Lavandula angustifolia mill. (Lamiaceae) ethanol extract and its main constituents as promising agents for the treatment of metabolic disorders: chemical profile, in vitro biological studies, and molecular docking.

Lavandula angustifolia Mill. (lavender) is one of the most used medicinal plants. Herein, we chemically characterised and investigated the antioxidant properties and the capability to inhibit key enzymes for the treatment of type 2 diabetes (TD2) and obesity such as pancreatic lipase, α-glucosidase, and α-amylase of the ethanolic extract of two lavender samples (La1 and La2) from southern Italy. Both extracts significantly inhibited α-glucosidase, while La1 inhibited α-amylase and lipase more effectively than La2. To investigate whether these properties could be due to a direct interaction of the main constituents of the extracts with the targeted enzymes, molecular docking studies have been performed. As a result, the selected compounds were able to interact with the key residues of the binding site of the three proteins, thus supporting biological data. Current findings indicate the new potential of lavender ethanolic extract for the development of novel agents for T2D and obesity.

In vitro anti-proliferative and anti-bacterial properties of new C7 benzoate derivatives of pinocembrin.

In the present work, the in vitro anti-proliferative and anti-bacterial activities of three semi-synthetic benzoate pinocembrin derivatives, isolated from the aerial parts of Glycyrrhiza glabra L., were investigated. As occurs in most natural compounds, the bioavailability of pinocembrin is very poor, therefore it should be improved by chemical strategies aimed to prolong its shelf life and, consequently, its activity. On this basis, three benzoate derivatives of pinocembrin (a1-a3) were synthesised and assayed in order to ascertain their biological value. Among them, compound a1 showed the highest anti-proliferative activity on a wide panel of cancer cell lines, as well as low toxic effects on non-malignant breast cells. The calculated IC50 values in HeLa and SKBR3 cells were 8.5 and 12.7 µM, respectively. Briefly, a1 treatment increased ROS levels, induced mitochondrial membrane damage leading to necrotic death of HeLa cells. Moreover, a1 displayed a promising anti-bacterial activity.

3-Amino-alkylated indoles: unexplored green products acting as anti-inflammatory agents.

Aim: Over the years, indole has proved to be a versatile scaffold for the design of molecules acting as anti-inflammatory agents. Materials & Methods: A small library of 3-amino-alkylated indoles has been obtained by an optimized Mannich green approach. The anti-inflammatory activity of the new 3-amino-alkylated indoles, GLYC 0-10, was evaluated in RAW 264.7 macrophages. Results: The anti-inflammatory activity of the new 3-amino-alkylated indoles, GLYC 0-10, was evaluatedn and, among them, GLYC 4, 5 and 9 displayed the greatest inhibitory effects on nitric oxide production, with IC50 values of 5.41, 4.22 and 6.3 µM, respectively. Conclusion: Our outcomes, overall, highlight the importance of the indole substitution in the anti-inflammatory activity of these compounds, exerted by acting on the interlinked NF-κB/ERK1/2 pathways.

Sericin/Poly(ethylcyanoacrylate) Nanospheres by Interfacial Polymerization for Enhanced Bioefficacy of Fenofibrate: In Vitro and In Vivo Studies.

Fenofibrate is a lipophilic drug used in hypercholesterolemia and hypertriglyceridemia as a lipid-regulating agent; however, it is characterized by poor water solubility and low dissolution rate, which result in a low oral bioavailability. In the present study, sericin/poly(ethylcyanoacrylate) nanospheres are synthesized by interfacial polymerization in aqueous media and investigated as a novel sericin-based delivery system for improved and enhanced oral bioefficacy of fenofibrate. The incorporation of sericin into the prepared cyanoacrylate nanoparticles and their spherical shape are confirmed by Lowry assay and scanning electron microscopy, respectively. Hydrophilic and mucoadhesive properties of the synthesized nanospheres are also evaluated. Finally, both in vitro release and in vivo studies are performed and the oral absorbable amount of fenofibrate is calculated to be higher than 70% when incorporated into the polymeric material, reducing the levels of total cholesterol (TC), triacylglycerols (TG), very low-density lipoproteins (VLDL), and low-density lipoproteins (LDL) compared to fenofibrate alone.

The mitochondrial citrate carrier (CIC) is present and regulates insulin secretion by human male gamete.

The mechanisms through which sperm manage their energy metabolism are poorly understood. The present study provides biochemical and morphological evidence that mitochondrial citrate carrier (CIC) is present in ejaculated human sperm and is restricted to the midpiece. The inhibition of CIC with the specific substrate analog 1,2,3-benzenetricarboxylate resulted in the reduction of cholesterol efflux, protein tyrosine phosphorylation, phospho-AKT, phospho-p60src, hyperactivated motility and acrosome reaction, suggesting a role for this mitochondrial carrier in sperm physiology. Furthermore, inhibition of CIC by 1,2,3-benzenetricarboxylate resulted in a reduction of glucose-stimulated insulin secretion and autocrine insulin secretion by sperm. Remarkably, blocking CIC also reduced glucose-6-phosphate dehydrogenase activity, probably in accordance with its regulation on insulin secretion. Capacitation and glucose metabolism were stimulated by glucose as well as citrate, the specific substrate of CIC, implying a similar action because glucose and citrate both induced insulin secretion by sperm. In the present finding, we discovered a new site of action for CIC in the regulation of metabolism, and it may be assumed that CIC works with other factors in the regulation of sperm energy metabolism to sustain capacitation process and acrosome reaction.

Functional and structural role of amino acid residues in the matrix alpha-helices, termini and cytosolic loops of the bovine mitochondrial oxoglutarate carrier.

The mitochondrial oxoglutarate carrier belongs to the mitochondrial carrier family and exchanges oxoglutarate for malate and other dicarboxylates across the mitochondrial inner membrane. Here, single-cysteine mutant carriers were engineered for every residue in the amino- and carboxy-terminus, cytoplasmic loops, and matrix alpha-helices and their transport activity was measured in the presence and absence of sulfhydryl reagents. The analysis of the cytoplasmic side of the oxoglutarate carrier showed that the conserved and symmetric residues of the mitochondrial carrier motif [DE]XX[RK] localized at the C-terminal end of the even-numbered transmembrane alpha-helices are important for the function of the carrier, but the non-conserved cytoplasmic loops and termini are not. On the mitochondrial matrix side of the carrier most residues of the three matrix alpha-helices that are in the interface with the transmembrane alpha-helical bundle are important for function. Among these are the residues of the symmetric [ED]G motif present at the C-terminus of the matrix alpha-helices; the tyrosines of the symmetric YK motif at the N-terminus of the matrix alpha-helices; and the hydrophobic residues M147, I171 and I247. The functional role of these residues was assessed in the structural context of the homology model of OGC. Furthermore, in this study no evidence was found for the presence of a specific homo-dimerisation interface on the surface of the carrier consisting of conserved, asymmetric and transport-critical residues.

The biochemical properties of the mitochondrial thiamine pyrophosphate carrier from Drosophila melanogaster.

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty. In the present study, we report the functional identification of two D. melanogaster genes distantly related to the human and yeast thiamine pyrophosphate carrier (TPC) genes as well as the corresponding expression pattern throughout development. Furthermore, the functional characterization of the D. melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described. DmTpc1p was over-expressed in bacteria, the purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. Reconstituted DmTpc1p transports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides. The expression of DmTpc1p in Saccharomyces cerevisiaeTPC1 null mutant abolishes the growth defect on fermentable carbon sources. The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and/or ADP.

Functional and structural role of amino acid residues in the odd-numbered transmembrane alpha-helices of the bovine mitochondrial oxoglutarate carrier.

The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle and gluconeogenesis. To establish amino acid residues that are important for function, each residue in the transmembrane alpha-helices H1, H3 and H5 was replaced systematically by a cysteine in a fully functional mutant carrier that was devoid of cysteine residues. The transport activity of the mutant carriers was measured in the presence and absence of sulfhydryl reagents. The observed effects were rationalized by using a comparative structural model of the OGC. Most of the residues that are critical for function are found at the bottom of the cavity and they belong to the signature motifs P-X-[DE]-X-X-[KR] that form a network of three inter-helical salt bridges that close the carrier at the matrix side. The OGC deviates from most other carriers, because it has a conserved leucine (L144) rather than a positively charged residue in the signature motif of the second repeat and thus the salt bridge network is lacking one salt bridge. Incomplete salt-bridge networks due to hydrophobic, aromatic or polar substitutions are observed in other dicarboxylate, phosphate and adenine nucleotide transporters. The interaction between the carrier and the substrate has to provide the activation energy to trigger the re-arrangement of the salt-bridge network and other structural changes required for substrate translocation. For substrates such as malate, which has only two carboxylic and one hydroxyl group, a reduction in the number of salt bridges in the network may be required to lower the energy barrier for translocation. Another group of key residues, consisting of T36, A134, and T233, is close to the putative substrate binding site and substitutions or modifications of these residues may interfere with substrate binding and ion coupling. Residues G32, A35, Q40, G130, G133, A134, G230, and S237 are potentially engaged in inter-helical interactions and they may be involved in the movements of the alpha-helices during translocation.

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study.

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.