Green ultrasound-assisted extraction of lichen substances from Hypotrachyna cirrhata. Ethyl lactate, a better extracting agent than methanol toxic organic solvent?

For the first time, we report a green extraction of lichen substances assisted by high power ultrasounds from Hypotrachyna cirrhata using ethyl lactate. This sustainable alternative was comparable, both in isolation and detection of lichen substances, to methanol. In the metabolomic analysis, a total of 77 lichen substances were detected comprising depsides, depsidones, dibenzofurans, organic acids, and lipids. Although the UHPLC/ESI/MS profiles were similar, the antioxidant activity was higher for the ethyl lactate extract. Ethyl lactate can replace toxic organic solvents, such as methanol, in order to provide more sustainable green chemistry methods.

A sustainable application for the extraction of lichen metabolites from Usnea cornuta: nontargeted metabolomics and antioxidant activity.

In this study, isolation and purification of lichen substances from Usnea cornuta were performed using conventional solvents, green solvents and green technologies. In addition, several lichen compounds were tentatively identified by UHPLC/ESI/MS/MS and usnic acid, diffractaic and galbinic acids were quantified as well. Limonene, ethyl lactate and methanol, were compared regarding their extraction properties and antioxidant capacities, determined by DPPH, ORAC, and FRAP assays. In the ethyl lactate, methanol and limonene extracts, 28 compounds in all, were detected for the first time by high resolution UHPLC-MS/MS fingerprinting. Untargeted metabolomics tentatively identified 14 compounds from the methanolic extract, 4 from limonene extract, and 20 metabolites from ethyl lactate extract. The green extract of ethyl lactate showed a similar antioxidant capacity to toxic methanol extract, except at ORAC assay where it was higher. Therefore, ethyl lactate can replace methanol, to provide more sustainable green chemistry methods.

Theobroma cacao L. compounds: Theoretical study and molecular modeling as inhibitors of main SARS-CoV-2 protease.

Cocoa beans contain antioxidant molecules with the potential to inhibit type 2 coronavirus (SARS-CoV-2), which causes a severe acute respiratory syndrome (COVID-19). In particular, protease. Therefore, using in silico tests, 30 molecules obtained from cocoa were evaluated. Using molecular docking and quantum mechanics calculations, the chemical properties and binding efficiency of each ligand was evaluated, which allowed the selection of 5 compounds of this series. The ability of amentoflavone, isorhoifolin, nicotiflorin, naringin and rutin to bind to the main viral protease was studied by means of free energy calculations and structural analysis performed from molecular dynamics simulations of the enzyme/inhibitor complex. Isorhoifolin and rutin stand out, presenting a more negative binding ΔG than the reference inhibitor N-[(5-methylisoxazol-3-yl)carbonyl]alanyl-l-valyl-N~1~-((1R,2Z)-4-(benzyloxy)-4-oxo-1-{[(3R)-2-oxopyrrolidin-3-yl]methyl}but-2-enyl)-L-leucinamide (N3). These results are consistent with high affinities of these molecules for the major SARS-CoV-2. The results presented in this paper are a solid starting point for future in vitro and in vivo experiments aiming to validate these molecules and /or test similar substances as inhibitors of SARS-CoV-2 protease.

In Vitro Anthelmintic Evaluation of Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce: Fingerprint Analysis of Extracts by UHPLC-Orbitrap Mass Spectrometry.

In the present work, the anthelmintic activity (AA) of ethanolic extracts obtained from Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce was evaluated using the third-stage-larval (L3) exsheathment inhibition test (LEIT) and egg hatch test (EHT) on Haemonchus contortus. Extracts were tested at concentrations of 0.3, 0.6, 1.2, 2.5, 5.0, 10, 20, and 40 mg/mL. The larval exsheathment inhibition (LEI) results showed that G. sepium achieved the highest average inhibition of 91.2%, compared with 44.6% for P. dulce and 41.0% for L. leucocephala at a concentration of 40 mg/mL; the corresponding IC50 values were 22.4, 41.7, and 43.3 mg/mL, respectively. The rates of egg hatching inhibition (EHI) at a concentration of 5 mg/mL were 99.5% for G. sepium, 64.2% for P. dulce, and 54% for L. leucocephala; the corresponding IC50 values were 1.9 mg/mL for G. sepium, 3.9 mg/mL for P. dulce, and 4.3 mg/mL for L. leucocephala. The species extracts studied here were also analyzed by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry (UHPLC-Q/Orbitrap/MS/MS), resulting in the compounds' identification associated with AA. Glycosylated flavonoids and methoxyphenols were observed in all three species: fatty acids in G. sepium and P. dulce; phenylpropanoids, anthraquinone glycosides, amino acids and glycosylated phenolic acids in G. sepium; and flavonoids in L. leucocephala. Comparatively, G. sepium presented a greater diversity of compounds potentially active against the control of gastrointestinal nematodes, which was associated with the results obtained in the applied tests.

Mulinum crassifolium Phil; Two New Mulinanes, Gastroprotective Activity and Metabolomic Analysis by UHPLC-Orbitrap Mass Spectrometry.

Mulinum crassifolium Phil. (Apiaceae) is an endemic shrub from Chile commonly used as infusion in traditional medicine to treat diabetes, bronchial and intestinal disorders and stomach ailments, including ulcers. From the EtOAc extract of this plant, the new mulinane-type diterpenoids 3 and 5 were isolated along with three known diterpenoids. The gastroprotective effect of the infusion of the plant was assayed to support the traditional use and a fast HPLC analysis using high resolution techniques was performed to identify the bioactive constituents. The EtOAc extract and the edible infusion showed gastroprotective effect at 100 mg/kg in the HCl/EtOH induced gastric ulcer model in mice, reducing lesions by 33% and 74%, respectively. Finally, a metabolomic profiling based on UHPLC-ESI-MS/HRMS of the edible infusion was performed and thirty-five compounds were tentatively identified including quercetin, caffeic acid, apigenine glucoside, p-coumaric acid, chlorogenic acids, and caffeoylquinic acids, which have been associated previously with gastroprotective and antiulcer properties. This scientific evidence can support the contribution of polyphenols in the gastroprotective activity of the edible infusion of this plant, and can validate at least in part, its ethnopharmacological use.

UHPLC-ESI-ORBITRAP-MS analysis of the native Mapuche medicinal plant palo negro (Leptocarpha rivularis DC. - Asteraceae) and evaluation of its antioxidant and cholinesterase inhibitory properties.

UHPLC/ESI/MS identification of organic compounds is the first step in the majority of screening techniques for the characterization of biologically active metabolites in natural sources. This paper describes a method for the fast identification and characterisation of secondary metabolites in Leptocarpha rivularis DC. (Palo negro) extracts by HPLC/UV (DAD)-Mass Spectrometry (HPLC/MS). The plant is used for the treatment of several diseases since pre-hispanic Mapuche times. Thirty-seven compounds were detected in the aqueous edible extract for the first time including 4 sesquiterpenes, 10 flavonoids, 9 oxylipins, 2 organic acids, and 11 phenolic acids. In addition, phenolic content antioxidant and cholinesterase inhibitory activities were measured for the first time using the edible infusion. The total polyphenol content of the infusion was 230.76 ± 2.5 mmol GAE/kg dry weight, while the antioxidant activity was 176.51 ± 28.84; 195.28 ± 4.83; and 223.92 ± 2.95 mmol TE/kg dry weight, for the DPPH, ABTS, and FRAP assays, respectively. The cholinesterase inhibitory activity was 7.38 ± 0.03 and 5.74 ± 0.06 mmol GALAE/kg, for the inhibition of acetylcholinesterase AChE and BChE, respectively, showing that this plant is a candidate for the isolation of compounds that can be useful for the treatment of neurodegenerative diseases. Furthermore, this plant could serve also as a raw material for the production of dietary supplements, due to its content of polyphenolic compounds.

Secondary Metabolite Profiling of Species of the Genus Usnea by UHPLC-ESI-OT-MS-MS.

Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria, which are considered among the slowest growing organisms, with strong tolerance to adverse environmental conditions. There are about 400 genera and 1600 species of lichens and those belonging to the Usnea genus comprise about 360 of these species. Usnea lichens have been used since ancient times as dyes, cosmetics, preservatives, deodorants and folk medicines. The phytochemistry of the Usnea genus includes more than 60 compounds which belong to the following classes: depsides, depsidones, depsones, lactones, quinones, phenolics, polysaccharides, fatty acids and dibenzofurans. Due to scarce knowledge of metabolomic profiles of Usnea species (U. barbata, U. antarctica, U. rubicunda and U. subfloridana), a study based on UHPLC-ESI-OT-MS-MS was performed for a comprehensive characterization of their secondary metabolites. From the methanolic extracts of these species a total of 73 metabolites were identified for the first time using this hyphenated technique, including 34 compounds in U. barbata, 21 in U. antarctica, 38 in U. rubicunda and 37 in U. subfloridana. Besides, a total of 13 metabolites were not identified and reported so far, and could be new according to our data analysis. This study showed that this hyphenated technique is rapid, effective and accurate for phytochemical identification of lichen metabolites and the data collected could be useful for chemotaxonomic studies.

Neuroprotective Effect of a New 7,8-Dihydroxycoumarin-Based Fe2+/Cu2+ Chelator in Cell and Animal Models of Parkinson's Disease.

Disturbed iron homeostasis, often coupled to mitochondrial dysfunction, plays an important role in the progression of common neurodegenerative diseases such as Parkinson's disease (PD). Recent studies have underlined the relevance of iron chelation therapy for the treatment of these diseases. Here we describe the synthesis, chemical, and biological characterization of the multifunctional chelator 7,8-dihydroxy-4-((methylamino)methyl)-2H-chromen-2-one (DHC12). Metal selectivity of DHC12 was Cu2+ ∼ Fe2+ > Zn2+ > Fe3+. No binding capacity was detected for Hg2+, Co2+, Ca2+, Mn2+, Mg2+, Ni2+, Pb2+, or Cd2+. DHC12 accessed cells colocalizing with Mitotracker Orange, an indication of mitochondrial targeting. In addition, DHC12 chelated mitochondrial and cytoplasmic labile iron. Upon mitochondrial complex I inhibition, DHC12 protected plasma membrane and mitochondria against lipid peroxidation, as detected by the reduced formation of 4-hydroxynonenal adducts and oxidation of C11-BODIPY581/591. DHC12 also blocked the decrease in mitochondrial membrane potential, detected by tetramethylrhodamine distribution. DHC12 inhibited MAO-A and MAO-B activity. Oral administration of DHC12 to mice (0.25 mg/kg body weight) protected substantia nigra pars compacta (SNpc) neurons against MPTP-induced death. Taken together, our results support the concept that DHC12 is a mitochondrial-targeted neuroprotective iron-copper chelator and MAO-B inhibitor with potent antioxidant and mitochondria protective activities. Oral administration of low doses of DHC12 is a promising therapeutic strategy for the treatment of diseases with a mitochondrial iron accumulation component, such as PD.

An unusual mulinane diterpenoid from the Chilean plant Azorella trifurcata (Gaertn) Pers.

Four new mulinane-type diterpenoids besides the known compounds mulin-11,13-dien-20-oic acid, 13α-hydroxyazorellane, 13ß-hydroxyazorellane, mulinolic acid, azorellanol, and mulin-11,13-dien-18-acetoxy-16,20-dioic acid were isolated from the Chilean plant Azorella trifurcata. One of the new metabolites isolated, 7α-acetoxy-9-epi-13ß-hydroxymulinane, possesses a new trans-syn-trans arrangement in a tricyclic ring system not previously encountered in nature. Among the mulinane diterpenoids isolated, mulin-11,13-dien-20-oic acid showed the gastroprotective effect on HCl-EtOH-induced gastric lesions in mice (ED50 = 55 mg kg(-1)). Regarding the mode of gastroprotective action for this active compound, its effect was reduced by pre-treatment of the mice with indomethacin and N-ethylmaleimide, suggesting that prostaglandins and sulfhydryl compounds are positively involved in the gastroprotective activity using this model.