Chemoenzymatic Synthesis of Complex Phenylpropanoid Derivatives by the Botrytis cinerea Secretome and Evaluation of Their Wnt Inhibition Activity.

In this study, a series of complex phenylpropanoid derivatives were obtained by chemoenzymatic biotransformation of ferulic acid, caffeic acid, and a mixture of both acids using the enzymatic secretome of Botrytis cinerea. These substrates were incubated with fungal enzymes, and the reactions were monitored using state-of-the-art analytical methods. Under such conditions, a series of dimers, trimers, and tetramers were generated. The reactions were optimized and scaled up. The resulting mixtures were purified by high-resolution semi-preparative HPLC combined with dry load introduction. This approach generated a series of 23 phenylpropanoid derivatives, 11 of which are described here for the first time. These compounds are divided into 12 dimers, 9 trimers (including a completely new structural scaffold), and 2 tetramers. Elucidation of their structures was performed with classical spectroscopic methods such as NMR and HRESIMS analyses. The resulting compound series were analyzed for anti-Wnt activity in TNBC cells, with several derivatives demonstrating specific inhibition.

Generation of Stilbene Antimicrobials against Multiresistant Strains of Staphylococcus aureus through Biotransformation by the Enzymatic Secretome of Botrytis cinerea.

The biotransformation of a mixture of resveratrol and pterostilbene was performed by the protein secretome of Botrytis cinerea. Several reaction conditions were tested to overcome solubility issues and to improve enzymatic activity. Using MeOH as cosolvent, a series of unusual methoxylated compounds was generated. The reaction was scaled-up, and the resulting mixture purified by semipreparative HPLC-PDA-ELSD-MS. Using this approach, 15 analogues were isolated in one step. Upon full characterization by NMR and HRMS analyses, eight of the compounds were new. The antibacterial activities of the isolated compounds were evaluated in vitro against the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The selectivity index was calculated based on cytotoxic assays performed against human liver carcinoma cells (HepG2) and the human breast epithelial cell line (MCF10A). Some compounds revealed remarkable antibacterial activity against multidrug-resistant strains of S. aureus with moderate human cell line cytotoxicity.

Chemo-Diversification of Plant Extracts Using a Generic Bromination Reaction and Monitoring by Metabolite Profiling.

A generic procedure for direct bromination of polyphenol in crude plant extracts was developed to generate multiple "unnatural" halogenated natural products for further bioassay evaluation. To better control the halogenation procedure, the bromination was optimized with a flavonoid standard, and the reactions were monitored by high-performance liquid chromatography photometric diode array coupled to the evaporative light scattering detection (ELSD). ELSD detection was successfully used for a relative yield estimation of the compounds obtained. From the halogenation of hesperitin (11), five brominated compounds were obtained. After optimization, the reaction was successfully applied to the methanolic extract of Citrus sinensis peels, a typical waste biomass and also to the methanolic extract of the medicinal plant Curcuma longa. In both cases, the methanolic extracts were profiled by NMR for a rapid estimation of the polyphenol versus primary metabolite content. An enriched secondary metabolites extract was obtained using vacuum liquid chromatography and submitted to bromination. Metabolite profiling performed by ultrahigh purity liquid chromatography time-of-flight high-resolution mass spectrometry revealed the presence of various halogenated products. To isolate these compounds, the reactions were scaled up, and six halogenated analogues were isolated and fully characterized by NMR and high-resolution electrospray ionization mass spectrometry analyses. The antibacterial properties of these compounds were evaluated using in vitro bioassays against multiresistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. Some of the halogenated derivatives obtained presented moderate antibacterial properties.

Anti-inflammatory and antiproliferative diterpenoids from Plectranthus scutellarioides.

Chemical investigation of the dichloromethane extract of the aerial parts of Plectranthus scutellarioides led to the isolation and characterization of 10 diterpenoids with an abietane skeleton and one cembrane-type diterpenoid. Among them, six have not yet been described in the literature. Their structures were established by 1D and 2D NMR, UV and IR spectroscopy, and HRESIMS. The relative configuration was determined by Gauge-Independent Atomic Orbital NMR chemical shift calculations supported by the advanced statistical method DP4 plus and further confirmed by electronic circular dichroism. The isolated constituents were evaluated for their in vitro NF-κB inhibitory activity, as well as for their cytotoxic effects in human multiple myeloma cancer stem cells and RPMI 8226 tumor cell line. Coleon O, coleon G, lanugone K and 6-acetylfredericone B showed the highest inhibitory effect against NF-κB, displaying IC50 of 11.2, 11.0, 4.5 and 9.7 µM, respectively. Coleon O exhibited also a significant activity towards human multiple myeloma cancer stem cells and RPMI 8226 cells with IC50 of 9.2 and 8.4 µM, respectively.

Zebrafish-based identification of the antiseizure nucleoside inosine from the marine diatom Skeletonema marinoi.

With the goal of identifying neuroactive secondary metabolites from microalgae, a microscale in vivo zebrafish bioassay for antiseizure activity was used to evaluate bioactivities of the diatom Skeletonema marinoi, which was recently revealed as being a promising source of drug-like small molecules. A freeze-dried culture of S. marinoi was extracted by solvents with increasing polarities (hexane, dichloromethane, methanol and water) and these extracts were screened for anticonvulsant activity using a larval zebrafish epilepsy model with seizures induced by the GABAA antagonist pentylenetetrazole. The methanolic extract of S. marinoi exhibited significant anticonvulsant activity and was chosen for bioassay-guided fractionation, which associated the bioactivity with minor constituents. The key anticonvulsant constituent was identified as the nucleoside inosine, a well-known adenosine receptor agonist with previously reported antiseizure activities in mice and rat epilepsy models, but not reported to date as a bioactive constituent of microalgae. In addition, a UHPLC-HRMS metabolite profiling was used for dereplication of the other constituents of S. marinoi. Structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution spectrometry. These results highlight the potential of zebrafish-based screening and bioassay-guided fractionation to identify neuroactive marine natural products.

A new antifungal and antiprotozoal bibenzyl derivative from Gavilea lutea.

A new bibenzyl derivative (4), together with two glycosylated flavonoids (1 and 2), batatasin III (3) and the phenanthrene isohircinol (5) were isolated from the aerial parts of Gavilea lutea. Their structures were elucidated on the basis of spectroscopic studies including 1D and 2D NMR, UV, IR and HRESIMS. All isolated compounds were evaluated for their antifungal activity towards Candida albicans. The new compound 4 showed inhibitory activity with a MIQ of 50 µg. In addition, compound 4 exhibited a selective activity (IC50 = 2.3 µg/mL) against Leishmania donovani.

Bioactive Natural Products Prioritization Using Massive Multi-informational Molecular Networks.

Natural products represent an inexhaustible source of novel therapeutic agents. Their complex and constrained three-dimensional structures endow these molecules with exceptional biological properties, thereby giving them a major role in drug discovery programs. However, the search for new bioactive metabolites is hampered by the chemical complexity of the biological matrices in which they are found. The purification of single constituents from such matrices requires such a significant amount of work that it should be ideally performed only on molecules of high potential value (i.e., chemical novelty and biological activity). Recent bioinformatics approaches based on mass spectrometry metabolite profiling methods are beginning to address the complex task of compound identification within complex mixtures. However, in parallel to these developments, methods providing information on the bioactivity potential of natural products prior to their isolation are still lacking and are of key interest to target the isolation of valuable natural products only. In the present investigation, we propose an integrated analysis strategy for bioactive natural products prioritization. Our approach uses massive molecular networks embedding various informational layers (bioactivity and taxonomical data) to highlight potentially bioactive scaffolds within the chemical diversity of crude extracts collections. We exemplify this workflow by targeting the isolation of predicted active and nonactive metabolites from two botanical sources (Bocquillonia nervosa and Neoguillauminia cleopatra) against two biological targets (Wnt signaling pathway and chikungunya virus replication). Eventually, the detection and isolation processes of a daphnane diterpene orthoester and four 12-deoxyphorbols inhibiting the Wnt signaling pathway and exhibiting potent antiviral activities against the CHIKV virus are detailed. Combined with efficient metabolite annotation tools, this bioactive natural products prioritization pipeline proves to be efficient. Implementation of this approach in drug discovery programs based on natural extract screening should speed up and rationalize the isolation of bioactive natural products.

Generation of Antifungal Stilbenes Using the Enzymatic Secretome of Botrytis cinerea.

The protein secretome of Botrytis cinerea was used to perform the biotransformation of resveratrol, pterostilbene, and a mixture of both. Metabolite profiling by UHPLC-HRMS revealed the presence of compounds with unusual molecular formula, suggesting the existence of new products. To isolate these products, the reactions were scaled-up, and 21 analogues were isolated and fully characterized by NMR and HRESIMS analyses. The reaction with pterostilbene afforded five new compounds, while the reaction with a mixture of pterostilbene and resveratrol afforded seven unusual stilbene dimers. The antifungal properties of these compounds were evaluated using in vitro bioassays against Plasmopara viticola. The cytological effects of the isolated antifungal compounds on the ultrastructure of P. viticola were also evaluated.

Stage Characterization and Marginal Bone Loss Evaluation Up to 96 Months of Crestal Sinus Augmentation With Sequential Drills: A Retrospective Study.

INTRODUCTION: The 2-stage crestal approach to augment the maxillary sinus is a little-used technique. The aim of this retrospective study was to assess events characterizing stages of this technique after implant placement in the posterior maxilla with residual bone height less than 4 mm and evaluate the marginal bone loss (MBL) changes over time. MATERIAL AND METHODS: Thirty-three patients underwent unilateral sinus augmentations using the trancrestal technique with mineralized allograft. Six-months (6 m) after first surgery, if skeletal subsidence prevented insertion of a 10-mm-length implant, additional grafting was performed during implant (n = 33) insertion. Radiographs were taken before grafting (baseline), immediately after and at 6 months; immediately after and 6 months after implant placement; and at follow-up (24-96 m). RESULTS: One implant was lost (ISR = 96.97%). Of the remaining 32 patients, 14 (A group) underwent standard implant placement, whereas 18 (B group) underwent additional grafting immediately before implant placement. Given that B-group patients initially obtained lower crestal bone height after first surgery, additional grafting procedures provided greater crestal height in the B group. A significant relationship between ending (eMBL) and 6mMBL was found in both groups, with greater values in the B group. However, in both groups, eMBL was always greater if 6mMBL was greater than 0.44 mm. DISCUSSION: Results suggest a high and low skeletal-reactivity patient categorization. In both patient categories, MBL greatly depends upon 6-month values. Investigations are necessary to relate sinus size with results obtained by this 2-stage crestal approach. CONCLUSIONS: The 2-stage crestal sinus lift procedure not only provides predictable results, but also allows low skeletal-reactivity patient recovery.

Metabolite profiling of polyphenols in a Terminalia chebula Retzius ayurvedic decoction and evaluation of its chemopreventive activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of Terminalia chebula fruit is an ayurvedic remedy whose prolonged oral administration is prized as a generic intestinal and hepatic detoxifying agent. Its administration is suggested also under the perspective of a reduced risk of cancer, metabolic and cardiovascular diseases. AIM OF THE STUDY: To evaluate the phytochemical profile and the chemopreventive potential of Terminalia chebula fruit decoction prepared according to the ayurvedic decoction recipe. MATERIALS AND METHODS: The quali- and quantitative metabolite profiling of polyphenols was obtained using HPLC-UV/DAD and HPLC-ESI-MS. The crude decoction and purified compounds were tested for their capability to interact with the EphA2-ephrin-A1 system and for their antimutagenic properties against dietary and environmental mutagens (AA, 2-NF, NaN3, and heterocyclic amines IQ, MeIQ, MeIQx, Glu-P1, Glu-P2,) in the Ames-Salmonella/microsome assay, with and without enzymatic induction. RESULTS: The decoction was found to contain 3,4,6-tri-O-galloyl-d-glucose (55.87 mg/g), chebulic acid (54.03 mg/g), ß-punicalagin (41.25mg/g), corilagin (40.31 mg/g), α-punicalagin (35.55 mg/g), chebulagic acid (29.09 mg/g), gallic acid (27.96 mg/g) 1,3,4,6-tri-O-galloyl-ß-d-glucose (24.25mg/g) chebulinic acid (20.23 mg/g), 1,2,3,4,6-penta-O-galloyl-d-glucose (13.53 mg/g), ellagic acid (8.00 mg/g), 1,6-di-O-galloyl-d-glucose (4.16 mg/g). An inhibitory effect was recorded in both Salmonella typhimurium TA98 and TA100 strains against the mutagenic activity of heterocyclic amines (22-61%), promutagen AA (91-97%) and directly acting mutagen 2-NF (52%) with but not against NaN3 (7%). Galloyl derivatives allowed an inhibition of mutagenicity induced by MeIQ above 80% at 0.01 mol/plate. Both decoction and purified compounds were able to modulate the EphA2-ephrinA1 system, suggesting a potential multiple chemopreventive mechanism. CONCLUSIONS: The traditional ayurvedic decoction of Terminalia chebula may harbour a potential as a safe and low-cost chemopreventive agent at the intestinal level, if administered according to the ayurvedic specifications. Moreover, its recourse may enhance the presence of some polyphenolic constituents.

Crestal sinus lift with sequential drills and simultaneous implant placement in sites with <5 mm of native bone: a multicenter retrospective study.

PURPOSE: The aim of this multicenter retrospective clinical study was to evaluate the survival rate of implants placed in the posterior maxilla with a residual bone height of <5 mm. MATERIALS AND METHODS: One hundred seventeen patients, recruited from 6 different centers, had 134 implants placed below the maxillary sinus. The patient population consisted of 52 men and 65 women ranging in age from 39 to 78 years (mean age, 53.2 years). Sinus lift procedures were performed following a crestal approach using a specific sequence of drills (Cosci's technique). All implants were submerged. Periapical radiographs were obtained with a paralleling technique and were digitized. The pattern of bone remodeling was subsequently evaluated. RESULTS: The average (±SD) follow-up time was 48.2 months (±29.30 months; range, 24 to 120 months). Of the original 134 implants placed, 5 implants (3.7%) failed. The implant survival rate was 96.3%. The average residual bone height was 3.46 mm (±0.91 mm) at baseline. The average height of the alveolar crest in the treated implant sites was 9.94 ± 2.29 mm. The radiographic bone gain was 6.48 ± 2.38 mm. CONCLUSION: The investigation suggests that this crestal drill approach can be a successful sinus lifting procedure in a severe atrophic maxilla with <5 mm of crestal bone height.