Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators.

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABAA receptors with an α1ß2γ2 subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC50 values of 0.5, 4.6, and 1.4 µM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABAA receptors of the α1ß2γ2S and α1ß2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC50 (α1ß2γ2S) = 43.6 µM; Emax = 809% and EC50 (α1ß2) = 57.6 µM; Emax = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

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.

Pharmacognostic Evaluation of Ten Species of Medicinal Importance of Cecropia: Current Knowledge and Therapeutic Perspectives.

This work covers a systematic review of literature about the genus Cecropia from 1978 to 2020, emphasizing the analysis of 10 of the most relevant species and their associated biological activities. Cecropia is a neotropical genus, which comprises about 61 native species in the American continent where it is known to be part of the traditional medicine of numerous countries. Secondary metabolites described for this genus showed an elevated structural and functional diversity, where polyphenols have been the most abundant. Based on this diversity, Cecropia phytochemicals represent an important source of potential therapeutic agents yet to be exploited. This review also highlights the effectiveness of combining chemometrics and ultra-performance liquid chromatography-tandem mass spectrometry as a novel approach to successfully single out Cecropia species phytochemicals. While the medicinal use of Cecropia species is officially recognized in National Pharmacopoeias and Formularies of several Latin American countries, it is important to recognize that these phytomedicines are complex mixtures requiring a thorough understanding of their chemical composition and their correlation with biological activities to guarantee their quality, safety, and efficacy.

Assessment of the antinociceptive and anti-inflammatory activities of the stem methanol extract of Diplotropis purpurea.

Context: Since there is still a great need to search for plant species with antinociceptive and anti-inflammatory activities, Diploptropis purpurea (Rich.) Amshoff (Fabaceae) is studied for the first time. Objective: This evaluates the analgesic and anti-inflammatory activities of the stem methanol extract of Diplotropis purpurea (MEDP). Material and methods: The anti-inflammatory and analgesic effects of MEDP of D. purpurea were evaluated in vivo. The antinociceptive activity was assessed in CD1 male mice were treated by oral gavage with 500 mg/kg of MEDP 30 min before submitting to acetic acid-induced abdominal writhing, hot-plate, and formalin tests. Paws oedema induced by carrageenan, histamine or serotonin were performed in male Sprague-Dawley rats to determinate the anti-inflammatory activity. Results: Oral administration of MEDP produced significant antinociceptive effects on the inflammatory phase in the formalin test [12.0 s versus 72.5 s in carboxymethyl cellulose (CMC) control group]. MEDP produced an analgesic effect in the hot-plate model, although the effect was modest compared to tramadol (40 and 60%, respectively). The oral administration of MEDP in a dose of 500 mg/kg showed maximum inhibition (75.1%) after 0.5 h in carrageenan-induced oedema, but it did not modify histamine or serotonin-induced oedemas. Discussion and conclusion: In the peripheral nociception model, acetic acid-induced abdominal writhing, the MEDP did not show a protective effect, but its analgesic effects were evident in the inflammatory phase of the formalin test and in the hot-plate model. These results show that the anti-inflammatory effect was accompanied by a reduction in the perception of painful stimuli.

Eudesmane Sesquiterpenes from Verbesina lanata with Inhibitory Activity against Grapevine Downy Mildew.

An in-house library of more than 3000 extracts of plant and fungal origin was screened against some major plant pathogens. As one of the hits, an ethyl acetate extract from inflorescences of Verbesina lanata showed significant inhibitory activity in vitro against grapevine downy mildew (Plasmopara viticola), with a MIC100 value of 35 µg/mL. An emulsifiable concentrate formulation with 50 mg/g of the extract was developed for in vivo evaluation. A suspension of the formulation containing 1 mg/mL of extract lowered leaf surface infection of grapevine seedling by 82% compared to the nontreated control. With the aid of HPLC-based activity profiling, the antifungal activity was correlated with a series of lipophilic compounds. Preparative isolation by a combination of chromatographic techniques afforded 16 eudesmane sesquiterpenes including eight new congeners. Nine compounds were obtained in sufficient quantities to be tested in vitro and were found to inhibit the zoospore activity of P. viticola with MIC100 values ranging from 4 to 50 µg/mL. The two major compounds, 6ß-cinnamoyloxy-4ß,9ß,15-trihydroxyeudesmane (9) and 6ß-cinnamoyloxy-1ß,15-dihydroxyeudesm-4-en-3-one (13), showed MIC100 values of 5 and 31 µg/mL, respectively.

Cheminformatic characterization of natural products from Panama.

In this work, we discuss the characterization and diversity analysis of 354 natural products (NPs) from Panama, systematically analyzed for the first time. The in-house database was compared to NPs from Brazil, compounds from Traditional Chinese Medicine, natural and semisynthetic collections used in high-throughput screening, and compounds from ChEMBL. An analysis of the "global diversity" was conducted using molecular properties of pharmaceutical interest, three molecular fingerprints of different design, molecular scaffolds, and molecular complexity. The global diversity was visualized using consensus diversity plots that revealed that the secondary metabolites in the Panamanian flora have a large scaffold diversity as compared to other composite databases and also have several unique scaffolds. The large scaffold diversity is in agreement with the broad range of biological activities that this collection of NPs from Panama has shown. This study also provided further quantitative evidence of the large structural complexity of NPs. The results obtained in this study support that NPs from Panama are promising candidates to identify selective molecules and are suitable sources of compounds for virtual screening campaigns.

Neoflavonoids as Inhibitors of HIV-1 Replication by Targeting the Tat and NF-κB Pathways.

Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter gene. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Seven 4-phenylchromen-2-one derivatives showed HIV transcriptional inhibitory activity but only the phenylchrome-2-one 10 inhibited NF-κB and displayed anti-Tat activity simultaneously. Compounds 10, 14, and 25, inhibited HIV replication in both targets at concentrations <25 µM. The assays of these synthetic 4-phenylchromen-2-ones may aid in the investigation of some aspects of the anti-HIV activity of such compounds and could serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential anti-HIV therapeutic drug.

Chemical Composition and Biological Activity of Essential Oils from Different Species of Piper from Panama.

The chemical composition of leaf essential oils from 11 species of Piper from Panama was analyzed by a combination GC-FID and GC-MS procedures. Six of them had sesquiterpene hydrocarbons as major constituents, three were characterized by monoterpene hydrocarbons, one by a diterpene, and one by a phenylpropanoid, dillapiole. The main components identified in each species were: cembratrienol (25.4 %) in Piper augustum; ß-pinene (26.6 %) in Piper corrugatum; α-pinene (19.4 %) in Piper curtispicum; trans-ß-farnesene (63.7 %) in Piper darienense; p-cymene (43.9 %) in Piper grande; dillapiole (57.7 %) in Piper hispidum; linalool (14.5 %), α-phellandrene (13.8 %), and limonene (12.2 %) in Piper jacquemontianum; ß-caryophyllene (45.2 %) in Piper longispicum; linalool (16.5 %), α-phellandrene (11.8 %), limonene (11.4 %), and p-cymene (9.0 %) in Piper multiplinervium; ß-selinene (19.0 %), ß-elemene (16.1 %), and α-selinene (15.5 %) in Piper reticulatum; and germacrene D (19.7 %) in Piper trigonum. The essential oils of P. hispidum and P. longispicum at a concentration of 250 µg/mL showed larvicidal activity against Aedes aegypti, while the oils from P. curtispicum, P. multiplinervium, P. reticulatum, and P. trigonum were inactive (LC100 ≥ 500 µg/mL). The essential oils of P. grande, P. jacquemontianum, and P. multiplinervium showed no significant antifungal activity (MIC > 250 µg/mL) against several yeasts and filamentous fungal strains.

Chemical Adulterants in Herbal Medicinal Products: A Review.

Many herbal medicinal products have been found to contain synthetic prescription drugs as chemical adulterants. This has become evident by the number of toxicity cases and adverse reactions reported in which casualties were reported via analytical techniques that detected the presence of chemical adulterants in them, which could be responsible for their toxicity. The adulteration of herbal medicinal products with synthetic drugs continues to be a serious problem for regulatory agencies. This review provides up to date information on cases of toxicity, major chemical adulterants in herbal medicinal products, and current analytical techniques used for their detection.

Anti-Candida Cassane-Type Diterpenoids from the Root Bark of Swartzia simplex.

A dichloromethane extract of the roots from the Panamanian plant Swartzia simplex exhibited a strong antifungal activity in a bioautography assay against a genetically modified hypersusceptible strain of Candida albicans. At-line HPLC activity based profiling of the crude extract enabled a precise localization of the antifungal compounds, and dereplication by UHPLC-HRESIMS indicated the presence of potentially new metabolites. Transposition of the HPLC reversed-phase analytical conditions to medium-pressure liquid chromatography (MPLC) allowed an efficient isolation of the major constituents. Minor compounds of interest were isolated from the MPLC fractions using semipreparative HPLC. Using this strategy, 14 diterpenes (1-14) were isolated, with seven (5-10, 14) being new antifungal natural products. The new structures were elucidated using NMR spectroscopy and HRESIMS analysis. The absolute configurations of some of the compounds were elucidated by electronic circular dichroism spectroscopy. The antifungal properties of these compounds were evaluated as their minimum inhibitory concentrations in a dilution assay against both hypersusceptible and wild-type strains of C. albicans and by assessment of their antibiofilm activities. The potential cytological effects on the ultrastructure of C. albicans of the antifungal compounds isolated were evaluated on thin sections by transmission electron microscopy.

Anti-malarial activity and HS-SPME-GC-MS chemical profiling of Plinia cerrocampanensis leaf essential oil.

BACKGROUND: Plinia cerrocampanensis is an endemic plant of Panama. The leaf essential oil of this plant has shown antibacterial activity. However, anti-malarial activity and chemical profiling by HS-SPME-GC-MS of this essential oil have not been reported before. METHODS: Anti-malarial activity of the essential oil (EO) was evaluated in vitro against chloroquine-sensitive HB3 and chloroquine-resistant W2 strains of Plasmodium falciparum. Synergistic effect of chloroquine and the EO on parasite growth was evaluated by calculating the combination index. A methodology involving headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) was developed to investigate the composition of Plinia cerrocampanensis EO. RESULTS: Plinia cerrocampanensis EO showed a high anti-malarial activity and a synergistic interaction with chloroquine. The Plinia cerrocampanensis EO inhibited P. falciparum growth in vitro at an IC50 of 7.3 µg/mL. Chloroquine together with the EO decreased the IC50 of chloroquine from 0.1 µg/mL to 0.05 µg/mL, and of the EO from 7.3 µg/mL to 1.1 µg/mL. The measured combination index was 0.58, which clearly indicates that the EO acts synergistically with chloroquine. Since the EO maintained its inhibitory activity on the chloroquine-sensitive strain of the parasite, it could be acting by a different mechanism of action than chloroquine. The best HS-SPME-GC-MS analytical conditions were obtained when the temperature of extraction was 49°C, incubation time 14 min, and the time of extraction 10 min. This method allowed for the identification of 53 volatile constituents in the EO, including new compounds not reported earlier. CONCLUSIONS: The anti-malarial activity exhibited by the Plinia cerrocampanensis EO may lend support for its possible use as an alternative for anti-malarial therapy.

Isolation of major components from the roots of Godmania aesculifolia and determination of their antifungal activities.

From the methanol root extract of Godmania aesculifolia, a species selected in a multinational OAS program aimed at discovering antifungal compounds from Latin American plants, a new chavicol diglycoside (1), the known 3,4-dihydroxy-2-(3-methylbut-2-en-1-yl)-3,4-dihydronaphthalen-1(2H)-one (2), and lapachol (3) were isolated and characterized by 1D and 2D NMR and MS techniques. Only 3 exhibited fairly good activity against a panel of clinical isolates of Cryptococcus neoformans (MIC50 between 7.8 and 31.2 µg/mL) and moderate activities against Candida spp. and non-albicans Candida spp.

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.

3-Phenylcoumarins as inhibitors of HIV-1 replication.

We have synthesized fourteen 3-phenylcoumarin derivatives and evaluated their anti-HIV activity. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase gene as reporter. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Six compounds displayed NF-κB inhibition, four resulted Tat antagonists and three of them showed both activities. Three compounds inhibited HIV replication with IC50 values < 25 µM. The antiviral effect of the 4-hydroxycoumarin derivative 19 correlates with its specific inhibition of Tat functions, while compound 8, 3-(2-chlorophenyl)coumarin, seems to act through a mechanism unrelated to the molecular targets considered in this research.

Antimalarial natural products drug discovery in Panama.

CONTEXT: Malaria is still a major public health problem. The biodiversity of the tropics is extremely rich and represents an invaluable source of novel bioactive molecules. For screening of this diversity more sensitive and economical in vitro methods are needed, Flora of Panama has been studied based on ethnomedical uses for discovering antimalarial compounds. OBJECTIVE: This review aims to provide an overview of in vitro screening methodologies for antimalarial drug discovery and to present results of this effort in Panama during the last quarter century. METHODS: A literature search in SciFinder and PubMed and original publications of Panamanian scientists was performed to gather all the information on antimalarial drug discovery from the Panamanian flora and in vitro screening methods. RESULTS AND CONCLUSIONS: A variety of colorimetric, staining, fluorometric, and mass spectrometry and radioactivity-based methods have been provided. The advantages and limitations of these methods are also discussed. Plants used in ethnomedicine for symptoms of malaria by three native Panamanian groups of Amerindians, Kuna, Ngöbe Buglé and Teribes are provided. Seven most active plants with IC(50) values < 10 µg/mL were identified Talisia nervosa Radlk. (Sapindaceae), Topobea parasitica Aubl.(Melastomataceae), Monochaetum myrtoideum Naudin (Melastomataceae), Bourreria spathulata (Miers) Hemsl.(Boraginaceae), Polygonum acuminatum Kunth (Polygonaceae), Clematis campestris A. St.-Hil. (Ranunculaceae) and Terminalia triflora (Griseb.) Lillo (Combretaceae). Thirty bioactive compounds belonging to a variety of chemical classes such as spermine and isoquinoline alkaloids, glycosylflavones, phenylethanoid glycosides, ecdysteroids, quercetin arabinofuranosides, clerodane-type diterpenoids, sipandinolid, galloylquercetin derivatives, gallates, oleamide and mangiferin derivatives.

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.

A quarter century of pharmacognostic research on Panamanian flora: a review.

Panama is a unique terrestrial bridge of extreme biological importance. It is one of the "hot spots" and occupies the fourth place among the 25 most plant-rich countries in the world, with 13.4 % endemic species. Panamanian plants have been screened for a wide range of biological activities: as cytotoxic, brine shrimp-toxic, antiplasmodial, antimicrobial, antiviral, antioxidant, immunosuppressive, and antihypertensive agents. This review concentrates on ethnopharmacological uses of medicinal plants employed by three Amerindian groups of Panama and on selected plants with novel structures and/or interesting bioactive compounds. During the last quarter century, a total of approximately 390 compounds from 86 plants have been isolated, of which 160 are new to the literature. Most of the work reported here has been the result of many international collaborative efforts with scientists worldwide. From the results presented, it is immediately obvious that the Panamanian flora is still an untapped source of new bioactive compounds.

Identification of oleamide in Guatteria recurvisepala by LC/MS-based Plasmodium falciparum thioredoxin reductase ligand binding method.

Our current research on applications of mass spectrometry to natural product drug discovery against malaria aims to screen plant extracts for new ligands to Plasmodium falciparum thioredoxin reductase (PfTrxR) followed by their identification and structure elucidation. PfTrxR is involved in the antioxidant defense and redox regulation of the parasite and is validated as a promising target for therapeutic intervention against malaria. In the present study, detannified methanol extracts from Guatteria recurvisepala, Licania kallunkiae, and Topobea watsonii were screened for ligands to PfTrxR using ultrafiltration and liquid chromatography/mass spectrometry-based binding experiments. The PfTrxR ligand identified in the extract of Guatteria recurvisepala displayed a relative binding affinity of 3.5-fold when incubated with 1 µM PfTrxR. The ligand corresponding to the protonated molecule m/z 282.2792 [M+ H]+ was eluted at a retention time of 17.95 min in a 20-min gradient of 95% B consisting of (A) 0.1%formic acid in 95% H2O-5% ACN, and (B) 0.1% formic acid in 95% ACN-5% H2O in an LC-QTOF-MS.Tandem MS of the protonated molecule m/z 282.2792 [M + H]+, C18H36NO (DBE: 2; error: 1.13 ppm) resulted in two daughter ions m/z 265.2516[M + H-NH3]+ (DBE: 3; error: 0.35 ppm) and m/z 247.2405 [M + H-NH3-H2O] +, (DBE: 4; error:2.26 ppm). The PfTrxR ligand was identified as oleamide and confirmed by comparison of the retention time, molecular formula, accurate mass,and double bond equivalence with the standard oleamide. This is the first report on the identification of oleamide as a PfTrxR ligand from Guatteria recurvisepala R. E. Fr. and the corresponding in vitro activity against P. falciparum strain K1 (IC50 4.29 µg/mL).

Structure elucidation and NMR assignments of two new triterpenoids from the stems of Paragonia pyramidata (Bignoniaceae).

Phytochemical investigation of dichloromethane (DCM) extract from the stems of Paragonia pyramidata var. pyramidata L. Rich. (Bur.) resulted in the isolation and characterization of two new triterpenoids 3ß,19ß-dihydroxylup-12, 20(29)-diene-28-oic acid (1) and 3ß,19ß-dihydroxylup-12-en-28-oic acid (2), three known triterpenoids lupeol (3), spinosic acid A (4) and oleanolic acid (5), together with four known steroids (20R)-22E-24-ethylcholesta-4,22-dien-3-one (6), (20R)-24-ethylcholest-4-en-3-one (7), stigmasterol (8) and ß-sitosterol (9). HREIMS, GC-MS and NMR experiments including HSQC, HMBC, COSY and NOESY were used for the determination of the structures and NMR spectral assignments. This is the first report about the chemical constituents for this plant.