Constituents from the Polar Extract of Pisolithus arhizus and Their Anti-inflammatory Activity.

The phytochemical study of the Pisolithus arhizus fruiting body methanol extract led to the isolation of six new triterpenoids (1-6) and one new naphthalenoid pulvinic acid derivative (7), together with five known compounds, including norbadione A (8). Their structure was established from 1D and 2D NMR spectroscopy and HRESIMS analyses. The absolute configuration of the triterpenoids was determined by circular dichroism. The two pulvinic acid derivatives 7 and 8, showing the highest activity in modulating IL-6 secretion, were tested for their effect on COX-2, STAT3, and p-STAT3 proteins; both compounds were able to downregulate p-STAT3.

Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F1 Fo ATP synthase.

Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.

Abietane Diterpenoids from the Hairy Roots of Salvia corrugata.

Salvia corrugata Vahl. is an interesting source of abietane and abeo-abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain transformed roots of S. corrugata and to evaluate the production of terpenoids in comparison with in vivo root production. Hairy roots were initiated from leaf explants by infection with ATCC 15834 Agrobacterium rhizogenes onto hormone-free Murashige and Skoog (MS) solid medium. Transformation was confirmed by polymerase chain reaction analysis of rolC and virC1 genes. The biomass production was obtained in hormone-free liquid MS medium using Temporary Immersion System bioreactor RITA®. The chromatographic separation of the methanolic extract of the untransformed roots afforded horminone, ferruginol, 7-O-acetylhorminone and 7-O-methylhorminone. Agastol and ferruginol were isolated and quantified from the hairy roots. The amount of these metabolites indicated that the hairy roots of S. corrugata can be considered a source of these compounds.

Labdane Diterpenoids from Salvia tingitana Etl. Synergize with Clindamycin against Methicillin-Resistant Staphylococcus aureus.

Quorum-sensing (QS) is a regulatory mechanism in bacterial communication, important for pathogenesis control. The search for small molecules active as quorum-sensing inhibitors (QSI) that can synergize with antibiotics is considered a good strategy to counteract the problem of antibiotic resistance. Here the antimicrobial labdane diterpenoids sclareol (1) and manool (2) extracted from Salvia tingitana were considered as potential QSI against methicillin-resistant Staphylococcus aureus. Only sclareol showed synergistic activity with clindamycin. The quantification of these compounds by LC-MS analysis in the organs and in the calli of S. tingitana showed that sclareol is most abundant in the flower spikes and is produced by calli, while manool is the major labdane of the roots, and is abundant also in the leaves. Other metabolites of the roots were abietane diterpenoids, common in Salvia species, and pentacyclic triterpenoids, bearing a γ-lactone moiety, previously undescribed in Salvia. Docking simulations suggested that 1 and 2 bind to key residues, involved in direct interactions with DNA. They may prevent accessory gene regulator A (AgrA) binding to DNA or AgrA activation upon phosphorylation, to suppress virulence factor expression. The antimicrobial activity of these two compounds probably achieves preventing upregulation of the accessory gene regulator (agr)-regulated genes.

In Silico Identification of Tripeptides as Lead Compounds for the Design of KOR Ligands.

The kappa opioid receptor (KOR) represents an attractive target for the development of drugs as potential antidepressants, anxiolytics and analgesics. A robust computational approach may guarantee a reduction in costs in the initial stages of drug discovery, novelty and accurate results. In this work, a virtual screening workflow of a library consisting of ~6 million molecules was set up, with the aim to find potential lead compounds that could manifest activity on the KOR. This in silico study provides a significant contribution in the identification of compounds capable of interacting with a specific molecular target. The main computational techniques adopted in this experimental work include: (i) virtual screening; (ii) drug design and leads optimization; (iii) molecular dynamics. The best hits are tripeptides prepared via solution phase peptide synthesis. These were tested in vivo, revealing a good antinociceptive effect after subcutaneous administration. However, further work is due to delineate their full pharmacological profile, in order to verify the features predicted by the in silico outcomes.

Trace Elements in Edible Flowers from Italy: Further Insights into Health Benefits and Risks to Consumers.

The use of edible flowers in cooking dates back to ancient times, but recently it is gaining success among the consumers, increasingly attentive to healthy and sustainable foods of high quality, without neglecting taste, flavour, and visual appeal. The present study aims to deepen the knowledge regarding the mineral composition of edible flowers, an aspect not widely investigated in scientific literature. The concentrations of Cd, Co, Cu, Fe, Mn, Ni, Pb, Sr, V, and Zn have been determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) in flowers belonging to a wide variety of species. The study highlights that some floral species are characterized by significantly higher concentrations of certain trace elements, e.g., the flowers of Acmella oleracea for Mn, those of basil (Ocimum basilicum) and of pumpkins (Cucurbita moschata and C. pepo) for Cu and Sr, and those of orange daylily (Hemerocallis fulva) for Ni. Potentially toxic elements are present at low concentrations, often below the limit of the detection for Cd, Co, Ni, V. In all samples, Cd and Pb are well below the maximum permitted levels in foodstuffs. It can be concluded that the edible flowers analyzed can be considered a good source of essential elements and do not present risks for the consumer health as for the mineral composition.

NMR Metabolite Profiling for the Characterization of Vessalico Garlic Ecotype and Bioactivity against Xanthomonas campestris pv. campestris.

The Italian garlic ecotype "Vessalico" possesses distinct characteristics compared to its French parent cultivars Messidor and Messidrôme, used for sowing, as well as other ecotypes in neighboring regions. However, due to the lack of a standardized seed supply method and cultivation protocol among farmers in the Vessalico area, a need to identify garlic products that align with the Vessalico ecotype arises. In this study, an NMR-based approach followed by multivariate analysis to analyze the chemical composition of Vessalico garlic sourced from 17 different farms, along with its two French parent cultivars, was employed. Self-organizing maps allowed to identify a homogeneous subset of representative samples of the Vessalico ecotype. Through the OPLS-DA model, the most discriminant metabolites based on values of VIP (Variable Influence on Projections) were selected. Among them, S-allylcysteine emerged as a potential marker for distinguishing the Vessalico garlic from the French parent cultivars by NMR screening. Additionally, to promote sustainable agricultural practices, the potential of Vessalico garlic extracts and its main components as agrochemicals against Xanthomonas campestris pv. campestris, responsible for black rot disease, was explored. The crude extract exhibited a MIC of 125 µg/mL, and allicin demonstrated the highest activity among the tested compounds (MIC value of 31.25 µg/mL).

Sustainable Agrochemical Exploitation of Ligurian Aromatic Plant Ecotypes.

The characterization of local improved varieties as well as the reduction of synthetic chemical fertilizers are sustainable approaches in the vision of a new precision Farming. Aim of our study was to improve the geographical characterization of local ecotypes and to identify peculiar features of new crops in terms of bioactive compounds. NMR and LC-MS metabolite profiling approaches followed by multivariate data analysis were applied to characterize local rosemary and garlic ecotypes. With the aim of applying for a protected designation of origin, orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify representative sensory quality indicators for Vessalico garlic and rosemary "Eretto Liguria" local ecotypes, Variable Influence on Projections (VIP) values of OPLS-DA indicated six metabolites as quality indicators for Vessalico garlic and sixteen metabolites as quality indicators for rosemary "Eretto Liguria". Finally, to discover and utilize new ecotypes in a sustainable way, Vessalico garlic extracts antiviral activity, previously evaluated against Tomato brown rugose fruit virus (ToBRFV), a Tobamovirus affecting tomato crops, was extended to Pepino mosaic virus (PepMV) with positive results.

Targeted metabolite profiling of Salvia rosmarinus Italian local ecotypes and cultivars and inhibitory activity against Pectobacterium carotovorum subsp. carotovorum.

Introduction: The development of agriculture in terms of sustainability and low environmental impact is, at present, a great challenge, mainly in underdeveloped and marginal geographical areas. The Salvia rosmarinus "Eretto Liguria" ecotype is widespread in Liguria (Northwest Italy), and farmers commonly use it by for cuttings and for marketing. In the present study, this ecotype was characterized in comparison with other cultivars from the same geographical region and Campania (Southern Italy), with a view to application and registration processes for the designation of protected geographical indications. Moreover, the possibility of using the resulting biomass after removing cuttings or fronds as a source of extracts and pure compounds to be used as phytosanitary products in organic farming was evaluated. Specifically, the potential of rosemary extracts and pure compounds to prevent soft rot damage was then tested. Methods: A targeted NMR metabolomic approach was employed, followed by multivariate analysis, to characterize the rosemary accessions. Bacterial soft rot assay and disk diffusion test were carried out to evaluate the activity of extracts and isolated compounds against Pectobacterium carotovorum subsp. carotovorum. Enzymatic assay was performed to measure the in vitro inhibition of the pectinase activity produced by the selected pathogen. Molecular docking simulations were used to explore the possible interaction of the selected compounds with the pectinase enzymes. Results and Discussion: The targeted metabolomic analysis highlighted those different geographical locations can influence the composition and abundance of bioactive metabolites in rosemary extracts. At the same time, genetic factors are important when a single geographical area is considered. Self-organizing maps (SOMs) showed that the accessions of "Eretto Liguria" appeared well characterized when compared to the others and had a good content in specialized metabolites, particularly carnosic acid. Soft rotting Enterobacteriaceae belonging to the Pectobacterium genus represent a serious problem in potato culture. Even though rosemary methanolic extracts showed a low antibacterial activity against a strain of Pectobacterium carotovorum subsp. carotovorum in the disk diffusion test, they showed ability in reducing the soft rot damage induced by the bacterium on potato tissue. 7-O-methylrosmanol, carnosol and isorosmanol appeared to be the most active components. In silico studies indicated that these abietane diterpenoids may interact with P. carotovorum subsp. carotovorum pectate lyase 1 and endo-polygalacturonase, thus highlighting these rosemary components as starting points for the development of agents able to prevent soft rot progression.

Anti-Biofilm Activity of Carnosic Acid from Salvia rosmarinus against Methicillin-Resistant Staphylococcus aureus.

The Salvia rosmarinus "Eretto Liguria" ecotype was studied as a source of valuable bioactive compounds. LC-MS analysis of the methanolic extract underlined the presence of diterpenoids, triterpenoids, polyphenolic acids, and flavonoids. The anti-virulence activity of carnosic acid along with the other most abundant compounds against methicillin-resistant Staphylococcus aureus (MRSA) was evaluated. Only carnosic acid induced a significant reduction in the expression of agrA and rnaIII genes, which encode the key components of quorum sensing (QS), an intracellular signaling mechanism controlling the virulence of MRSA. At a concentration of 0.05 mg/mL, carnosic acid inhibited biofilm formation by MRSA and the expression of genes involved in toxin production and made MRSA more susceptible to intracellular killing, with no toxic effects on eukaryotic cells. Carnosic acid did not affect biofilm formation by Pseudomonas aeruginosa, a human pathogen that often coexists with MRSA in complex infections. The selected ecotype showed a carnosic acid content of 94.3 ± 4.3 mg/g. In silico analysis highlighted that carnosic acid potentially interacts with the S. aureus AgrA response regulator. Our findings suggest that carnosic acid could be an anti-virulence agent against MRSA infections endowed with a species-specific activity useful in multi-microbial infections.

Exploring the Anticancer Potential of Premna resinosa (Hochst.) Leaf Surface Extract: Discovering New Diterpenes as Heat Shock Protein 70 (Hsp70) Binding Agents.

Premna, a genus consisting of approximately 200 species, predominantly thrives in tropical and subtropical areas. Many of these species have been utilized in ethnopharmacology for diverse medicinal applications. In Saudi Arabia, Premna resinosa (Hochst.) Schauer (Lamiaceae) grows wildly, and its slightly viscid leaves are attributed to the production of leaf accession. In this study, we aimed to extract the surface accession from fresh leaves using dichloromethane to evaluate the anticancer potential. The plant exudate yielded two previously unknown labdane diterpenes, Premnaresone A and B, in addition to three already described congeners and four known flavonoids. The isolation process was accomplished using a combination of silica gel column chromatography and semi-preparative HPLC, the structures of which were identified by NMR and HRESIMS analyses and a comparison with the literature data of associated compounds. Furthermore, we employed a density functional theory (DFT)/NMR approach to suggest the relative configuration of different compounds. Consequently, we investigated the possibility of developing new chaperone inhibitors by subjecting diterpenes 1-5 to a Surface Plasmon Resonance-screening, based on the knowledge that oridonin, a diterpene, interacts with Heat Shock Protein 70 (Hsp70) 1A in cancer cells. Additionally, we studied the anti-proliferative activity of compounds 1-5 on human Jurkat (human T-cell lymphoma) and HeLa (epithelial carcinoma) cell lines, where diterpene 3 exhibited activity in Jurkat cell lines after 48 h, with an IC50 of 15.21 ± 1.0 µM. Molecular docking and dynamic simulations revealed a robust interaction between compound 3 and Hsp70 key residues.

The Flavone Cirsiliol from Salvia x jamensis Binds the F1 Moiety of ATP Synthase, Modulating Free Radical Production.

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F1Fo-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H2O2 production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress.

Sclareol modulates free radical production in the retinal rod outer segment by inhibiting the ectopic f1fo-atp synthase.

We have previously shown that the retinal rod outer segments (OS) produce reactive oxygen species in the function of illumination in vitro, establishing a relationship among the extra-mitochondrial oxidative phosphorylation and phototransduction. This source of oxidative stress in the OS can be modulated by polyphenols, acting as inhibitors of F1Fo-ATP synthase. The present study aimed at exploring whether sclareol, a diterpene, interacts with F1Fo-ATP synthase mitigating the light-induced free radical production in the rod OS. Characterization of bovine retinal sections was conducted by immunogold analysis. Reactive oxygen intermediates production, oxygen consumption, the activity of the four respiratory complexes and ATP synthesis were evaluated in purified bovine rod OS. Molecular docking analyses were also conducted. Sclareol reduced free radical production by light-exposed rod OS. Such antioxidant effect was associated with an inhibition of the respiratory complexes and oxygen consumption (OCR), in coupled conditions. Sclareol also inhibited the rod OS ATP synthetic ability. Since the inhibitor effect on respiratory complexes and OCR is not observed in uncoupled conditions, it is supposed that the modulating effect of sclareol on the ectopic oxidative phosphorylation in the rod OS targets specifically the F1Fo-ATP synthase. This hypothesis is confirmed by the in silico molecular docking analyses, which shows that sclareol binds the F1 moiety of ATP synthase with high affinity. In conclusion, a beneficial effect of sclareol can be envisaged as a modulator of oxidative stress in the photoreceptor, a risk factor for the degenerative retinal diseases, suggestive of its potential beneficial action also in vivo.