Anticancer Activity and Molecular Targets of Piper cernuum Substances in Oral Squamous Cell Carcinoma Models.

Oral squamous cell carcinoma (OSCC) is a worldwide public health problem, with high morbidity and mortality rates. The development of new drugs to treat OSCC is paramount. Piper plant species have shown many biological activities. In the present study, we show that dichloromethane partition of Piper cernuum (PCLd) is nontoxic in chronic treatment in mice, reduces the amount of atypia in tongues of chemically induced OSCC, and significantly increases animal survival. To identify the main active compounds, chromatographic purification of PCLd was performed, where fractions 09.07 and 14.05 were the most active and selective. These fractions promoted cell death by apoptosis characterized by phosphatidyl serine exposition, DNA fragmentation, and activation of effector caspase-3/7 and were nonhemolytic. LC-DAD-MS/MS analysis did not propose matching spectra for the 09.07 fraction, suggesting compounds not yet known. However, aporphine alkaloids were annotated in fraction 14.05, which are being described for the first time in P. cernuum and corroborate the observed cytotoxic activity. Putative molecular targets were determined for these alkaloids, in silico, where the androgen receptor (AR), CHK1, CK2, DYRK1A, EHMT2, LXRß, and VEGFR2 were the most relevant. The results obtained from P. cernuum fractions point to promising compounds as new preclinical anticancer candidates.

Combined application of DP4+ and ANN-PRA to determine the relative configuration of natural products: The alpha-bisabol case study.

The combination of computational methods and experimental data from Nuclear Magnetic Resonance (NMR) is a considerably valuable tool in the elucidation of new natural product structures and, also, in the structural revision of previously reported compounds. Until recently, only classical statistical parameters were used, for example, linear correlation coefficient (R2 ), mean absolute error (MAE), or root mean square deviation (RMSD), as a way to statistically "validate" the structure pointed out by experimental NMR spectra. Regarding the resolution of the relative configuration of organic molecules, novel tools were available in the last few years to assist in the NMR elucidation process. The most relevant are DP4+, which is based on a Bayesian probability, and ANN-PRA, which is based on artificial neural networks. The combined application of these tools has become the most accurate and important alternative to solve structural and stereochemical problems in natural product chemistry. Therefore, herein, in this case study, we intended to promote these novel tools, exploring the strengths and limitations of each approach in resolving the relative configuration of the sesquiterpene alpha-bisabol. We also highlighted the advantages of the complementary use of H- and C-DP4+ to obtain optimal results in the differentiation of the stereoisomers, validating the proposal with ANN-PRA method.

Exploring Chemical Diversity of Phorbas Sponges as a Source of Novel Lead Compounds in Drug Discovery.

Porifera, commonly referred to as marine sponges, are acknowledged as major producers of marine natural products (MNPs). Sponges of the genus Phorbas have attracted much attention over the years. They are widespread in all continents, and several structurally unique compounds have been identified from this species. Terpenes, mainly sesterterpenoids, are the major secondary metabolites isolated from Phorbas species, even though several alkaloids and steroids have also been reported. Many of these compounds have presented interesting biological activities. Particularly, Phorbas sponges have been demonstrated to be a source of cytotoxic metabolites. In addition, MNPs exhibiting cytostatic, antimicrobial, and anti-inflammatory activities have been isolated and structurally characterized. This review provides an overview of almost 130 secondary metabolites from Phorbas sponges and their biological activities, and it covers the literature since the first study published in 1993 until November 2021, including approximately 60 records. The synthetic routes to the most interesting compounds are briefly outlined.

Metabolomic fingerprinting of Brazilian marine sponges: a case study of Plakinidae species from Fernando de Noronha Archipelago.

Marine sponges from the Plakinidae family are well known for hosting cytotoxic secondary metabolites and the Brazilian Atlantic coast and its oceanic islands have been considered as a hotspot for the discovery of new Plakinidae species. Herein, we report the chemical profile among cytotoxic extracts obtained from four species of Plakinidae, collected in Fernando de Noronha Archipelago (PE, Northeastern Brazil). Crude organic extracts of Plakinastrella microspiculifera, Plakortis angulospiculatus, Plakortis insularis, and Plakortis petrupaulensis showed strong antiproliferative effects against two different cancer cell lines (HCT-116: 86.7-100%; MCF-7: 74.9-89.5%) at 50 µg/mL, by the MTT assay. However, at a lower concentration (5 µg/mL), high variability in inhibition of cell growth was observed (HCT-116: 17.3-68.7%; MCF-7: 0.00-55.5%), even within two samples of Plakortis insularis which were collected in the west and east sides of the Archipelago. To discriminate the chemical profile, the samples were investigated by UHPLC-HRMS under positive ionization mode. The produced data was uploaded to the Global Natural Products Social Molecular Networking and organized based on spectral similarities for purposes of comparison and annotation. Compounds such as dipeptides, nucleosides and derivatives, polyketides, and thiazine alkaloids were annotated and metabolomic differences were perceived among the species. To the best of our knowledge, this is the first assessment for cytotoxic activity and chemical profiling for Plakinastrella microspiculifera, Plakortis insularis and Plakortis petrupaulensis, revealing other biotechnologically relevant members of the Plakinidae family.

Biotechnological Potential of Eugenol and Thymol Derivatives Against Staphylococcus aureus from Bovine Mastitis.

Bovine mastitis is an infectious disease that affects the mammary gland of dairy cattle with considerable economic losses. Staphylococcus aureus is the main microorganism involved in this highly contagious process, and the treatment is only using antibiotics. Currently, the search for new treatment and/or compounds is still in need due to microbial resistance. In this work, we evaluated the potential of eugenol and thymol derivatives against S. aureus strains from bovine mastitis. On that purpose, nine derivatives were synthesized from eugenol and thymol (1-9), and tested against 15 strains of S. aureus from subclinical bovine mastitis. Initially, the strains were evaluated for the biofilm production profile, and those with strong adherence were selected to the antimicrobial sensitivity determination in the Minimum Inhibitory Concentration (MIC) assays. Herein the compounds toxicity was also evaluated by in silico analysis using Osiris DataWarrior® software. The results showed that 60% of the strains were considered strongly adherent and three strains (S. aureus 4271, 4745 and 4746) were selected for the MIC tests. Among the nine eugenol and thymol derivatives tested, four were active against the evaluated strains (MIC = 32 µg mL-1) within CLSI standard values. In silico analysis showed that all derivatives had cLopP < 5, cLogS > - 4 and TPSA < 140 Å2, and similar theoretical toxicity parameters to some antibiotics currently on the market. These molecules also showed negative drug-likeness values, pointing to the originality of these structures and theoretical feasibility on escaping of resistance mechanism and act against resistant strains. Thus, these eugenol derivatives may be considered as promising for the development of new treatments against bovine mastitis and future exploring on this purpose.

Micro- and nano-sized amine-terminated magnetic beads in a ligand fishing assay.

Functionalized micro- and nano-sized magnetic beads (MBs) have been widely used as versatile supports for proteins, enzymes, and drugs. Immobilized protein on MB surfaces has been successfully applied for ligand fishing assays allowing for direct identification of active ligands from complex mixtures, such as natural products and synthetic libraries. MBs with different properties such as different core compositions, sizes, coatings, and surface modifications are available commercially. Studies have been conducted to understand the role of these properties for ligand fishing assays. Here we evaluated, for the first time, the effect of MB size on the ligand fishing assay for acetylcholinesterase from Electrophorus electricus (AChE). For this purpose, four commercially available amine-terminated magnetic particles with diameters ranging from 4.5 nm to 106 µm were evaluated to fish out galantamine, a well-known AChE inhibitor, from an aqueous solution. All MBs were efficient at using glutaraldehyde to covalently immobilize AChE. The particles with diameters of about 1 µm (small microparticles) presented a higher protein mass capacity per milligram of particle than did those with diameters of about 4.5 nm (nanoparticles) and those with diameters of about 106 µm (large microparticles). The influence of these supports on the produced AChE-MBs with regards to hydrolysis turnover and ligand fishing was evaluated and is fully discussed.

Anatomical, Histochemical and Biological Studies of Clusia grandiflora Splitg. (Clusiaceae)

Abstract Clusia grandiflora belongs to an important botanical family which is known for its medicinal value, however there are few reports in literature about the species, highlighting the relevance of this study. Anatomical studies with leaves and stems were performed using traditional techniques. In this investigation it was identified particularities of the species such as the presence of vascular system arranged in an opened arc-shaped with the flexed ends towards the inside of the arch with accessory bundles in the petiole. In histochemical studies, performed with different reagents, alkaloids, phenols substances, carbohydrates and lipids were located. The cytotoxic activity of the extracts was performed by tetrazole salt and showed promising results for ethanolic extracts of stems (IC50 human colon cells of 24.30 μg/ mL) and leaves (IC50 ascites gastric cells of 44.15 μg/ mL), without cell membrane disruption of erythrocytes. The antibacterial activity was evaluated by tryptic soy agar and minimal inhibitory concentration assays and showed positive results for Pseudomonas aeruginosa and Escherichia coli, with better result for adventitious roots (32 μg/ mL and 16 μg/ mL, respectively), stems (64 μg/ mL and 32 μg/ mL, respectively) and leaves (64 μg/ mL and 32 μg/ mL, respectively) ethanolic extracts. Thus, these studies were able to characterize the species and show its potential as promising source of active substances.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

BACKGROUND: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. OBJECTIVE: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. METHODS: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. RESULTS: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. CONCLUSION: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

Isolation of a larvicidal compound from Piper solmsianum C.DC. (Piperaceae).

The Aedes aegypti mosquito is one of the major vectors of arboviruses. These diseases have re-emerged and the insecticides used nowadays are toxic to mammals and environment and have only been effective in the short-term. In this context, natural products are an alternative. The genus Piper has many active compounds against arthropods, including neolignans. The present study evaluated the larvicidal potential of the n-hexanic extract of Piper solmsianum and eupomatenoid-6, identified by GC-MS and NMR techniques, from this extract against Ae. aegypti. The crude extract (100 µg/mL) killed 80% and 98.3% of larvae in the first and third day, respectively. Eupomatenoid-6 exhibited LD50 of 19.33 µM and LD90 of 28.68 µM and was then assayed in human fibroblast cells (MRC5), showing an IC50 of 39.30 µM with estimated LD50 of 42.26 mmol/kg. Our results indicate eupomatenoid-6 as a potent insecticide with relatively low toxicity for mammals.

An Overview of Neolignans of the Genus Piper L.: Isolation Methods and Biological Activities.

BACKGROUND: The genus Piper L. has the shikimic acid pathway predominantly expressed, biosynthesizing many cinnamic acid derivatives (CAD). OBJECTIVE: Neolignans comprise an important class of CAD that exhibit a wide range of pharmacological properties such as antibacterial, antitumor, insecticidal, anti-inflammatory, antioxidant, smooth muscle relaxant, neuroprotective, antiprotozoal and against platelet aggregation factor. These substances have been extracted and isolated from Piper species using different technics. RESULTS: The present review aims to summarize extraction and isolation methods and biological activities of the different types of neolignans covering the period from 1968 to January 2016.