Pharmacokinetics and Metabolism Investigation of Oleocanthal.

Oleocanthal is a secoiridoid found in olive oil, which lately gained great scientific interest due to its important pharmacological spectrum and biological properties. However, limited data exist on the metabolic fate of oleocanthal in vivo, a commonly underestimated aspect in natural products research. Especially, its pharmacokinetic (PK) characteristics have never been described so far. Thus, in the current study, a mouse-based protocol was designed, and oleocanthal was administered intraperitoneally in a standard dose of 5 mg/kg. In order to determine the PK parameters of oleocanthal or its metabolites, plasma samples were collected at 10 time points. Extraction and analysis protocols were developed and applied for the recovery and detection of oleocanthal in plasma, as well as the identification of its metabolites, using LC-HRMS/MS. Oleocanthal was not detected, proving the short lifetime of the compound in vivo, and 13 metabolites were identified. Among them, oleocanthalic acid and tyrosol sulfate were proposed as oleocanthal's biomarkers, in vivo. This is the first report associating oleocanthalic acid with oleocanthal administration in vivo, while its PK parameters, Tmax (T0) and Cmax (926 µg/mL), were also determined. The current study enlightens bioavailability and metabolism aspects of oleocanthal and suggests the association of specific metabolites with the biological effects attributed to oleocanthal administration. More studies are needed to give better insights into the metabolism and the mechanism of action of secoiridoids as well as to respond to identification challenges related to secoiridoid in vivo setups.

Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

Targeting protein kinases that regulate signalling pathways in inflammation is an effective pharmacological approach to alleviate uncontrolled inflammatory diseases. In this context, the natural product indirubin and its 6-bromo-substituted analogue 6-bromoindirubin-3 -glycerol-oxime ether (6BIGOE; 1) were identified as potent inhibitors of glycogen synthase kinase-3ß (GSK-3ß). These inhibitors suppress the release of pro-inflammatory cytokines and prostaglandins (PG) from human monocytes. However, indirubin derivatives target several protein kinases such as cyclin-dependent kinases (CDKs) which has been a major concern for their application in inflammation therapy. Here, we report on a library of 13 5-bromo-substituted indirubin derivatives that have been designed to improve potency and target selectivity. Side-by-side comparison of reference compound 1 (6BIGOE) with 5-bromo derivatives revealed its isomer 2 (5BIGOE), as the most potent derivative able to supress pro-inflammatory cytokine and PG release in lipopolysaccharide-stimulated human monocytes. Analysis of protein kinase inhibition in intact monocytes, supported by our in silico findings, proposed higher selectivity of 1 for GSK-3ß inhibition with lesser potency against CDKs 8 and 9. In contrast, 2 supressed the activity of these CDKs with higher effectiveness than GSK-3ß, representing additional targets of indirubins within the inflammatory response. Encapsulation of 1 and 2 into polymer-based nanoparticles (NP) improved their pharmacological potential. In conclusion, the 5- and 6-brominated indirubins 1 and 2 as dual GSK-3ß and CDK8/9 inhibitors represent a novel concept for intervention with inflammatory disorders.

The Bioactivity of a Hydroxytyrosol-Enriched Extract Originated after Direct Hydrolysis of Olive Leaves from Greek Cultivars.

Nowadays, olive leaf polyphenols have been at the center of scientific interest due to their beneficial effects on human health. The most abundant polyphenol in olive leaves is oleuropein. The biological properties of oleuropein are mainly due to the hydroxytyrosol moiety, a drastic catechol group, whose biological activity has been mentioned many times in the literature. Hence, in recent years, many nutritional supplements, food products, and cosmetics enriched in hydroxytyrosol have been developed and marketed, with unexpectedly positive results. However, the concentration levels of hydroxytyrosol in olive leaves are low, as it depends on several agricultural factors. In this study, a rapid and easy methodology for the production of hydroxytyrosol-enriched extracts from olive leaves was described. The proposed method is based on the direct acidic hydrolysis of olive leaves, where the extraction procedure and the hydrolysis of oleuropein are carried out in one step. Furthermore, we tested the in vitro bioactivity of this extract using cell-free and cell-based methods, evaluating its antioxidant and DNA-protective properties. Our results showed that the hydroxytyrosol-enriched extract produced after direct hydrolysis of olive leaves exerted significant in vitro antioxidant and geno-protective activity, and potentially these extracts could have various applications in the pharmaceutical, food, and cosmetic industries.

Next-generation sequencing reveals altered gene expression and enriched pathways in triple-negative breast cancer cells treated with oleuropein and oleocanthal.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by poor prognosis and limited treatment options. Oleuropein and oleocanthal are bioactive chemicals found in extra-virgin olive oil; they have been shown to have anti-cancer potential. In this study, we examined the inhibitory effects of these two natural compounds, on MDA-MB-231 and MDA-MB-468 TNBC cell lines. The human TNBC MDA-MB-231 and MDA-MB-468 cell lines were treated with oleuropein or oleocanthal at ranging concentrations for 48 h. After determining the optimum concentration to reach IC50, using the sulforhodamine B assay, total RNA was extracted after 12, 24, and 48 h from treated and untreated cells. Poly(A)-RNA selection was conducted, followed by library construction and RNA sequencing. Differential gene expression (DEG) analysis was performed to identify DEGs between treated and untreated cells. Pathway analysis was carried out using the KEGG and GO databases. Oleuropein and oleocanthal considerably reduced the proliferation of TNBC cells, with oleocanthal having a slightly stronger effect than oleuropein. Furthermore, multi-time series RNA sequencing showed that the expression profile of TNBC cells was significantly altered after treatment with these compounds, with temporal dynamics and groups of genes consistently affected at all time points. Pathway analysis revealed several significant pathways associated with TNBC, including cell death, apoptotic process, programmed cell death, response to stress, mitotic cell cycle process, cell division, and cancer progression. Our findings suggest that oleuropein and oleocanthal have potential therapeutic benefits for TNBC and can be further investigated as alternative treatment options.

Synergistic Pulmonoprotective Effect of Natural Prolyl Oligopeptidase Inhibitors in In Vitro and In Vivo Models of Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a highly morbid inflammatory lung disease with limited pharmacological interventions. The present study aims to evaluate and compare the potential pulmonoprotective effects of natural prolyl oligopeptidase (POP) inhibitors namely rosmarinic acid (RA), chicoric acid (CA), epigallocatechin-3-gallate (EGCG) and gallic acid (GA), against lipopolysaccharide (LPS)-induced ARDS. Cell viability and expression of pro-inflammatory mediators were measured in RAW264.7 cells and in primary murine lung epithelial and bone marrow cells. Nitric oxide (NO) production was also assessed in unstimulated and LPS-stimulated RAW264.7 cells. For subsequent in vivo experiments, the two natural products (NPs) with the most favorable effects, RA and GA, were selected. Protein, cell content and lipid peroxidation levels in bronchoalveolar lavage fluid (BALF), as well as histopathological changes and respiratory parameters were evaluated in LPS-challenged mice. Expression of key mediators involved in ARDS pathophysiology was detected by Western blotting. RA and GA favorably reduced gene expression of pro-inflammatory mediators in vitro, while GA decreased NO production in macrophages. In LPS-challenged mice, RA and GA co-administration improved respiratory parameters, reduced cell and protein content and malondialdehyde (MDA) levels in BALF, decreased vascular cell adhesion molecule-1 (VCAM-1) and the inducible nitric oxide synthase (iNOS) protein expression, activated anti-apoptotic mechanisms and down-regulated POP in the lung. Conclusively, these synergistic pulmonoprotective effects of RA and GA co-administration could render them a promising prophylactic/therapeutic pharmacological intervention against ARDS.

Drug delivery of 6-bromoindirubin-3'-glycerol-oxime ether employing poly(D,L-lactide-co-glycolide)-based nanoencapsulation techniques with sustainable solvents.

BACKGROUND: Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. RESULTS: For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around - 15 to - 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen's egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. CONCLUSIONS: Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential.

The antimicrobial effect of Rosmarinus officinalis extracts on oral initial adhesion ex vivo.

OBJECTIVE: In the last few decades, there has been a growing worldwide interest in the use of plant extracts for the prevention of oral diseases. The main focus of this interest lies in the identification and isolation of substances that limit the formation of microbial biofilm which plays a major role in the development of caries, periodontitis, and peri-implantitis. In this clinical ex vivo study, we investigated the antimicrobial effects of Rosmarinus officinalis extract against oral microorganisms within in situ initial oral biofilms. MATERIALS AND METHODS: Initial in situ biofilm samples (2 h) from six healthy volunteers were treated ex vivo with R. officinalis extract at concentrations of 20 mg/ml and 30 mg/ml. The number of viable bacterial cells was determined by counting the colony-forming units. All surviving bacteria were isolated in pure cultures and identified using MALDI-TOF and biochemical testing procedures. Additionally, live/dead staining in combination with epifluorescence microscopy was used for visualizing the antimicrobial effects in the initial biofilms. RESULTS: The number of colony-forming units in the R. officinalis-treated biofilms was significantly lower than in the untreated controls (p < 0.001). The reduction range of log10 was 1.64-2.78 and 2.41-3.23 for aerobic and anaerobic bacteria, respectively. Regarding the bacterial composition, large intra- and interindividual variability were observed. Except for Campylobacter spp., the average amount of all bacterial taxa was lower after treatment with R. officinalis than in the untreated biofilms. A total of 49 different species were detected in the untreated biofilms, while only 11 bacterial species were detected in the R. officinalis-treated biofilms. Live/dead staining confirmed that the R. officinalis-treated biofilms had significantly lower numbers of surviving bacteria than the untreated biofilms. CONCLUSIONS: The treatment with R. officinalis extract has a significant potential to eliminate microbial oral initial biofilms. CLINICAL RELEVANCE: The results of this study encourage the use of R. officinalis extracts in biofilm control and thus in the treatment of caries and periodontitis as a herbal adjuvant to synthetic substances.

Oleuropein-Induced Acceleration of Cytochrome P450-Catalyzed Drug Metabolism: Central Role for Nuclear Receptor Peroxisome Proliferator-Activated Receptor α.

Oleuropein (OLE), the main constituent of Olea europaea, displays pleiotropic beneficial effects in health and disease, which are mainly attributed to its anti-inflammatory and cardioprotective properties. Several food supplements and herbal medicines contain OLE and are available without a prescription. This study investigated the effects of OLE on the main cytochrome P450s (P450s) catalyzing the metabolism of many prescribed drugs. Emphasis was given to the role of peroxisome proliferator-activated receptor α (PPARα), a nuclear transcription factor regulating numerous genes including P450s. 129/Sv wild-type and Ppara-null mice were treated with OLE for 6 weeks. OLE induced Cyp1a1, Cyp1a2, Cyp1b1, Cyp3a14, Cyp3a25, Cyp2c29, Cyp2c44, Cyp2d22, and Cyp2e1 mRNAs in liver of wild-type mice, whereas no similar effects were observed in Ppara-null mice, indicating that the OLE-induced effect on these P450s is mediated by PPARα. Activation of the pathways related to phosphoinositide 3-kinase/protein kinase B (AKT)/forkhead box protein O1, c-Jun N-terminal kinase, AKT/p70, and extracellular signal-regulated kinase participates in P450 induction by OLE. These data indicate that consumption of herbal medicines and food supplements containing OLE could accelerate the metabolism of drug substrates of the above-mentioned P450s, thus reducing their efficacy and the outcome of pharmacotherapy. Therefore, OLE-induced activation of PPARα could modify the effects of drugs due to their increased metabolism and clearance, which should be taken into account when consuming OLE-containing products with certain drugs, in particular those of narrow therapeutic window. SIGNIFICANCE STATEMENT: This study indicated that oleuropein, which belongs to the main constituents of the leaves and olive drupes of Olea europaea, induces the synthesis of the major cytochrome P450s (P450s) metabolizing the majority of prescribed drugs via activation of peroxisome proliferator-activated receptor α. This effect could modify the pharmacokinetic profile of co-administered drug substrates of the P450s, thus altering their therapeutic efficacy and toxicity.

Natural and Nature-Derived Products Targeting Human Coronaviruses.

The ongoing pandemic of severe acute respiratory syndrome (SARS), caused by the SARS-CoV-2 human coronavirus (HCoV), has brought the international scientific community before a state of emergency that needs to be addressed with intensive research for the discovery of pharmacological agents with antiviral activity. Potential antiviral natural products (NPs) have been discovered from plants of the global biodiversity, including extracts, compounds and categories of compounds with activity against several viruses of the respiratory tract such as HCoVs. However, the scarcity of natural products (NPs) and small-molecules (SMs) used as antiviral agents, especially for HCoVs, is notable. This is a review of 203 publications, which were selected using PubMed/MEDLINE, Web of Science, Scopus, and Google Scholar, evaluates the available literature since the discovery of the first human coronavirus in the 1960s; it summarizes important aspects of structure, function, and therapeutic targeting of HCoVs as well as NPs (19 total plant extracts and 204 isolated or semi-synthesized pure compounds) with anti-HCoV activity targeting viral and non-viral proteins, while focusing on the advances on the discovery of NPs with anti-SARS-CoV-2 activity, and providing a critical perspective.

Exploitation of Vitis vinifera, Foeniculum vulgare, Cannabis sativa and Punica granatum By-Product Seeds as Dermo-Cosmetic Agents.

In the current study, by-product seed pastes (VSPs) from Vitis vinifera, Foeniculum vulgare, Cannabis sativa and Punica granatum, generated during the oil production process, were investigated for their potential exploitation as dermo-cosmetic agent. The extraction pipeline of all the raw materials was developed with emphasis on green methodologies and employed on laboratory scale based on industry-adopted techniques. Two different protocols were applied, Supercritical Fluid Extraction (SFE) and Ultrasound Assisted Extraction (UAE); the by-product pastes were defatted with supercritical CO2 and n-Hexane, respectively. Then, two SFE extracts (CO2 with 10% and 20% of ethanol as co-solvent) and two UAE extracts (with ethanol and ethanol/water 1:1 v/v) were obtained from each raw material. The providing yield range was between 2.6 to 76.3 mg/g raw material. The extracts were analyzed with High-Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD) and Liquid Chromatography coupled with High-Resolution Mass Spectrometer (LC-HRMS), and the major compounds, were identified. All the extracts were evaluated for their antioxidant and inhibition activity against collagenase, elastase and tyrosinase enzymes. Grapevine by-product extracts found rich in proanthocyanidins and presented the higher inhibition activity. A holistic green experimental methodology is proposed for the obtainment of extracts from significant medicinal plants by-products that provides us with promising results concerning dermo-cosmetic properties, especially for grape seeds extracts.

Palmatine, a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica, Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin.

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER+/HER2- breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC50 values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.

Glycyrrhiza glabra-Enhanced Extract and Adriamycin Antiproliferative Effect on PC-3 Prostate Cancer Cells.

Prostate cancer is the second most commonly diagnosed cancer in men worldwide, which is almost incurable, once it progresses into the metastatic stage. Adriamycin (ADR) is a known chemotherapeutic agent that causes severe side effects. In recent years, studies in natural plant products have revealed their anticancer activities. In particular, Glycyrrhiza glabra enhanced extract (GGE), commonly known as licorice, has been reported to exert antiproliferative properties against cancer cells. In this study, the cytotoxic potential of GGE was assessed in PC-3 cells, when it is administrated alone or in combination with Adriamycin. PC-3 cells were treated with GGE and/or ADR, and the inhibition of cell proliferation was evaluated by the MTT assay. Cell cycle alterations and apoptosis rate were measured through flow cytometry. Expression levels of autophagy-related genes were evaluated with specific ELISA kits, Western blotting, and real-time PCR, while NMR spectrometry was used to identify the implication of specific metabolites. Our results demonstrated that GGE alone or in co-treatment with ADR shows antiproliferative properties against PC-3 cells, which are mediated by both apoptosis and autophagy mechanisms.

Biomimetic Synthesis of Oleocanthal, Oleacein, and Their Analogues Starting from Oleuropein, A Major Compound of Olive Leaves.

Oleocanthal and oleacein are known for a wide range of beneficial activities in human health and the prevention of diseases. The inability to isolate significant and pure amounts of these natural compounds and their demanding synthesis lead to the development of an efficient, five-step, three-pot procedure. The synthesis is performed by a convenient biomimetic approach, starting from oleuropein, an abundant raw material in olive leaves, through the mixed anhydride of oleoside. The method is stereocontrolled and provides an efficient approach to the synthesis of various oleocanthal analogues; thus, a small library of four compounds was prepared with 35-45% overall yield.

Behavioral and Neurochemical Effects of Extra Virgin Olive Oil Total Phenolic Content and Sideritis Extract in Female Mice.

The aim of this study was to determine the cognitive and behavioral effects of extra virgin olive oil total phenolic content (TPC) and Sideritis (SID) extracts in female mice, and identify the associated neurochemical changes in the hippocampus and the prefrontal cortex. All animals received intraperitoneal low or high doses of TPC, SID or vehicle treatment for 7 days and were subjected to the Open Field (OF), Novel Object Recognition (NOR) and Tail Suspension Test (TST). The prefrontal cortex and hippocampus were dissected for analysis of neurotransmitters and aminoacids with high performance liquid chromatography with electrochemical detection (HPLC-ED). Both TPC doses enhanced vertical activity and center entries in the OF, which could indicate an anxiolytic-like effect. In addition, TPC enhanced non-spatial working memory and, in high doses, exerted antidepressant effects. On the other hand, high SID doses remarkably decreased the animals' overall activity. Locomotor and exploratory activities were closely associated with cortical increases in serotonin turnover induced by both treatments. Cognitive performance was linked to glutamate level changes. Furthermore, TPC reduced cortical taurine levels, while SID reduced cortical aspartate levels. TPC seems to have promising cognitive, anxiolytic and antidepressant effects, whereas SID has sedative effects in high doses. Both extracts act in the brain, but their specific actions and properties merit further exploration.

Indirubin Analogues Inhibit Trypanosoma brucei Glycogen Synthase Kinase 3 Short and T. brucei Growth.

The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short (TbGSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 µM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of TbGSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3'-oxime bearing an extra bulky substituent on the 3' oxime [(6-BIO-3'-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin-TbGSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3'-bulky-substituted indirubin-treated parasites and parasites silenced for TbGSK3s by RNA interference suggested that the above-described compounds may target TbGSK3s in vivo To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of TbGSK3s, we investigated the intracellular localization of TbGSK3s. TbGSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3'-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery.

A Biomimetic, One-Step Transformation of Simple Indolic Compounds to Malassezia-Related Alkaloids with High AhR Potency and Efficacy.

Malassezia furfur isolates from diseased skin preferentially biosynthesize compounds which are among the most active known aryl-hydrocarbon receptor (AhR) inducers, such as indirubin, tryptanthrin, indolo[3,2-b]carbazole, and 6-formylindolo[3,2-b]carbazole. In our effort to study their production from Malassezia spp., we investigated the role of indole-3-carbaldehyde (I3A), the most abundant metabolite of Malassezia when grown on tryptophan agar, as a possible starting material for the biosynthesis of the alkaloids. Treatment of I3A with H2O2 and use of catalysts like diphenyldiselenide resulted in the simultaneous one-step transformation of I3A to indirubin and tryptanthrin in good yields. The same reaction was first applied on simple indole and then on substituted indoles and indole-3-carbaldehydes, leading to a series of mono- and bisubstituted indirubins and tryptanthrins bearing halogens, alkyl, or carbomethoxy groups. Afterward, they were evaluated for their AhR agonist activity in recombinant human and mouse hepatoma cell lines containing a stably transfected AhR-response luciferase reporter gene. Among them, 3,9-dibromotryptanthrin was found to be equipotent to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an AhR agonist, and 3-bromotryptanthrin was 10-times more potent than TCDD in the human HG2L7.5c1 cell line. In contrast, 3,9-dibromotryptanthrin and 3-bromotryptanthrin were ∼4000 and >10,000 times less potent than TCDD in the mouse H1L7.5c3 cell line, respectively, demonstrating that they are species-specific AhR agonists. Involvement of the AhR in the action of 3-bromotryptanthrin was confirmed by the ability of the AhR antagonists CH223191 and SR1 to inhibit 3-bromotryptanthrin-dependent reporter gene induction in human HG2L7.5c1 cells. In conclusion, I3A can be the starting material used by Malassezia for the production of both indirubin and tryptanthrin through an oxidation mechanism, and modification of these compounds can produce some highly potent, efficacious and species-selective AhR agonists.

Silymarin Enriched Extract (Silybum marianum) Additive Effect on Doxorubicin-Mediated Cytotoxicity in PC-3 Prostate Cancer Cells.

Silymarin-enriched extract (SEE) is obtained from Silybum marianum (Asteraceae). Doxorubicin (DXR) is a widely used chemotherapeutical yet with severe side effects. The goal of the present study was to assess the pharmacologic effect of SEE and its bioactive components silibinin and silychristine when administrated alone or in combination with DXR in the human prostate cancer cells (PC-3). PC-3 cells were treated with SEE, silibinin (silybins A and B), silychristine, alone, and in combination with DXR, and cell proliferation was assessed by the MTT assay. Cell cycle, apoptosis, and autophagy rate were assessed by flow cytometry. Expression levels of autophagy-related genes were quantified by qRT-PCR, ELISA and western blot while transmission electron microscopy was performed to reveal autophagic structures. Finally, NMR spectrometry was used to identify specific metabolites related to autophagy. SEE inhibited PC-3 cell proliferation in a dose-dependent manner while the co-treatment (DXR-SEE) revealed an additive cytotoxic effect. Cell cycle, apoptosis, and autophagy variations were observed in addition to altered expression levels of autophagy related genes (LC3, p62, NBR1, Beclin1, ULK1, AMBRA1), while several modifications in autophagic structures were identified after DXR-SEE co-treatment. Furthermore, treated cells showed a different metabolic profile, with significant alterations in autophagy-related metabolites such as branched-chain amino acids. In conclusion, the DXR-SEE co-treatment provokes perturbations in the autophagic mechanism of prostate cancer cells (PC-3) compared to DXR treatment alone, causing an excessive cell death. These findings propose the putative use of SEE as an adjuvant cytotoxic agent.

Compounds from Olea europaea and Pistacia lentiscus inhibit oral microbial growth.

BACKGROUND: In view of the increasing antibiotic resistance, the introduction of natural anti-infective agents has brought a new era in the treatment of bacterially derived oral diseases. METHODS: The aim of this study was to investigate the antimicrobial potential of five natural constituents of Olea europaea (oleuropein, maslinic acid, hydroxytyrosol, oleocanthal, oleacein) and three compounds of Pistacia lentiscus (24Z-isomasticadienolic acid, oleanolic acid, oleanonic aldehyde) against ten representative oral bacterial species and a Candida albicans strain. After the isolation and quality control of natural compounds, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assay were performed. RESULTS: Among all O. europaea-derived constituents, maslinic acid was the most active (MIC = 4.9-312 µg mL- 1, MBC = 9.8-25 µg mL- 1) one against oral streptococci and anaerobic pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra), while oleuropein, hydroxytyrosol, oleocanthal and oleacein showed milder, yet significant effects against P. gingivalis and F. nucleatum. Among all P. lentiscus compounds, oleanolic acid was the most effective one against almost all microorganisms with MIC values ranging from 9.8 µg mL- 1 (P. gingivalis) to 625 µg mL- 1 (F. nucleatum, P. micra). In the presence of 24Z-isomasticadienolic acid, a mean inhibitory concentration range of 2.4 µg mL- 1 to 625 µg mL- 1 was observed for strict anaerobia. The MIC value for 24Z-isomasticadienolic acid was estimated between 39 µg mL- 1 (Streptococcus sobrinus, Streptococcus oralis) and 78 µg mL- 1 (Streptococcus mutans). All tested compounds showed no effects against Prevotella intermedia. CONCLUSIONS: Overall, maslinic acid and oleanolic acid exerted the most significant inhibitory activity against the tested oral pathogens, especially streptococci and anaerobic oral microorganisms.

A Three-Step, Gram-Scale Synthesis of Hydroxytyrosol, Hydroxytyrosol Acetate, and 3,4-Dihydroxyphenylglycol.

Hydroxytyrosol and two other polyphenols of olive tree, hydroxytyrosol acetate and 3,4-dihydroxyphenylglycol, are known for a wide range of beneficial activities in human health and prevention from diseases. The inability to isolate high, pure amounts of these natural compounds and the difficult and laborious procedures for the synthesis of them led us to describe herein an efficient, easy, cheap, and scaling up synthetic procedure, from catechol, via microwave irradiation.

Rapid isolation and characterization of crocins, picrocrocin, and crocetin from saffron using centrifugal partition chromatography and LC-MS.

This study demonstrates a simple method for one-step isolation of the main secondary metabolites of a hydroalcoholic extract of Crocus sativus stigmas (saffron) using step-gradient centrifugal partition chromatography. The analysis was performed in dual and elution-extrusion mode, using five biphasic systems of the solvents heptane/ethyl acetate/butanol/ethanol/water in ratios of 4:10:0:4:10, 1:13:0:4:10, 1:12:1:4:10, 1:10:3:4:10, and 1:7:6:4:10. Five major crocins, picrocrocin, and crocetin were directly isolated in one step. Scaling up to preparative level, allowed the recovery of significantly high quantities of pure compounds, especially trans-crocin-4, saffron's principal crocin. Comparing dual-mode and elution-extrusion, in dual-mode, the trans-crocin-4 containing fractions were co-eluted with a high amount of free ß-d-glucose. In contrast, absence of free ß-d-glucose was observed in the corresponding trans-crocin-4 fractions obtained by the second method denoting its superiority against dual-mode. Initiating analysis with the 4th solvent-system afforded selective isolation of trans-crocin-4, with reduction in experimental time and solvent consumption. Structure elucidation was performed by nuclear magnetic resonance spectroscopy, liquid chromatography with mass spectrometry, and high-resolution tandem mass spectrometry. The proposed methodology comprises an integrated approach for the purification and characterization of biologically active saffron components in a fast, selective, and environmentally friendly manner.