Effect of Environmental Factors on Plectranthus Neochilus Volatile Composition: A GC-MS-Based Metabolomics Approach

Plectranthus neochilus Schltr. is an aromatic species, commonly used for digestive, antispasmodic, and analgesic purposes. Although many studies have reported the chemical composition of its essential oil, variations in the volatile profile were observed, which may be due to multiple factors linked to growth and field conditions. In order to detect metabolic variations in this species, we employed a GC-MS-based untargeted metabolomics approach analyzing samples of four P. neochilus individuals collected over a year. From all analyses, 24 mass features were detected and 21 were identified according to their respective chromatographic peaks. All features varied among samples, particularly (2E)-hexenal, 3-octanone and δ-3-carene, which showed the highest coefficient of variation percentage in our study. Although the four individuals presented the same peaks in the chromatograms, significant differences in the intensity of specific mass features were detected between individuals throughout the year. Time of sampling did not affect P. neochilus volatile composition; the chemical profile remained constant throughout the day. Seasonal trends were observed for the species. Winter months coincided with a drop in the intensity of most components. Air temperature showed a positive correlation with some feature intensities, while myrcene and α-thujene resulted in a positive and a negative correlation with rainfall, respectively. This study was the first attempt to correlate metabolic variation and environmental factors in P. neochilus. Our approach was successful in identifying the composition and variation of the headspace volatiles of P. neochilus leaves.

In vitro Inhibitory Action of the Essential Oils of Origanum Vulgare and Rosmarinus Officinalis against Aspergillus Fumigatus

Aspergillus fumigatus is the main etiological agent of aspergillosis. Considering azole antifungal drug resistance in A. fumigatus, which compromises treatment, new alternatives are needed. Among them, essential oils (EOs) can be an alternative treatment, having shown positive results in inhibiting phytopathogenic fungi in vitro. We aimed to determine the in vitro antifungal activity of Origanum vulgare L. subsp. hirtum (Link) (oregano) and Rosmarinus officinalis L. (rosemary) EOs alone and in association (O. vulgare+R. officinalis) against A. fumigatus. EOs were analyzed by gas chromatography (GC-FID and GC/MS systems), and analyses showed that the major components of O. vulgare EO were carvacrol (67.8%), p-cymene (14.8%), and thymol (3.9%); for R. officinalis, they were the monoterpenes 1,8-cineole (49.1%), camphor (18.1%) and α-pinene (8.1). For biological assays, five EO concentrations, 0.2; 0.4; 0.6; 0.8 and 1.0%, were used in disk diffusion and agar dilution tests for 21 days. In disk diffusion, O. vulgare EO alone and in association (O. vulgare+R. officinalis) showed fungicidal activity at all concentrations. In agar dilution, inhibitory action was demonstrated from 0.6% for O. vulgare EO and in association (O. vulgare+R. officinalis). R. officinalis EO at 1.0% showed no fungal growth, determining the minimum inhibitory concentration (MIC). The present study demonstrated inhibitory actions of O. vulgare and R. officinalis EOs in A. fumigatus. GC analyses corroborated the literature regarding their antibacterial and antifungal effects. However, further in vitro and in vivo studies are needed to evaluate EOs as alternative antifungals for treating aspergillosis.

Anticipate, Target and Characterize: MS²-anticipated C-glycosylflavones from Erythrococca anomala

We herein report on the first chemical assessment of Erythrococca anomala (Juss. ex Poir.) Prain (Euphorbiaceae), a genus that was – to the best of our knowledge – not studied yet from a phytochemical perspective. A molecular networking strategy was implemented to rapidly identify the known specialized metabolites from untargeted MS/MS analyses of E. anomala leaves ethanolic extract. This strategy allowed for the identification of diverse C-glycosyl flavones and a cursory examination of MS/MS spectra could extend the GNPS-provided annotation to pinpoint the structural novelty of further derivatives. The isolation of the sought-after structures could be streamlined based on MS-guidance and their structures, determined through extensive NMR analyses, displayed structural features in line with MS²-based predictions. Anticipating sharp structural features at an early stage of the dereplication process through a critical assessment of the tandem mass spectrometric landmarks was essential to embark on the isolation of the newly reported structures owing to the elevated number of flavonoid glycosides isomers thereof formerly known, which would have deterred us from isolating them without the support of additional tandem mass spectrometric information. The isolation of the main components of the ethanolic extract completed the currently provided chemical report on E. anomala, also resulting in the description of a new phenylethanoid derivative (3) and of a new orcinol-based dimer (4). Anomaloflavone (1) exhibit significant activities with minimal inhibitory concentration values of 25 µg/mL against Staphylococcus aureus and Mycobacterium smegmatis while failing to exert an antibacterial activity against Pseudomonas aeruginosa, while being devoid of cytotoxicity against SiHa cells.

Thymol and Piperine-Loaded Poly(D,L-lactic-co-glycolic acid) Nanoparticles Modulate Inflammatory Mediators and Apoptosis in Murine Macrophages

This study aimed at preparing and characterizing thymol, eugenol, and piperine-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles and evaluating the effect on inflammatory mediators secretion and apoptosis in Raw 264.7 macrophage cells. Nanoparticles were produced by the solvent evaporation technique. Dynamic light scattering and scanning electron microscopy were used to study the physicochemical characteristics. Raw 264.7 macrophage cells were used as a model for in vitro assays. The 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium assay was used to determine the cytotoxicity of the formulated nanoparticles. An annexin V apoptosis detection kit was used to assess apoptosis. Nitric oxide production was determined using the Griess reagent, and the inflammatory mediators level was evaluated with Th1/Th2 cytokine and fluorometric cyclooxygenase kits. The loaded nanoparticles showed a particle size around 190 nm with a low polydispersity between 0.069 and 0.104 and a zeta potential between–1.2 and–9.5 mV. Reduced cytotoxicity of nanoparticles compared to free molecules against Raw 264.7 macrophage cells was observed and seemed to occur through a mechanism associated with apoptosis. A decrease in cyclooxygenase enzyme activity with an increasing concentration was observed. Both free molecules and nanoparticles showed their capacity to modulate the inflammatory process mostly by inhibiting the investigated inflammatory cytokines. The data presented in this study indicate that thymol and piperine-loaded poly(D,L-lactic-co-glycolic acid nanoparticles could serve as a novel anti-inflammatory colloidal drug delivery system with reduced toxicity. However, further study should be considered to optimize the formulation’s loading capacity and thereby probably enhance their bioactivity in treating inflammatory diseases.

Triterpenoids from Alisma orientale and their NF-кB Inhibitory Activity

The rhizome of Alisma orientale (sam.) Juz. is used in clinics for eliminating dampness, reducing edema, and promoting diuresis. This study aimed to elucidate the compounds and investigate their nuclear factor-kappa inhibitory activities in human embryonic kidney 293 cells. A new triterpene, alismaketone B (1); a new natural nortriterpene, noralisolic acid A (2); and 13 known protostane-type triterpenes were isolated from the rhizome of A. orientale. The new structures and their absolute configurations were established using HRESIMS, NMR, and electronic circular dichroism experiments. All isolated compounds were evaluated for their inhibitory activity on NF-κB. The compounds 8, 9, 10, and 14 showed moderate NF-κB inhibitory activities with their IC50 values being 64.7, 32.3, 47.3, and 37.3 μM, respectively.

Effects of Chronic Administration of P-Cymene in an Animal Model of LPS-Induced Autism

p-Cymene is a monoterpene found in over 100 plant species. It shows a range of biological activity, including anti-inflammatory and antimicrobial effects. It is possibly a new therapeutic alternative for autism spectrum disorder characterized by deficits in interaction and behavioral abnormalities. These symptoms can occur in response to maternal immune activation through prenatal exposure to lipopolysaccharide. Thus, this study aimed to evaluate the behavioral, memory, and biochemical effects of chronic administration of p-cymene in an animal model of autism by prenatal maternal exposure to lipopolysaccharide. Twenty-four pregnant Wistar rats were used, who received 100 μg/kg of lipopolysaccharide or saline intraperitoneally (i.p.) on the 9.5 gestational day. After birth, the male offspring remained with the mothers until weaning and underwent model validation tests on postnatal day 30. From postnatal day 31 on, chronic administration, via i.p., of saline (1 mL/kg), risperidone (0.2 mg/kg), or p-cymene (100 mg/kg) for 22 days was performed. The animals were submitted to behavioral (postnatal day 52) and memory tests (postnatal days 52–53) and subsequently sacrificed (postnatal day 54) when their brain structures were removed for quantification of proinflammatory cytokines (TNF-α, interleukin 1β, and interleukin 6). Prenatal exposure to lipopolysaccharide significantly increased episodes of stereotyped movement (p=0.0001) and decreased parameters of social interaction in offspring, including sniffing, following, mounting, and allowing mounting (p=0.0043, p<0.0001, p=0.0009, and p=0.0200, respectively). Chronic p-cymene treatment was not significant for behavioral, memory, and biochemical tests. However, due to their pharmacokinetic characteristics, p-cymene nanomaterials’ formulation may be an exciting alternative to be tested for further results.

Neuroprotective Effects of Delta-9-Tetrahydrocannabinol against FeSO4- and H2O2-Induced Cell Damage on Dopaminergic Neurons in Primary Mesencephalic Cell Culture

Delta-9-Tetrahydrocannabinol and other phytocannabinoids have been previously demonstrated to possess neuroprotective effects in murine mesencephalic cell culture models of Parkinson’s disease, in which increased levels of superoxide radicals led to the loss of dopaminergic neurons. In these models, delta-9-tetrahydrocannabinol did not scavenge these radicals but displayed antioxidative capacity by increasing glutathione levels. Based on these findings, in the present study, we investigated whether the neuroprotective effect of delta-9-tetrahydrocannabinol can also be detected in FeSO4- and H2O2-stressed cells. Mesencephalic cultures were concomitantly treated with FeSO4 (350 μM) or H2O2 (150 μM) and delta-9-tetrahydrocannabinol (0.01, 0.1, 1, 10 μM) on the 12th days in vitro for 48 h. On the 14th DIV, dopaminergic neurons were stained immunocytochemically by tyrosine hydroxylase, and fluorescently using crystal violet, Hoechst 33342, and JC-1. FeSO4 and H2O2 significantly reduced the number of dopaminergic neurons by 33 and 36%, respectively, and adversely affected the morphology of surviving neurons. Moreover, FeSO4, but not H2O2, significantly decreased the fluorescence intensity of crystal violet and Hoechst 33342, and reduced the red/green ratio of JC-1. Co-treatment with delta-9-tetrahydrocannabinol at the concentrations 0.01 and 0.1 μM significantly rescued dopaminergic neurons in FeSO4 and H2O2-treated cultures by 16 and 30%, respectively. delta-9-Tetrahydrocannabinol treatment also led to a higher fluorescence intensity of crystal violet and Hoechst 33342, and increased the red/green fluorescence ratio of JC-1 when concomitantly administered with FeSO4 but not H2O2. To conclude, delta-9-tetrahydrocannabinol rescues dopaminergic neurons against FeSO4- and H2O2-induced neurotoxicity. Using fluorescence dyes, this effect seems to be mediated partially by restoring mitochondrial integrity and decreasing cell death, particularly in FeSO4-treated cultures.

Screening of 20 Pantanal Wetland Plants for Anti-Candida Activity using HPLC-DAD-MS/MS and Bioautography to Characterize Active Compounds

The Pantanal wetland harbors a rich flora with uncharted pharmacological potential. This study evaluated 20 Brazilian Pantanal plants against Candida albicans, C. parapsilosis, C. tropicalis, and C. krusei. Fungal susceptibility was determined by agar diffusion and broth microdilution; active compounds were identified by bioautography and HPLC-DAD-MS/MS. Sesbania virgata, Polygala molluginifolia, and Cantinoa mutabilis extracts and their chloroform and ethyl acetate (EtOAc) fractions exhibited the best activity against all Candida species tested. The EtOAc fraction of P. molluginifolia proved to be more efficient in inhibiting C. parapsilosis and C. krusei growth (Minimum inhibitory concentration of 125 and 62.5 μg/mL, respectively). Bioautography of this fraction revealed two active bands, characterized by HPLC-DAD-MS/MS as a mixture of podophyllotoxin derivatives blumenol, besides some flavonoids. This work demonstrated antifungal potential of P. molluginifolia podophyllotoxin derivatives and the versatility of bioautography with HPLC-DAD-MS/MS to identify the bioactive compounds.

Cytokinin Mediated Increased In Vitro Production of Secondary Metabolites with Special Reference to Solasodine in Solanum erianthum

Steroid alkaloid solasodine is a nitrogen analogue of diosgenin and has great importance in the production of steroidal medicines. Solanum erianthum D. Don (Solanaceae) is a good source of solasodine. The aim of this study was to evaluate the effect of different cytokinins on the production of secondary metabolites, especially solasodine in the in vitro culture of S. erianthum. For solasodine estimation, field-grown plant parts and in vitro tissues were extracted thrice and subjected to high-performance liquid Chromatography. Quantitative analysis of different secondary metabolites showed that the amount was higher in the in vitro regenerated plantlets compared to callus and field-grown plants. The present study critically evaluates the effect of the type of cytokinin used in the culture medium on solasodine accumulation in regenerated plants. The highest solasodine content (46.78±3.23 mg g-1) was recorded in leaf extracts of the in vitro grown plantlets in the presence of 6-γ,γ-dimethylallylamino purine in the culture medium and the content was 3.8-fold higher compared to the mother plant.

Pachypodostyflavone, a New 3-methoxy Flavone and Other Constituents with Antifilarial Activities from the Stem Bark of Duguetia staudtii

A new flavone derivative named pachypodostyflavone (1), along with 8 known compounds (2–9) and a mixture of β-sitosterol and stigmasterol were isolated from the stem bark of Duguetia staudtii (Annonaceae), based on a bioassay-guided fractionation. Their structures were determined using high-resolution mass spectrometry and NMR spectroscopic data, as well as by comparison with the literature values of their analogs. Selected isolated compounds were evaluated for their in vitro antifilaricidal activities on Onchocerca ochengi microfilariae and adult worms. Inhibition of motility was evaluated spectroscopically on microfilaria and adult male worms. Viability was determined on adult female worms by the MTT/ Formazan assay. Auranofin at 10 µM and 2% DMSO were used as positive and negative controls, respectively. Compounds 1 and 7 showed potent anti-onchocerca activities with 100% activity at 250 µg/mL on both O. ochengi adult male and female worms, while compound 5 displayed 100% activity at 30 µg/mL.