement activity towards the tested receptors compared to either 2 or 3 alone, which in turn provides an explanation for the strong displacement activity of the n-hexane extract. Due to the unavailability of an NMR or X-ray crystal structure of bound neolignans with the CB1 and CB2 receptors, a docking study was performed to predict ligand-protein interactions at a molecular level and to delineate structure-activity relationships (SAR) of the neolignan analogs with the CB1 and CB2 receptors. The putative binding modes of neolignans 1-3 and previously reported related analogs (4, 4a, 5, 5a, 6, 6a, and 6b) into the active site of the CB1 and CB2 receptors were assessed for the first time via molecular docking and binding free-energy (∆G) calculations. The docking and ∆G results revealed the importance of a hydroxyl moiety in the molecules that forms strong H-bonding with Ser383 and Ser285 within CB1R and CB2R, respectively. The impact of a shift from a hydroxyl to the methoxy group on experimental binding affinity to CB1R versus CB2R was explained through ∆G data and the orientation of the alkyl chain within the CB1R. This comprehensive SAR, influenced by the computational study and the observed in vitro displacement binding affinities, has indicated the potential of magnolia neolignans for developing new CB agonists for potential use as analgesics, anti-inflammatory agents, or anxiolytics.

Cannabinoid and Opioid Receptor Affinity and Modulation of Cancer-Related Signaling Pathways of Machaeriols and Machaeridiols from Machaerium Pers.

Machaeriols and machaeridiols are unique hexahydrodibenzopyran-type aralkyl phytocannabinoids isolated from Machaerium Pers. Earlier studies of machaeriol A (1) and B (2) did not show any affinity for cannabinoid receptor 1 (CB1 or CNR1), although they are structural analogs of psychoactive hexahydrocannabinol. This study comprehensively reports on the affinities of isolated Machaerium Pers. compounds, namely machaeriol A-D (1-4) and machaeridiol A-C (5-7), against cannabinoid (CB1 and CB2) and opioid (κ, δ and µ) receptors. Among the isolated compounds, machaeriol D (4) and machaeridiol A-C (5-7) showed some selective binding affinity for the CB2 receptor, using a radioligand binding assay, with Ki values of >1.3, >1.77, >2.18 and >1.1 µM, respectively. On the other hand, none of the compounds showed any binding to the CB1 receptor. Due to recent reports on the anticancer potential of the endocannabinoid system, compounds 1-7 were tested against a battery of luciferase reporter gene vectors that assess the activity of many cancer-related signaling pathways, including Stat3, Smad2/3, AP-1, NF-κB, E2F, Myc, Ets, Notch, FoxO, Wnt, Hedgehog and pTK in HeLa and T98G glioblastoma cells. Complete dose-response curves have been determined for each compound in both of these cell lines, which revealed that machaeridiol 6 displayed activities (IC50 in µM in HeLa and T98G cells) towards Stat3 (4.7, 1.4), Smad2/3 (1.2, 3.0), AP-1 (5.9, 4.2), NF-κB (0.5, 4.0), E2F (5.7, 0.7), Myc (5.3, 2.0), ETS (inactive, 5.9), Notch (5.3, 4.6), Wnt (4.2, inactive) and Hedgehog (inactive, 5.0). Furthermore, a combination study between machaeriol C (3) and machaeridiol B (6) displayed additive effects for E2F, ETS, Wnt and Hedgehog pathways, where these compounds individually were either minimally active or inactive. None of the compounds inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity for luciferase enzyme inhibition at the 50 µM concentration in both of these cell lines. The significance of the inhibition of these signaling pathways via machaeridiol 5-7 and their cross-talk potential has been discussed.

Natural Products Inhibitors of Monoamine Oxidases-Potential New Drug Leads for Neuroprotection, Neurological Disorders, and Neuroblastoma.

Monoamine oxidase inhibitors (MAOIs) are an important class of drugs prescribed for treatment of depression and other neurological disorders. Evidence has suggested that patients with atypical depression preferentially respond to natural product MAOIs. This review presents a comprehensive survey of the natural products, predominantly from plant sources, as potential new MAOI drug leads. The psychoactive properties of several traditionally used plants and herbal formulations were attributed to their MAOI constituents. MAO inhibitory constituents may also be responsible for neuroprotective effects of natural products. Different classes of MAOIs were identified from the natural product sources with non-selective as well as selective inhibition of MAO-A and -B. Selective reversible natural product MAOIs may be safer alternatives to the conventional MAOI drugs. Characterization of MAO inhibitory constituents of natural products traditionally used as psychoactive preparations or for treatment of neurological disorders may help in understanding the mechanism of action, optimization of these preparations for desired bioactive properties, and improvement of the therapeutic potential. Potential therapeutic application of natural product MAOIs for treatment of neuroblastoma is also discussed.

Synthesis and Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways.

Three unique 5,6-seco-hexahydrodibenzopyrans (seco-HHDBP) machaeridiols A−C, reported previously from Machaerium Pers., have displayed potent activities against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium, and E. faecalis (VRE). In order to enrich the pipeline of natural product-derived antimicrobial compounds, a series of novel machaeridiol-based analogs (1−17) were prepared by coupling stemofuran, pinosylvin, and resveratrol legends with monoterpene units R-(−)-α-phellandrene, (−)-p-mentha-2,8-diene-1-ol, and geraniol, and their inhibitory activities were profiled against MRSA ATCC 1708, VRE ATCC 700221, and cancer signaling pathways. Compounds 5 and 11 showed strong in vitro activities with MIC values of 2.5 µg/mL and 1.25 µg/mL against MRSA, respectively, and 2.50 µg/mL against VRE, while geranyl analog 14 was found to be moderately active (MIC 5 µg/mL). The reduction of the double bonds of the monoterpene unit of compound 5 resulted in 17, which had the same antibacterial potency (MIC 1.25 µg/mL and 2.50 µg/mL) as its parent, 5. Furthermore, a combination study between seco-HHDBP 17 and HHDBP machaeriol C displayed a synergistic effect with a fractional inhibitory concentrations (FIC) value of 0.5 against MRSA, showing a four-fold decrease in the MIC values of both 17 and machaeriol C, while no such effect was observed between vancomycin and 17. Compounds 11 and 17 were further tested in vivo against nosocomial MRSA at a single intranasal dose of 30 mg/kg in a murine model, and both compounds were not efficacious under these conditions. Finally, compounds 1−17 were profiled against a panel of luciferase genes that assessed the activity of complex cancer-related signaling pathways (i.e., transcription factors) using T98G glioblastoma multiforme cells. Among the compounds tested, the geranyl-substituted analog 14 exhibited strong inhibition against several signaling pathways, notably Smad, Myc, and Notch, with IC50 values of 2.17 µM, 1.86 µM, and 2.15 µM, respectively. In contrast, the anti-MRSA actives 5 and 17 were found to be inactive (IC50 > 20 µM) across the panel of these cancer-signaling pathways.

Crosstalk of Cancer Signaling Pathways by Cyclic Hexapeptides and Anthraquinones from Rubia cordifolia.

The anticancer activities of Rubia cordifolia and its constituents have been reported earlier, but their influence on the crosstalk of complex cancer-related signaling metabolic pathways (i.e., transcription factors; TF) has not yet been fully investigated. In this study, R. cordifolia root extract was subjected to the cancer signaling assay based bioactivity-guided fractionation, which yielded the following compounds viz., three anthraquinones, namely alizarin (1), purpurin (2), and emodin (3); two lignans, namely eudesmin (4) and compound 5; and two cyclic hexapeptides, namely deoxybouvardin RA-V (6), and a mixture of 6+9 (RA-XXI). The structures of the isolated compounds were determined by NMR spectroscopy and HRESIMS. The isolated compounds 1, 2, 3, 6, and a mixture of 6+9 were tested against a panel of luciferase reporter genes that assesses the activity of a wide-range of cancer-related signaling pathways. In addition, reference anthraquinones viz., chrysophanol (11), danthron (12), quinizarin (13), aloe-emodin (14), and α-lapachone (15) were also tested. Among the tested compounds, the cyclic hexapeptide 6 was found to be very active against several signaling pathways, notably Wnt, Myc, and Notch with IC50 values of 50, 75, and 93 ng/mL, respectively. Whereas, the anthraquinones exhibited very mild or no inhibition against these signaling pathways. Compound 6 being the most active, we tested it for stability in simulated intestinal (SIF) and gastric fluids (SGF), since the stability in biological fluid is a key short-coming of cyclic hexapeptides. The anticancer activity of 6 was found to remain unchanged before and after the treatment of simulated gastric/intestinal fluids, indicating that RA-V was stable. As a result, it could be bioavailable when orally used in therapeutics and possibly a drug candidate for cancer treatment. The mechanism for the preferential inhibition of these pathways and the possible crosstalk effect with other previously reported signaling pathways has been discussed.

Ethiopian Medicinal Plants Traditionally Used for the Treatment of Cancer; Part 3: Selective Cytotoxic Activity of 22 Plants against Human Cancer Cell Lines.

Medicinal plants have been traditionally used to treat cancer in Ethiopia. However, very few studies have reported the in vitro anticancer activities of medicinal plants that are collected from different agro-ecological zones of Ethiopia. Hence, the main aim of this study was to screen the cytotoxic activities of 80% methanol extracts of 22 plants against human peripheral blood mononuclear cells (PBMCs), as well as human breast (MCF-7), lung (A427), bladder (RT-4), and cervical (SiSo) cancer cell lines. Active extracts were further screened against human large cell lung carcinoma (LCLC-103H), pancreatic cancer (DAN-G), ovarian cancer (A2780), and squamous cell carcinoma of the esophagus (KYSE-70) by using the crystal violet cell proliferation assay, while the vitality of the acute myeloid leukemia (HL-60) and histiocytic lymphoma (U-937) cell lines was monitored in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) microtiter assay. Euphorbia schimperiana, Acokanthera schimperi, Kniphofia foliosa, and Kalanchoe petitiana exhibited potent antiproliferative activity against A427, RT-4, MCF-7, and SiSo cell lines, with IC50 values ranging from 1.85 ± 0.44 to 17.8 ± 2.31 µg/mL. Furthermore, these four extracts also showed potent antiproliferative activities against LCLC-103H, DAN-G, A2780, KYSE-70, HL-60, and U-937 cell lines, with IC50 values ranging from 0.086 to 27.06 ± 10.8 µg/mL. Hence, further studies focusing on bio-assay-guided isolation and structural elucidation of active cytotoxic compounds from these plants are warranted.

Therapeutic Intervention of COVID-19 by Natural Products: A Population-Specific Survey Directed Approach.

To date very few promising leads from natural products (NP) secondary metabolites with antiviral and immunomodulatory properties have been identified for promising/potential intervention for COVID-19. Using in-silico docking studies and genome based various molecular targets, and their in vitro anti-SARS CoV-2 activities against whole cell and/or selected protein targets, we select a few compounds of interest, which can be used as potential leads to counteract effects of uncontrolled innate immune responses, in particular those related to the cytokine storm. A critical factor for prevention and treatment of SARS-CoV-2 infection relates to factors independent of viral infection or host response. They include population-related variables such as concurrent comorbidities and genetic factors critically relevant to COVID-19 health disparities. We discuss population risk factors related to SARS-CoV-2. In addition, we focus on virulence related to glucose-6-phosphate dehydrogenase deficiency (G6PDd), the most common human enzymopathy. Review of data on the response of individuals and communities with high prevalence of G6PDd to NP, prompts us to propose the rationale for a population-specific management approach to rationalize design of therapeutic interventions of SARS-CoV-2 infection, based on use of NP. This strategy may lead to personalized approaches and improve disease-related outcomes.

Ethiopian Medicinal Plants Traditionally Used for the Treatment of Cancer, Part 2: A Review on Cytotoxic, Antiproliferative, and Antitumor Phytochemicals, and Future Perspective.

This review provides an overview on the active phytochemical constituents of medicinal plants that are traditionally used to manage cancer in Ethiopia. A total of 119 articles published between 1968 and 2020 have been reviewed, using scientific search engines such as ScienceDirect, PubMed, and Google Scholar. Twenty-seven medicinal plant species that belong to eighteen families are documented along with their botanical sources, potential active constituents, and in vitro and in vivo activities against various cancer cells. The review is compiled and discusses the potential anticancer, antiproliferative, and cytotoxic agents based on the types of secondary metabolites, such as terpenoids, phenolic compounds, alkaloids, steroids, and lignans. Among the anticancer secondary metabolites reported in this review, only few have been isolated from plants that are originated and collected in Ethiopia, and the majority of compounds are reported from plants belonging to different areas of the world. Thus, based on the available bioactivity reports, extensive and more elaborate ethnopharmacology-based bioassay-guided studies have to be conducted on selected traditionally claimed Ethiopian anticancer plants, which inherited from a unique and diverse landscape, with the aim of opening a way forward to conduct anticancer drug discovery program.

Selective Interactions of O-Methylated Flavonoid Natural Products with Human Monoamine Oxidase-A and -B.

A set of structurally related O-methylated flavonoid natural products isolated from Senecio roseiflorus (1), Polygonum senegalense (2 and 3), Bhaphia macrocalyx (4), Gardenia ternifolia (5), and Psiadia punctulata (6) plant species were characterized for their interaction with human monoamine oxidases (MAO-A and -B) in vitro. Compounds 1, 2, and 5 showed selective inhibition of MAO-A, while 4 and 6 showed selective inhibition of MAO-B. Compound 3 showed ~2-fold selectivity towards inhibition of MAO-A. Binding of compounds 1-3 and 5 with MAO-A, and compounds 3 and 6 with MAO-B was reversible and not time-independent. The analysis of enzyme-inhibition kinetics suggested a reversible-competitive mechanism for inhibition of MAO-A by 1 and 3, while a partially-reversible mixed-type inhibition by 5. Similarly, enzyme inhibition-kinetics analysis with compounds 3, 4, and 6, suggested a competitive reversible inhibition of MAO-B. The molecular docking study suggested that 1 selectively interacts with the active-site of human MAO-A near N5 of FAD. The calculated binding free energies of the O-methylated flavonoids (1 and 4-6) and chalcones (2 and 3) to MAO-A matched closely with the trend in the experimental IC50's. Analysis of the binding free-energies suggested better interaction of 4 and 6 with MAO-B than with MAO-A. The natural O-methylated flavonoid (1) with highly potent inhibition (IC50 33 nM; Ki 37.9 nM) and >292 fold selectivity against human MAO-A (vs. MAO-B) provides a new drug lead for the treatment of neurological disorders.

Antimicrobial Constituents from Machaerium Pers.: Inhibitory Activities and Synergism of Machaeriols and Machaeridiols against Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococcus faecium, and Permeabilized Gram-Negative Pathogens.

Two new epimeric bibenzylated monoterpenes machaerifurogerol (1a) and 5-epi-machaerifurogerol (1b), and four known isoflavonoids (+)-vestitol (2), 7-O-methylvestitol (3), (+)-medicarpin (4), and 3,8-dihydroxy-9-methoxypterocarpan (5) were isolated from Machaerium Pers. This plant was previously assigned as Machaerium multiflorum Spruce, from which machaeriols A-D (6-9) and machaeridiols A-C (10-12) were reported, and all were then re-isolated, except the minor compound 9, for a comprehensive antimicrobial activity evaluation. Structures of the isolated compounds were determined by full NMR and mass spectroscopic data. Among the isolated compounds, the mixture 10 + 11 was the most active with an MIC value of 1.25 µg/mL against methicillin-resistant Staphylococcus aureus (MRSA) strains BAA 1696, -1708, -1717, -33591, and vancomycin-resistant Enterococcus faecium (VRE 700221) and E. faecalis (VRE 51299) and vancomycin-sensitive E. faecalis (VSE 29212). Compounds 6-8 and 10-12 were found to be more potent against MRSA 1708, and 6, 11, and 12 against VRE 700221, than the drug control ciprofloxacin and vancomycin. A combination study using an in vitro Checkerboard method was carried out for machaeriols (7 or 8) and machaeridiols (11 or 12), which exhibited a strong synergistic activity of 12 + 8 (MIC 0.156 and 0.625 µg/mL), with >32- and >8-fold reduction of MIC's, compared to 12, against MRSA 1708 and -1717, respectively. In the presence of sub-inhibitory concentrations on polymyxin B nonapeptide (PMBN), compounds 10 + 11, 11, 12, and 8 showed activity in the range of 0.5-8 µg/mL for two strains of Acinetobacter baumannii, 2-16 µg/mL against Pseudomonas aeruginosa PAO1, and 2 µg/mL against Escherichia coli NCTC 12923, but were inactive (MIC > 64 µg/mL) against the two isolates of Klebsiella pneumoniae.

Selective Inhibition of Human Monoamine Oxidase B by Acacetin 7-Methyl Ether Isolated from Turnera diffusa (Damiana).

The investigation of the constituents that were isolated from Turnera diffusa (damiana) for their inhibitory activities against recombinant human monoamine oxidases (MAO-A and MAO-B) in vitro identified acacetin 7-methyl ether as a potent selective inhibitor of MAO-B (IC50 = 198 nM). Acacetin 7-methyl ether (also known as 5-hydroxy-4', 7-dimethoxyflavone) is a naturally occurring flavone that is present in many plants and vegetables. Acacetin 7-methyl ether was four-fold less potent as an inhibitor of MAO-B when compared to acacetin (IC50 = 50 nM). However, acacetin 7-methyl ether was >500-fold selective against MAO-B over MAO-A as compared to only two-fold selectivity shown by acacetin. Even though the IC50 for inhibition of MAO-B by acacetin 7-methyl ether was ~four-fold higher than that of the standard drug deprenyl (i.e., SelegilineTM or ZelaparTM, a selective MAO-B inhibitor), acacetin 7-methyl ether's selectivity for MAO-B over MAO-A inhibition was greater than that of deprenyl (>500- vs. 450-fold). The binding of acacetin 7-methyl ether to MAO-B was reversible and time-independent, as revealed by enzyme-inhibitor complex equilibrium dialysis assays. The investigation on the enzyme inhibition-kinetics analysis with varying concentrations of acacetin 7-methyl ether and the substrate (kynuramine) suggested a competitive mechanism of inhibition of MAO-B by acacetin 7-methyl ether with Ki value of 45 nM. The docking scores and binding-free energies of acacetin 7-methyl ether to the X-ray crystal structures of MAO-A and MAO-B confirmed the selectivity of binding of this molecule to MAO-B over MAO-A. In addition, molecular dynamics results also revealed that acacetin 7-methyl ether formed a stable and strong complex with MAO-B. The selective inhibition of MAO-B suggests further investigations on acacetin 7-methyl as a potential new drug lead for the treatment of neurodegenerative disorders, including Parkinson's disease.

4-O-Methylhonokiol Influences Normal Cardiovascular Development in Medaka Embryo.

Although 4-O-Methylhonokiol (MH) effects on neuronal and immune cells have been established, it is still unclear whether MH can cause a change in the structure and function of the cardiovascular system. The overarching goal of this study was to evaluate the effects of MH, isolated from Magnolia grandiflora, on the development of the heart and vasculature in a Japanese medaka model in vivo to predict human health risks. We analyzed the toxicity of MH in different life-stages of medaka embryos. MH uptake into medaka embryos was quantified. The LC50 of two different exposure windows (stages 9⁻36 (0⁻6 days post fertilization (dpf)) and 25⁻36 (2⁻6 dpf)) were 5.3 ± 0.1 µM and 9.9 ± 0.2 µM. Survival, deformities, days to hatch, and larval locomotor response were quantified. Wnt 1 was overexpressed in MH-treated embryos indicating deregulation of the Wnt signaling pathway, which was associated with spinal and cardiac ventricle deformities. Overexpression of major proinflammatory mediators and biomarkers of the heart were detected. Our results indicated that the differential sensitivity of MH in the embryos was developmental stage-specific. Furthermore, this study demonstrated that certain molecules can serve as promising markers at the transcriptional and phenotypical levels, responding to absorption of MH in the developing embryo.

Efficacy of Prosopilosidine from Prosopis glandulosa var. glandulosa against Cryptococcus neoformans Infection in a Murine Model.

In this study, 2,3-dihydro-1H-indolizinium alkaloid-prosopilosidine (PPD), that was isolated from Prosopis glandulosa, was evaluated against C. neoformans in a murine model of cryptococcosis. In vitro and in vivo toxicity of indolizidines were also evaluated. Mice were infected via the tail vein with live C. neoformans. Twenty-four hours post-infection, the mice were treated with PPD once a day (i.p.) or twice a day (bid) orally, or with amphotericin B (Amp B) intraperitoneally (IP), or with fluconazole (Flu) orally for 5 days. The brains of all of the animals were aseptically removed and the numbers of live C. neoformans were recovered. In vitro toxicity of indolizidine alkaloids was determined in HepG2 cells. PPD showed to be potent in vivo activity against C. neoformans at a dose of 0.0625 mg/kg by eliminating ~76% of the organisms compared to ~83% with Amp B (1.5 mg/kg). In addition, PPD was found to be equally efficacious, but less toxic, at either 0.125 or 0.0625 mg/kg compared to Amp B (1.5 mg/kg) when it was administered bid (twice a day) by an i.p. route. When tested by an oral route, PPD (10 mg/kg) showed potent activity in this murine model of cryptococcosis with ~82% of organisms eliminated from the brain tissue, whereas Flu (15 mg/kg) reduced ~90% of the infection. In vitro results suggest that quaternary indolizidines were less toxic as compared to those of tertiary bases. PPD (20 mg/kg) did not cause any alteration in the plasma chemistry profiles. These results indicated that PPD was active in eliminating cryptococcal infection by oral and i.p. routes at lower doses compared to Amp B. or Flu.

Tetra-glucopyranosyl Diterpene ent-Kaur-16-en-19-oic Acid and ent-13(S)-Hydroxyatisenoic Acid Derivatives from a Commercial Extract of Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana and its diterpene glycosides are one of the main focuses of food companies interested in developing novel zero calorie sugar substitutes since the recognition of steviol glycosides as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Rebaudioside A, one of the major steviol glycosides of the leaves is more than 200 times sweeter than sucrose. However, its lingering aftertaste makes it less attractive as a table-top sweetener, despite its human health benefits. Herein, we report the purification of two novel tetra-glucopyranosyl diterpene glycosides 1 and 3 (rebaudioside A isomers) from a commercial Stevia rebaudiana leaf extract compounds, their saponification products compounds 2 and 4, together with three known compounds isolated in gram quantities. Compound 1 was determined to be 13-[(2-O-ß-d-glucopyranosyl-6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid-ß-d-glucopyranosy ester (rebaudioside Z), whereas compound 3 was found to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid -ß-d-glucopyranosy ester. Two new tetracyclic derivatives with no sugar at position C-19 were prepared from rebaudiosides 1 and 3 under mild alkaline hydrolysis to afford compounds 2 13-[(2-O-ß-d-glucopyranosyl-6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z1) and 4 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid. Three known compounds were purified in gram quantities and identified as rebaudiosides A (5), H (6) and J (7). Chemical structures were unambiguously elucidated using different approaches, namely HRESIMS, HRESI-MS/MS, and 1D-and 2D-NMR spectroscopic data. Additionally, a high-quality crystal of iso-stevioside was grown in methanol and its structure confirmed by X-ray diffraction.

Diversity-oriented natural product platform identifies plant constituents targeting Plasmodium falciparum.

BACKGROUND: A diverse library of pre-fractionated plant extracts, generated by an automated high-throughput system, was tested using an in vitro anti-malarial screening platform to identify known or new natural products for lead development. The platform identifies hits on the basis of in vitro growth inhibition of Plasmodium falciparum and counter-screens for cytotoxicity to human foreskin fibroblast or embryonic kidney cell lines. The physical library was supplemented by early-stage collection of analytical data for each fraction to aid rapid identification of the active components within each screening hit. RESULTS: A total of 16,177 fractions from 1300 plants were screened, identifying several P. falciparum inhibitory fractions from 35 plants. Although individual fractions were screened for bioactivity to ensure adequate signal in the analytical characterizations, fractions containing less than 2.0 mg of dry weight were combined to produce combined fractions (COMBIs). Fractions of active COMBIs had EC50 values of 0.21-50.28 and 0.08-20.04 µg/mL against chloroquine-sensitive and -resistant strains, respectively. In Berberis thunbergii, eight known alkaloids were dereplicated quickly from its COMBIs, but berberine was the most-active constituent against P. falciparum. The triterpenoids α-betulinic acid and ß-betulinic acid of Eugenia rigida were also isolated as hits. Validation of the anti-malarial discovery platform was confirmed by these scaled isolations from B. thunbergii and E. rigida. CONCLUSIONS: These results demonstrate the value of curating and exploring a library of natural products for small molecule drug discovery. Attention given to the diversity of plant species represented in the library, focus on practical analytical data collection, and the use of counter-screens all facilitate the identification of anti-malarial compounds for lead development or new tools for chemical biology.

Bioactive Formylated Flavonoids from Eugenia rigida: Isolation, Synthesis, and X-ray Crystallography.

Two new flavonoids, rac-6-formyl-5,7-dihydroxyflavanone (1) and 2',6'-dihydroxy-4'-methoxy-3'-methylchalcone (2), together with five known derivatives, rac-8-formyl-5,7-dihydroxyflavanone (3), 4',6'-dihydroxy-2'-methoxy-3'-methyldihydrochalcone (4), rac-7-hydroxy-5-methoxy-6-methylflavanone (5), 3'-formyl-2',4',6'-trihydroxy-5'-methyldihydrochalcone (6), and 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7), were isolated from the leaves of Eugenia rigida. The individual (S)- and (R)-enantiomers of 1 and 3, together with the corresponding formylated flavones 8 (6-formyl-5,7-dihydroxyflavone) and 9 (8-formyl-5,7-dihydroxyflavone), as well as 2',4',6'-trihydroxychalcone (10), 3'-formyl-2',4',6'-trihydroxychalcone (11), and the corresponding 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7) and 2',4',6'-trihydroxydihydrochalcone (12), were synthesized. The structures of the isolated and synthetic compounds were established via NMR, HRESIMS, and electronic circular dichroism data. In addition, the structures of 3, 5, and 8 were confirmed by single-crystal X-ray diffraction crystallography. The isolated and synthetic flavonoids were evaluated for their antimicrobial and cytotoxic activities against a panel of microorganisms and solid tumor cell lines.

New ent-Clerodane and Abietane Diterpenoids from the Roots of Kenyan Croton megalocarpoides Friis & M. G. Gilbert.

The roots of the endangered medicinal plant Croton megalocarpoides collected in Kenya were investigated and twenty-two compounds isolated. Among them were twelve new ent-clerodane (1-12) and a new abietane (13) diterpenoids, alongside the known crotocorylifuran (4 a), two known abietane and four known ent-trachylobane diterpenoids, and the triterpenoids, lupeol and acetyl aleurotolic acid. The structures of the compounds were determined using NMR, HRMS and ECD. The isolated compounds were evaluated against a series of microorganisms (fungal and bacteria) and also against Plasmodium falciparum, however no activity was observed.

Modulation of CYPs, P-gp, and PXR by Eschscholzia californica (California Poppy) and Its Alkaloids.

Eschscholzia californica, a native US plant, is traditionally used as a sedative, analgesic, and anxiolytic herb. With the rapid rise in the use of herbal supplements together with over-the-counter and prescription drugs, the risk for potential herb-drug interactions is also increasing. Most of the clinically relevant pharmacokinetic drug interactions occur due to modulation of cytochrome P450 enzymes (CYPs), P-glycoprotein, and the pregnane X receptor by concomitantly used herbs. This study aimed to determine the effects of an EtOH extract, aqueous extract (tea), basic CHCl3 fractions, and isolated major alkaloids, namely protopine (1), escholtzine (2), allocryptopine (3), and californidine (4), of E. californica on the activity of cytochrome P450s, P-glycoprotein and the pregnane X receptor. The EtOH extract and fractions showed strong time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19, and reversible inhibition of CYP 2D6. Among the alkaloids, escholtzine (2) and allocryptopine (3) exhibited time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19 (IC50 shift ratio > 2), while protopine (1) and allocryptopine (3) showed reversible inhibition of CYP 2D6 enzyme. A significant activation of the pregnane X receptor (> 2-fold) was observed with the EtOH extract, basic CHCl3 fraction, and alkaloids (except protopine), which resulted into an increased expression of mRNA and the activity of CYP 3A4 and CYP 1A2. The expression of P-glycoprotein was unaffected. However, aqueous extract (tea) and its main alkaloid californidine (4) did not affect cytochrome P450s, P-glycoprotein, or the pregnane X receptor. This data suggests that EtOH extract of E. californica and its major alkaloids have a potential of causing interactions with drugs that are metabolized by cytochrome P450s, while the tea seems to be safer.

Monoamine oxidase inhibitory constituents of propolis: kinetics and mechanism of inhibition of recombinant human MAO-A and MAO-B.

Propolis is the resinous material that bees gather from leaf buds, flowers and vegetables. Propolis extracts contain constituents with a broad spectra of pharmacological properties and are important ingredients of popular dietary supplements. Propolis extracts were evaluated in vitro for inhibition of recombinant human monoamine oxidase (MAO)-A and MAO-B. The dichloromethane extract of propolis showed potent inhibition of human MAO-A and MAO-B. Further fractionation identified the most active fractions as rich in flavonoids. Galangin and apigenin were identified as the principal MAO-inhibitory constituents. Inhibition of MAO-A by galangin was about 36 times more selective than MAO-B, while apigenin selectivity for MAO-A vs. MAO-B was about 1.7 fold. Apigenin inhibited MAO-B significantly more potently than galangin. Galangin and apigenin were further evaluated for kinetic characteristics and the mechanism for the enzymes' inhibition. Binding of galangin and apigenin with MAO-A and -B was not time-dependent and was reversible, as suggested by enzyme-inhibitor binding and dissociation-dialysis assay. The inhibition kinetics studies suggested that galangin and apigenin inhibited MAO-A and -B by a competitive mechanism. Presence of prominent MAO inhibitory constituents in propolis products suggests their potential for eliciting pharmacological effects that might be useful in depression or other neurological disorders. The results may also have important implications in drug-dietary supplement interactions.

Cytotoxicity and modulation of cancer-related signaling by (Z)- and (E)-3,4,3',5'-tetramethoxystilbene isolated from Eugenia rigida.

Bioassay-guided fractionation of the leaves of Eugenia rigida yielded three stilbenes, (Z)-3,4,3',5'-tetramethoxystilbene (1), (E)-3,4,3',5'-tetramethoxystilbene (2), and (E)-3,5,4'-trimethoxystilbene (3). Their structures were determined using 1D- and 2D-NMR spectroscopy and HRESIMS. The sterically hindered Z-stereoisomer 1, a new natural product, was prepared by time-dependent photoisomerization of the E-isomer (2) under UV irradiation at λ254 nm, while 2,3,5,7-tetramethoxyphenanthrene (5) was identified at λ365 nm by UHPLC/APCI-MS and NMR spectroscopy. Compounds 1-3 were tested against a panel of luciferase reporter gene assays that assess the activity of many cancer-related signaling pathways, and the Z-isomer (1) was found to be more potent than the E-isomer (2) in inhibiting the activation of Stat3, Smad3/4, myc, Ets, Notch, and Wnt signaling, with IC50 values between 40 and 80 µM. However, both compounds showed similar inhibition against Ap-1 and NF-κB signaling. In addition, 1 demonstrated cytotoxic activity toward human leukemia cells, solid tumor cells of epidermal, breast, and cervical carcinomas, and skin melanoma, with IC50 values between 3.6 and 4.3 µM, while 2 was weakly active against leukemia, cervical carcinoma, and skin melanoma cells. Interestingly, 2 showed antioxidant activity by inhibition of ROS generation to 50% at 33.3 µM in PMA-induced HL-60 cells, while 1 was inactive at 100 µM (vs Trolox 1.4 µM).