Luteolin Isolated from Juncus acutus L., a Potential Remedy for Human Coronavirus 229E.

The COVID-19 pandemic, caused by SARS-CoV-2, addressed the lack of specific antiviral drugs against coronaviruses. In this study, bioguided fractionation performed on both ethyl acetate and aqueous sub-extracts of Juncus acutus stems led to identifying luteolin as a highly active antiviral molecule against human coronavirus HCoV-229E. The apolar sub-extract (CH2Cl2) containing phenanthrene derivatives did not show antiviral activity against this coronavirus. Infection tests on Huh-7 cells, expressing or not the cellular protease TMPRSS2, using luciferase reporter virus HCoV-229E-Luc showed that luteolin exhibited a dose-dependent inhibition of infection. Respective IC50 values of 1.77 µM and 1.95 µM were determined. Under its glycosylated form (luteolin-7-O-glucoside), luteolin was inactive against HCoV-229E. Time of addition assay showed that utmost anti-HCoV-229E activity of luteolin was achieved when added at the post-inoculation step, indicating that luteolin acts as an inhibitor of the replication step of HCoV-229E. Unfortunately, no obvious antiviral activity for luteolin was found against SARS-CoV-2 and MERS-CoV in this study. In conclusion, luteolin isolated from Juncus acutus is a new inhibitor of alphacoronavirus HCoV-229E.

Isolation, identification, and activity evaluation of antioxidant components from Inula viscosa: A bioguided approach.

Oxidative stress is linked to several invasive diseases which causes significant clinical and economic impact, therefore, there is a need to develop new antioxidants. The natural products could play an important role in overcoming the current need. In the present work, the antioxidant bioassay-guided fractionation of the ethanolic extract of Inula viscosa leaves (Asteraceae) was performed using DPPH and ABTS assays affording three known compounds, which were successfully characterized as ilicic acid (1), taxifolin (2) and quercetin (3) based on 1D, 2D NMR. Compounds 2 and 3 were identified as the most active, displaying similar or higher potency against ABTS (value 41.27 for quercetin and 142.58 for taxifolin) and similar activity against DPPH (value 41.27 for quercetin and 142.58 for taxifolin) than the well-known reference, ascorbic acid (value 65.36 for quercetin and 58.43 for taxifolin) but less potency than the standard gallic acid. The discussion of SAR of the antioxidant potential revealed that the type of natural product is crucial for the activity and the substitution pattern on the flavonoid skeleton modulate the antioxidant profile. Our findings show that I. viscosa leaves may be a natural source of antioxidants and once again the role of flavonoids health benefits is more strongly endorsed.

Anti-Aging Potential of a Rosa centifolia Stem Extract with Focus on Phytochemical Composition by Bioguided Fractionation.

Agricultural practices generate huge amounts of by-products, often simply discarded as waste that must be processed at some cost. The natural by-products revalorisation as raw material to produce high-added value ingredients for various industrial sectors may pave the way towards more sustainable industrial practices, via an optimised utilisation of natural resources. Integrating the circular economy precepts to production systems is considered to be a more and more promising management solution to significantly reduce the environmental impact of economic activities. This article discusses the valorisation of Rosa centifolia stem to produce a natural extract with cosmetic anti-aging potential. To do so, the cosmetic potential of 30 extracts obtained by maceration of agricultural by-products in a hydroalcoholic solvent was evaluated: their activities, as well as their inhibitory activities of specific enzymes were assessed in vitro to identify those that could be used effectively as anti-ageing actives while meeting the consumer's expectations in terms of sustainability, naturality, transparency and traceability.[1] A hydroalcoholic extract of R. centifolia stem revealed itself particularly promising due to its valuable anti-hyaluronidase and antioxidant activities, and its interesting anti-elastase and anti-inflammatory potential. The bio-guided fractionation of this extract allows the characterisation of three major compounds, e. g., isoquercitrin, quercitrin and euscaphic acid, never identified in R. centifolia previously.

Bioguided fractionation of procyanidin B2 as potent anti coxsackie virus B and Herpes simplex from cypress (Cupressus sempervirens L.).

The methanolic extracts of cypress (Cupressus sempervirens L.) collected at three phenological stages were evaluated for their cytotoxicity on Vero cells by MTT test as well as on Herpes simplex (HSV-2) and coxsackie (CVB-3) viruses by plaque reduction assay. The methanolic extract exhibited the highest cytotoxicity against HSV-2 (IC50 = 20.40 µg/mL) and CVB-3 (IC50 = 47.50 µg/mL) at the flowering stage. This extract also exhibited a virucidal action both during the entry of viruses and the release of newly formed virions. The methanolic extract bioguided purification showed that the ethyl-acetate fraction was responsible for virucidal activity. This fraction was endowed with more important selectivity index of 8.15 for HSV-2 and 4.40 for CVB-3. The ethyl acetate fraction was subjected to thin layer chromatography fractionation and identification by HPLC-DAD-ESI-MSn. Results showed that the condensed tannin procyanidin B2 was identified for the first time responsible of the antiviral activity of cypress.

Bioguided Isolation of Active Compounds from Rhamnus alaternus against Methicillin-Resistant Staphylococcus aureus (MRSA) and Panton-Valentine Leucocidin Positive Strains (MSSA-PVL).

Despite intensified efforts to develop an effective antibiotic, S. aureus is still a major cause of mortality and morbidity worldwide. The multidrug resistance of bacteria has considerably increased the difficulties of scientific research and the concomitant emergence of resistance is to be expected. In this study we have investigated the in vitro activity of 15 ethanol extracts prepared from Moroccan medicinal plants traditionally used for treatment of skin infections. Among the tested species I. viscosa, C. oxyacantha, R. tinctorum, A. herba alba, and B. hispanica showed moderate anti-staphylococcal activity. However, R. alaternus showed promising growth-inhibitory effects against specific pathogenic bacteria especially methicillin-susceptible Staphylococcus aureus Panton-Valentine leucocidin positive (MSSA-PVL) and methicillin-resistant S. aureus (MRSA). The bioguided fractionation of this plant using successive chromatographic separations followed by nuclear magnetic resonance (NMR) and mass spectrometry (MS) including EIMS and HREIMS analysis yielded the emodin (1) and kaempferol (2). Emodin being the most active with MICs ranging between 15.62 and 1.95 µg/mL and showing higher activity against the tested strains in comparison with the crude extract, its mechanism of action and the structure-activity relationship were interestingly discussed. The active compound has not displayed toxicity toward murine macrophage cells. The results obtained in the current study support the traditional uses of R. alaternus and suggest that this species could be a good source for the development of new anti-staphylococcal agents.

A Study on the Phytotoxic Potential of the Seasoning Herb Marjoram (Origanum majorana L.) Leaves.

In the search of new alternatives for weed control, spices appear as an option with great potential. They are rich in bioactive natural products and edible, which might minimize toxicity hazard. Marjoram (Origanum majorana L.) is an aromatic herb that has been widely employed as a seasoning herb in Mediterranean countries. Although marjoram boasts a plethora of therapeutic properties (painkiller, antibiotic, treatment for intestinal disorders, etc.), the potential for its extracts for weed control is still to be more thoroughly explored. In order to determine their phytotoxic potential, marjoram leaves were subjected to different bioguided extraction processes, using water, ethyl acetate, acetone or methanol. The most active extract (acetone) was sequentially fractionated to identify its most active compounds. This fractionation led to the isolation and identification of 25 compounds that were classified as monoterpenes, diterpenes or flavonoids. Among them, a new compound named majoradiol and several compounds are described in marjoram for the first time. The phytotoxicity of the major compounds to etiolated wheat coleoptiles was compared against that of the commercial herbicide (Logran®), with similar or higher activity in some cases. These results confirm the extraordinary potential of the extracts from this edible plant to develop safer and more environmentally friendly herbicides.

131 -Oxophorbine protopheophorbide A from Ziziphus lotus as a novel mesenchymal-epithelial transition factor receptor inhibitory lead for the control of breast tumor growth in vitro and in vivo.

The failure of chemotherapy especially in triple negative breast cancer (TNBC) patients has been correlated with the overexpression of the mesenchymal-epithelial transition factor (c-Met) receptor. Thus, the hepatocyte growth factor (HGF)/c-Met signaling axis has gained considerable attention as a valid molecular target for breast cancer therapy. This study reports for the first time the discovery of the 131 -oxophorbines pheophorbide A and protopheophorbide A along with chlorophyllide A from Ziziphus lotus, an edible typical Tunisian plant, as the potent antiproliferative compounds against the human breast cancer cells MDA-MB-231 and MCF-7. Compared to other compounds, protopheophorbide A exerted the highest light-independent antiproliferative effect against the metastatic TNBC MDA-MB-231 cells (IC50 = 6.5 µM). In silico, this compound targeted the kinase domain of multiple c-Met crystal structures. It potently inhibited the kinase domain phosphorylation of wild and mutant c-Met in Z-LYTE kinase assay. Protopheophorbide A inhibited HGF-induced downstream c-Met-dependent cell proliferation, survival, adhesion and migration through RAF/MEK/ERK and PI3K/PTEN/AKT signaling pathways modulation, ROS generation and activation of JNK and p38 pathways. Interestingly, this compound impaired the ability of the MDA-MB-231 cells to adhere at different extracellular matrix proteins by reducing the HGF-induced expression of integrins αv, ß3, α2, and ß1. Moreover, protopheophorbide A exhibited anti-migratory properties (IC50 = 2.2 µM) through impacting the expression levels of E-cadherin, vimentin, ß-catenin, FAK, Brk, Rac, and Src proteins. Importantly, treatment with protopheophorbide A significantly inhibited the MDA-MB-231 tumor growth in vivo. Our results suggest that protopheophorbide A could be a novel c-Met inhibitory lead with promise to control c-Met/HGF-dependent breast malignancies.

Assessment of the antiprotozoal activity of Pulicaria inuloides extracts, an Algerian medicinal plant: leishmanicidal bioguided fractionation.

The lack of an effective chemotherapy for treatment of protozoan disease urges a wide investigation for active compounds, and plant-derived compounds continue to provide key leads for therapeutic agents. The current study reports the in vitro antiprotozoal evaluation of the Algerian medicinal plant Pulicaria inuloides against Leishmania amazonensis, Trypanosoma cruzi, and Acanthamoeba castellanii str. Neff. All the extracts from the aerial part showed to be present a higher leishmanicidal activity than anti-Acanthamoeba or Trypanosoma. Therefore, bioguided fractionation of the active CHCl3 extract led to the isolation and characterization of the flavonol, quercetagetin-3,5,7,3'-tetramethyl ether (1) as the main component. The structure of compound 1 was established by extensive 1D and 2D NMR spectroscopic analysis (COSY, HSQC, HMBC, and ROESY experiments), chemical transformation (derivatives 2 and 3), and comparison with data in the literature. Compound 1 and derivatives 2 and 3 were further evaluated against the promastigote and amastigote stage of L. amazonensis. Compounds 1-3 exhibited moderate leishmanicidal activity with IC50 values ranging from 0.234 to 0.484 mM and from 0.006 to 0.017 mM for the promastigote and amastigote forms, respectively, as well as low toxicity levels on macrophages (CC50 ranging from 0.365 to 0.664 mM). This study represents the first report of the antiprotozoal evaluation of Pulicaria inuloides, and the results highlight this species as a promising source of leishmanicidal agents.

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.

Two antigenotoxic chalcone glycosides from Mentha longifolia subsp. longifolia.

CONTEXT: Mentha L. (Labiatae) species (mint) with their flavoring properties have been used in food industries for centuries. Besides they have a great importance in drug development and medicinal applications due to various bioactive compounds of several members of the genus. OBJECTIVE: The aim of this study was to isolate bioactive compounds with antimutagenic potential by bio-guided fractionation and determine their structures by spectroscopic methods. MATERIALS AND METHODS: The structural elucidation of the isolated compounds was done based on spectroscopic methods, including MALDI-MS, UV, IR, and 2D NMR experiments, and the bio-guided fractionation process was done by using the Ames/Salmonella test system. Henceforth, solely genotoxic and antigenotoxic potential of the new compounds were also confirmed up to 2 µM/plate by using the same test system. RESULTS: Two new chalcone glycosides: (ßR)-ß,3,2',6'-tetrahydroxy-4-methoxy-4'-O-rutinosyldihydrochalcone and (ßR)-ß,4,2',6'-tetrahydroxy-4'-O-rutinosyldihydrochalcone, were isolated from Mentha longifolia (L.) Hudson subsp. longifolia, together with known six flavonoid glycosides and one phenolic acid: apigenin-7-O-glucoside, luteolin-7-O-glucoside, apigenin-7-O-rutinoside, luteolin-7-O-rutinoside, apigenin-7-O-glucuronide, luteolin-7-O-glucuronide, rosmarinic acid. According to the antimutagenicity results, both new test compounds significantly inhibited the mutagenic activity of 9-aminoacridine in a dose-dependent manner at the tested concentrations from 0.8 to 2 µM/plate. (ßR)-ß,4,2',6'-Tetrahydroxy-4'-O-rutinosyldihydrochalcone showed the maximum inhibition rate as 75.94% at 2 µM/plate concentration. CONCLUSIONS: This is the first report that two new chalcone glycosides were isolated from Mentha longifolia subsp. longifolia and their antimutagenic potentials by using mutant bacterial tester strains. In conclusion, the two new chalcone glycosides showed a significant antigenotoxic effect on 9-aminoacridine-induced mutagenesis at tested concentrations.

Antimicrobial activity of spiculisporic acid isolated from endophytic fungus Aspergillus cejpii of Hedera helix against MRSA.

The surge in multidrug-resistant pathogens worldwide has jeopardized the clinical efficiency of many current antibiotics. This problem steered many researchers in their quest to discover new effective antimicrobial agents from natural origins including plants or their residing endophytes. In this work, we aimed to identify the endophytic fungi derived from Hedera helix L. and investigate their potential antimicrobial activity. Bioguided fractionation approach was conducted to isolate the pure compounds from the most active fungal fraction. Out of a total of six different isolated endophytic fungal strains, only Aspergillus cejpii showed the highest activity against all tested microbial strains. The most active fraction was the dichloromethane/methanol fraction (DCM:MeOH), where it showed significant activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, Acinetobacter baumannii, Salmonella typhi, and three drug-resistant clinical isolate strains including Methicillin-resistant Staphylococcus aureus (MRSA, H1), Pseudomonas aeruginosa (PS 16), and Acinetobacter baumannii (ACT 322) using tetracyline and kanamycin as the control antibiotics. Bioguided fractionation of the active fraction led to the isolation of the γ-butenolide, spiculisporic acid. Structure elucidation was carried out using 1H and 13C-NMR spectroscopic analysis. The compound showed good antimicrobial activities with minimum inhibitory concentration (MIC) values ranging from 3.9 to 31.25 µg/mL against all tested strains. Gas chromatography coupled to mass spectrometry (GC-MS) profiling was also carried out to identify the metabolites in the microbial crude extract. In conclusion, endophytic fungi, Aspergillus cejpii, isolated from Hedera helix L. roots showed promising antimicrobial activity which merits further in-depth investigations for potential utilization as a source of new antibiotics in the future. It can also be considered as a novel source for spiculisporic acid.

Antifungal Activity of Hop Leaf Extracts and Xanthohumol on Two Strains of Venturia inaequalis with Different Sensitivities to Triazoles.

Hop cones are well-known for their antimicrobial properties, attributed to their specialized metabolites. Thus, this study aimed to determine the in vitro antifungal activity of different hop parts, including by-products such as leaves and stems, and some metabolites against Venturia inaequalis, the causal agent of apple scab. For each plant part, two types of extracts, a crude hydro-ethanolic extract and a dichloromethane sub-extract, were tested on spore germination of two strains with different sensitivities to triazole fungicides. Both extracts of cones, leaves and stems were able to inhibit the two strains, whereas rhizomes did not show activity. The apolar sub-extract of leaves appeared as the most active modality tested with half maximal inhibitory concentrations (IC50) of 5 and 10.5 mg·L-1 on the sensitive strain and the strain with reduced sensitivity, respectively. Differences in activity level between strains were noticed for all active modalities tested. Sub-extracts of leaves were then separated into seven fractions by preparative HPLC and tested on V. inaequalis. One fraction, containing xanthohumol, was especially active on both strains. This prenylated chalcone was then purified by preparative HPLC and showed significant activity against both strains, with IC50 of 1.6 and 5.1 mg·L-1. Therefore, xanthohumol seems to be a promising compound to control V. inaequalis.

Mammea B/BA Isolated From the Seeds of Mammea americana L. (Calophyllaceae) is a Potent Inhibitor of Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus remains a pathogen of high concern in public health programs worldwide due to antibiotic resistance and emergence of highly virulent strains. Many phytochemicals have demonstrated activity against S. aureus and other Gram-positive bacteria, but the minimum inhibitory concentration (MIC) values comparable to commonly used antibiotics are needed. In the present study, bio-guided fractionation of the ethanol extract of seeds of Mammea americana L. (Calophyllaceae) throughout the antibacterial activity, against S. aureus strains that are sensitive and resistant to methicillin, led to the isolation of four coumarins identified as mammea B/BA, mammea B/BC, mammea A/AA cyclo D and mammea A/AA cyclo F, and a mixture of mammea B/BA cyclo F plus mammea B/BD cyclo F. The extract inhibited the growth of S. aureus with MIC values of 2-4 µg/ml and Mammea B/BA (MaBBA) presented MIC values in a range between 0.5 and 1.0 µg/ml in six methicillin-sensitive strains and eight methicillin-resistant strains evaluated. We consider MaBBA the most potent of all mammea coumarins reported to date, according to the literature review carried out at the time of writing of this article. Toxicity assessment in vivo against the nematode Caenorhabditis elegans and in vitro against human fibroblasts of the extract and the compound MaBBA indicated that both had low toxicity.

Trypanocidal effect of alcoholic extract of Castanedia santamartensis (Asteraceae) leaves is based on altered mitochondrial function.

The deficit of effective treatments for Chagas disease has led to searching for new substances with therapeutic potential. Natural products possess a wide variety of chemical structural motifs and are thus a valuable source of diverse lead compounds for the development of new drugs. Castanedia santamartensis is endemic to Colombia, and local indigenous communities often use it to treat skin sores from leishmaniasis; however, its mechanism of action against the infective form of Trypanosoma cruzi has not been determined. Thus, we performed chemical and biological studies of two alcoholic leaf extracts of C. santamartensis to identify their active fractions and relate them to a trypanocidal effect and evaluate their mechanism of action. Alcoholic extracts were obtained through cold maceration at room temperature and fractionated using classical column chromatography. Both ethanolic and methanolic extracts displayed activity against T. cruzi. Chemical studies revealed that kaurenoic acid was the major component of one fraction of the methanolic extract and two fractions of the ethanolic extract of C. santamartensis leaves. Moreover, caryophyllene oxide, kaurenol, taraxasterol acetate, pentadecanone, and methyl and ethyl esters of palmitate, as well as a group of phenolic compounds, including ferulic acid, caffeic acid, chlorogenic acid, myricetin, quercitrin, and cryptochlorogenic acid were identified in the most active fractions. Kaurenoic acid and the most active fractions CS400 and CS402 collapsed the mitochondrial membrane potential in trypomastigotes, demonstrating for the first time the likely mechanism against T. cruzi, probably due to interactions with other components of the fractions.

Bioprospection for new larvicides against Aedes aegypti based on ethnoknowledge from the Amazonian São Sebastião de Marinaú riverside community.

ETHNOPHARMACOLOGICAL RELEVANCE: Vector-borne diseases represent a huge global burden impacting health systems. Aedes aegypti is the main vector of arboviral diseases including dengue, Zika, chikungunya and urban yellow fever in both tropical and subtropical areas. Ethnopharmacological investigations provide potential avenues for developing new vector control strategies. AIM OF THE STUDY: The objective of this study is to document the São Sebastião de Marinaú riverside community's ethnoknowledge of local plants used to control mosquitoes and perform bioguided fractionation to isolate the compounds active against the arboviral disease vector Ae. aegypti. MATERIALS AND METHODS: Semi-structured interviews were conducted with residents of the Marinaú community located in the Caxiuanã National Forest, in the Amazon biome, Pará, Brazil. The plants used to control mosquitoes were subjected to phytochemical studies guided by Ae. aegypti assays. Extracts were obtained from seven species using distinct organic solvents. Active extracts and fractions were separated by chromatographic techniques. Isolated compounds were characterized by NMR, LC/MS and GC/MS. Sample activity against Ae. aegypti larvae and pupae was evaluated after 24, 48 and 72 h exposure. The extracts were also investigated against adult female mosquitoes. The LC50 values were determined by diluting each sample to obtain different concentrations in the respective activity range. RESULTS: The Marinaú community uses more than ten plants as a repellent, most of which are trees native to the region. The primary applications of these plants to protect against insect bites were: burning plants (fumigation), application of body oils and bathing in macerated plants. Carapa guianensis is the predominant species used as a repellent. Extracts from Diospyros guianensis fruits, Carapa guianensis seed shells and Aspidosperma nitidum wood demonstrated Ae. aegypti larvicidal activity. The C. guianensis seed shell extract demonstrated a residual larvicidal effect. Plumbagin, stigmasterol, ß-sitosterol, betulinic, ursolic and oleanolic acids, and betulin were identified in the D. guianensis extract. The plumbagin, ursolic and oleanolic acids displayed larvicidal activity. Oleanolic, ursolic and betulinic acids, and betulin were considered pupicidal. Aricine, the major alkaloid isolated from A. nitidum wood, also presented larvicidal activity. CONCLUSIONS: Ten plant species traditionally used by the Marinaú community to afford protection against mosquitoes were reported. C. guianensis, D. guianensis and A. nitidum extracts were considered larvicidal against Ae. aegypti. Four triterpenes stood out as very active compounds against pupae. Aricine, an indole alkaloid, displayed larvicidal activity. Therefore, traditional knowledge of Amazonian plants combined with bioguided fractionation constitutes a strategy for the development of eco-friendly insecticides to control Ae. aegypti, an arbovirus vector.

Psilostachyins as trypanocidal compounds: Bioguided fractionation of Ambrosia tenuifolia chemically modified extract.

This work focusses on the chemical diversification of an Ambrosia tenuifolia extract and its bioguided fractionation, aiming to unveil the chemical entity responsible for the trypanocidal activity. Besides, a revision of the phytochemical study of this species, based on previous reports of the antiparasitic psilostachyins A and C as main compounds, was conducted. To improve the biological properties of a plant extract through a simple chemical reaction, the oxidative diversification of the dichloromethane extract of this plant species was carried out. A bioguided fractionation of a chemically modified extract was performed by evaluating the inhibitory activity against Trypanosoma cruzi trypomastigotes. This experiment led to the isolation of one of the most active compounds. In general terms, epoxidized metabolites were obtained as a result of the oxidation of the major metabolite of the species. The trypanocidal activity of some tested metabolites overperformed the reference drug, benznidazole, displaying no cytotoxicity at trypanocidal concentrations. Key structure-activity relationships were obtained for designing previously undescribed antiparasitic sesquiterpene lactones.

Identification of 3,3'-O-dimethylellagic acid and apigenin as the main antiplasmodial constituents of Endodesmia calophylloides Benth and Hymenostegia afzelii (Oliver.) Harms.

BACKGROUND: Endodesmia calophylloides and Hymenostegia afzelii belong to the Guttiferae and Caesalpiniaceae plant families with known uses in African ethno-medicine to treat malaria and several other diseases. This study aimed at identifying antiplasmodial natural products from selected crude extracts from H. afzelii and E. calophylloides and to assess their cytotoxicity. METHODS: The extracts from H. afzelii and E. calophylloides were subjected to bioassay-guided fractionation to identify antiplasmodial compounds. The hydroethanol and methanol stem bark crude extracts, fractions and isolated compounds were assessed for antiplasmodial activity against the chloroquine-sensitive 3D7 and multi-drug resistant Dd2 strains of Plasmodium falciparum using the SYBR green I fluorescence-based microdilution assay. Cytotoxicity of active extracts, fractions and compounds was determined on African green monkey normal kidney Vero and murine macrophage Raw 264.7 cell lines using the Resazurin-based viability assay. RESULTS: The hydroethanolic extract of H. afzelii stem bark (HasbHE) and the methanolic extract of E. calophylloides stem bark (EcsbM) exhibited the highest potency against both Pf3D7 (EC50 values of 3.32 ± 0.15 µg/mL and 7.40 ± 0.19 µg/mL, respectively) and PfDd2 (EC50 of 3.08 ± 0.21 µg/mL and 7.48 ± 0.07 µg/mL, respectively) strains. Both extracts showed high selectivity toward Plasmodium parasites (SI > 13). The biological activity-guided fractionation led to the identification of five compounds (Compounds 1-5) from HasbHE and one compound (Compound 6) from EcsbM. Of these, Compound 1 corresponding to apigenin (EC50 Pf3D7, of 19.01 ± 0.72 µM and EC50 PfDd2 of 16.39 ± 0.52 µM), and Compound 6 corresponding to 3,3'-O-dimethylellagic acid (EC50 Pf3D7 of 4.27 ± 0.05 µM and EC50 PfDd2 of 1.36 ± 0.47 µM) displayed the highest antiplasmodial activities. Interestingly, both compounds exhibited negligible cytotoxicity against both Vero and Raw 264.7 cell lines with selectivity indices greater than 9. CONCLUSIONS: This study led to the identification of two potent antiplasmodial natural compounds, 3,3'-O-dimethylellagic acid and apigenin that could serve as starting points for further antimalarial drug discovery.

Specialized antiplasmodial secondary metabolites from Aspergillus niger 58, an endophytic fungus from Terminalia catappa.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia catappa L. (West Indian-Almond) is a medicinal plant used in traditional medicine for the treatment of infectious diseases. Moreover, various organic extracts prepared from this plant have been reported to exhibit antiplasmodial activity. AIM OF THE STUDY: The need for new antimalarials is still an urgency driven by the alarmingly high burden of malaria in endemic regions, with multitude of people dying annually. We have previously identified an endophytic fungus Aspergillus niger 58 harboured by T. catappa as having promising specialized secondary metabolites against the malaria parasites. In the present study, we report the antiplasmodial activity-guided chromatographic isolation of some metabolites secreted by this endophytic fungus. MATERIALS AND METHODS: The SYBR Green I-based fluorescence microtiter plate assay was used to monitor the growth of Plasmodium falciparum parasites in culture in the presence and absence of inhibitors and results were validated by microscopic analysis of Giemsa-stained culture smears. Giemsa-stain microscopy was also used to study the cell cycle stage-specific action of selected fractions. RESULTS: The results revealed that the multidimensional purification of the crude extract (IC50: 4.03 µg/mL) provided RPHPLC F17 (IC50: 0.09 µg/mL) and RPHPLC F18 (IC50: 0.1 µg/mL) with activity against P. falciparum 3D7 (Pf3D7) strain. Moreover, both fractions at IC99 (0.5 µg/mL) exhibited multi-stages action by targeting all the three stages of the life cycle of blood-stage Pf3D7. Two compounds, flavasperone (1) and aurasperone A (2) were isolated, of which aurasperone A exhibited good potency against Pf3D7 (IC50: 4.17 µM) and P. falciparum INDO (PfINDO) (IC50: 3.08 µM). CONCLUSION: Our study adds credence to the notion that endophytic extracts are potential storehouses for potent specialized secondary metabolites that can be harnessed to fight the malaria parasite and reduce the burden of this disease worldwide. An endophyte that can be cultured in laboratory with ability to secrete promising metabolites of medicinal value holds the promise of conserving Nature from the threat of annihilation of flora for medicinal purposes.

Bioguided identification of pentacyclic triterpenoids as anti-inflammatory bioactive constituents of Ocimum gratissimum extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Ocimum gratissimum is a plant spice widely used in African traditional medicine to treat pain-related conditions. However, the anti-inflammatory mechanisms underlying this activity and the main active ingredients in O. gratissimum have not yet been fully characterized. AIM OF THE STUDY: To isolate and identify the main anti-inflammatory active constituents of Ocimum gratissimum extract and their underlying mechanisms in murine macrophages. MATERIAL AND METHODS: Chromatographic techniques and spectroscopic data were used for compounds isolation and identification. Inflammatory conditions were produced in cultured RAW 264.7 macrophage cells by the application of lipopolysaccharide (LPS). The WST-1 assay was used to evaluate the cell viability, and the nitric oxide production was quantified by the Griess reagent method. The fluorometric cyclooxygenase (COX) activity assay kit was used to assess the activity of COX-1 and COX-2 enzymes. The levels of IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 cytokines and the apoptosis-inducing effect were measured by flow cytometer using the cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II and FITC Annexin V Apoptosis Detection kit, respectively. RESULTS: The results showed that the extract and fractions of Ocimum gratissimum inhibit nitric oxide production and the proliferation of Raw 264.7 macrophage cells. The bioguided fractionation led to the identification of pentacyclic triterpenes as anti-inflammatory bioactive compounds. Pomolic and tormentic acids being the most active, inhibiting the secretion of IFN-γ cytokine, COX enzyme, and inducing apoptosis in activated Raw 264.7 macrophage cells. CONCLUSIONS: This study revealed that pomolic and tormentic acids are the main active principles responsible at least in part for the anti-inflammatory effect of the extract of Ocimum gratissimum. Besides of providing more evidence for the traditional use of Ocimum gratissimum against inflammatory disorders, this study reveals the multitarget potential of pomolic and tormentic acids as promising future drugs against inflammatory diseases.

Exploring the Anti-Infective Value of Inuloxin A Isolated from Inula viscosa against the Brain-Eating Amoeba (Naegleria fowleri) by Activation of Programmed Cell Death.

Primary amoebic meningoencephalitis (PAM), caused by the pathogenic free-living amoeba Naegleria fowleri, is a rare but fatal disease. Nowadays, no fully effective therapy is available to erradicate or prevent this disease. Natural products could constitute a promising source of useful bioactive compounds in drug discovery. The present study is a characterization of main active compounds from the ethanolic extract of Inula viscosa (Asteraceae) leaves against N. fowleri trophozoites. Four compounds (1-4) were successfully identified by spectroscopic techniques, but only inuloxin A displayed a potential antiamoebic activity with an IC50 of 21.27 µM. The specificity of this compound toward the studied strain leads us to analyze the insight into its mechanism of action by performing in vitro assays of programmed cell death markers and to discuss the structure-activity relationship (SAR). The obtained results demonstrated that inuloxin A interferes with various processes leading to membrane damage, mitochondria alteration, chromatin condensation, and ROS accumulation, which highlight features specific to apoptosis. The current findings could be a promising step for developing new effective drugs against PAM.