Rhaponticum acaule (L) DC essential oil: chemical composition, in vitro antioxidant and enzyme inhibition properties.

BACKGROUND: α-glucosidase is a therapeutic target for diabetes mellitus (DM) and α-glucosidase inhibitors play a vital role in the treatments for the disease. Furthermore, xanthine oxidase (XO) is a key enzyme that catalyzes hypoxanthine and xanthine to uric acid which at high levels can lead to hyperuricemia which is an important cause of gout. Pancreatic lipase (PL) secreted into the duodenum plays a key role in the digestion and absorption of fats. For its importance in lipid digestion, PL represents an attractive target for obesity prevention. METHODS: The flowers essential oil of Rhaponticum acaule (L) DC (R. acaule) was characterized using gas chromatography-mass spectrometry (GC-MS). The antioxidant activities of R. acaule essential oil (RaEO) were also determined using 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), reducing power, phosphomolybdenum, and DNA nicking assays. The inhibitory power of RaEO against α-glucosidase, xanthine oxidase and pancreatic lipase was evaluated. Enzyme kinetic studies using Michaelis-Menten and the derived Lineweaver-Burk (LB) plots were performed to understand the possible mechanism of inhibition exercised by the components of this essential oil. RESULTS: The result revealed the presence of 26 compounds (97.4%). The main constituents include germacrene D (49.2%), methyl eugenol (8.3%), (E)-ß-ionone (6.2%), ß-caryophyllene (5.7%), (E,E)-α-farnesene (4.2%), bicyclogermacrene (4.1%) and (Z)-α-bisabolene (3.7%). The kinetic inhibition study showed that the essential oil demonstrated a strong α-glucosidase inhibiton and it was a mixed inhibitor. On the other hand, our results evidenced that this oil exhibited important xanthine oxidase inhibitory effect, behaving as a non-competitive inhibitor. The essential oil inhibited the turkey pancreatic lipase, with maximum inhibition of 80% achieved at 2 mg/mL. Furthermore, the inhibition of turkey pancreatic lipase by RaEO was an irreversible one. CONCLUSION: The results revealed that the RaEO is a new promising potential source of antioxidant compounds, endowed with good practical applications for human health.

Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2.

BACKGROUND: Heterodimeric phospholipase A2 from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity. METHODS: Molecular cloning of Sm-PLGV and heterologous expression in Escherichia coli of three recombinant forms was used to determine the role of the short chain on enzymatic activity. Infrared spectroscopy assisted 3D model building of the three recombinant constructs (phospholipases with and without the penta-peptide and Long chain only) allowed us to propose an explanation of the differences in specific activities and their interaction with various phospholipids. RESULTS: Nucleotide sequence of Sm-PLGV encodes 129 residues corresponding to the Long chain, the penta-peptide and the short chain. Although recombinant phospholipases without and with the penta-peptide have different specific activities, they display a similar substrate specificity on various phospholipid monolayers and similar bell-shaped activity profiles with maxima at high surface pressure. The absence of the short chain reduces significantly enzymatic and hemolytic activities. The 3D models pointed to an interaction of the short chain with the catalytic residues, what might explain the difference in activities of our constructs. CONCLUSION: Infrared spectroscopy data and 3D modeling confirm the experimental findings that highlight the importance of the short chain for the Sm-PLGV activity. GENERAL SIGNIFICANCE: New informations are given to further establish the structure-function relationships of the Sm-PLGV.

Inhibition of pancreatic lipase and amylase by extracts of different spices and plants.

The aim of this study is to search new anti-obesity and anti-diabetic agents from plant and spices crude extracts as alternative to synthetic drugs. The inhibitory effect of 72 extracts was evaluated, in vitro, on lipase and amylase activities. Aqueous extracts of cinnamon and black tea exhibited an appreciable inhibitory effect on pancreatic amylase with IC50 values of 18 and 87 µg, respectively. Aqueous extracts of cinnamon and mint showed strong inhibitory effects against pancreatic lipase with IC50 of 45 and 62 µg, respectively. The presence of bile salts and colipase or an excess of interface failed to restore the lipase activity. Therefore, the inhibition of pancreatic lipase, by extracts of spices and plants, belongs to an irreversible inhibition. Crude extract of cinnamon showed the strongest anti-lipase and anti-amylase activities which offer a prospective therapeutic approach for the management of diabetes and obesity.

Biochemical and monolayer characterization of Tunisian snake venom phospholipases.

The present study investigated the kinetic and interfacial properties of two secreted phospholipases isolated from Tunisian vipers'venoms: Cerastes cerastes (CC-PLA2) and Macrovipera lebetina transmediterranea (MVL-PLA2). Results show that these enzymes have great different abilities to bind and hydrolyse phospholipids. Using egg-yolk emulsions as substrate at pH 8, we found that MVL-PLA2 has a specific activity of 1473U/mg at 37°C in presence of 1mM CaCl2. Furthermore the interfacial kinetic and binding data indicate that MVL-PLA2 has a preference to the zwitterionic phosphatidylcholine monolayers (PC). Conversely, CC-PLA2 was found to be able to hydrolyse preferentially negatively charged head group phospholipids (PG and PS) and exhibits a specific activity 9 times more important (13333U/mg at 60°C in presence of 3mM CaCl2). Molecular models of both CC-PLA2 and MVL-PLA2 3D structures have been built and their electrostatic potentials surfaces have been calculated. A marked anisotropy of the overall electrostatic charge distribution leads to a significantly difference in the dipole moment intensity between the two enzymes explaining the great differences in catalytic and binding properties, which seems to be governed by the electrostatic and hydrophobic forces operative at the surface of the two phospholipases.

Cloning and molecular modeling of a thermostable carboxylesterase from the chicken uropygial glands.

Starting from total uropygial glands mRNAs, chicken uropygial carboxylesterase (cuCES) cDNA was synthesized by RT-PCR and cloned into the PGEM-T vector. Amino acid sequence of the cuCES is compared to that of human liver carboxylesterase 1 (hCES1). Given the high amino acid sequence homology between the two enzymes, a 3-D structure model of the chicken carboxylesterase was built using the structure of hCES1 as template. By following this model and utilizing molecular dynamics (MD) simulations, the resistance of the chicken carboxylesterase at high temperatures could be explained. The docking of substrate analogs into the cuCES active site was used to explain the fact that the chicken carboxylesterase cannot hydrolyze efficiently large substrate molecules.

A thermoactive secreted phospholipase A2 purified from the venom glands of Scorpio maurus: relation between the kinetic properties and the hemolytic activity.

A lipolytic activity was located in the scorpion venom glands (telsons), from which a phospholipase A2 (Sm-PLVG) was purified. Like known phospholipases A2 from scorpion venom, which are 14-18 kDa proteins, the purified Scorpio maurus-Phospholipase from Venom Glands (Sm-PLVG) has a molecular mass of 17 kDa containing long and short chains linked by disulfide bridge. It has a specific activity of 5500 U/mg measured at 47 °C and pH 8.5 using phosphatidylcholine as a substrate in presence of 8 mM NaTDC and 12 mM CaCl2. The NH2-terminal amino acid sequences of the purified Sm-PLVG showed similarities with those of long and short chains of some previously purified phospholipases from venom scorpions. Moreover, the Sm-PLVG exhibits hemolytic activity toward human, rabbit or rat erythrocytes. This hemolytic activity was related to its ability to interact with phospholipids' monolayer at high surface pressure. These properties are similar to those of phospholipases isolated from snake venoms.

A thermoactive uropygial esterase from chicken: purification, characterisation and synthesis of flavour esters.

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.

Purification, biochemical and kinetic properties of recombinant Staphylococcus aureus lipase.

We have compared the purification procedures as well as the biochemical and kinetic properties of wild type (wt-SAL3), untagged recombinant (rec(-His)SAL3), and tagged recombinant (rec(+His)SAL3) purified forms of Staphylococcus aureus lipase (SAL3). We used the pH-stat method (with emulsified tributyrin and olive oil as substrates) and the monomolecular film technique (with the three dicaprin isomers spread in the form of monomolecular films at the air-water interface). The data obtained showed that the recombinant expression process as well as the presence of a his-tag at the N-terminus of recombinant SAL3 affects significantly many biochemical and catalytic properties. The effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension.

Partial characterization of a novel amylase activity isolated from Tunisian Ficus carica latex.

CONTEXT: A large number of plants still need to be investigated through screening of amylases suitable for industry. In the present study, and for the first time, we describe the amylolytic activity of Saint Pedro Ficus carica L. (Moraceae) crude latex of Kahli and Bidhi varieties. OBJECTIVE: Effects of temperature, pH, metal ions, and inhibitors and compatibility with some commercial detergents were investigated for amylase activity. MATERIALS AND METHODS: Amylase activity was screened in crude latex using the DNS method and potato starch as a substrate. Analyses of amylolytic reaction products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were performed. RESULTS: Bidhi and Kahli amylases were active in optimal pH of 6.5 and 7 at 45°C, respectively, displaying a half life of 85 and 60 min, respectively, at 80°C, and they were very stable in a wide range of pH (4-12). Bidhi amylase activity increased to 260% by addition of 10(-3) mM Fe(2+) or 10(-2) mM Cu(2+), and was strongly inhibited by Mg(2+) and EDTA. In the presence of Ca(2+) and Mg(2+), Kahli amylase activity was dramatically enhanced by 220 and 260%, respectively. The compatibility of both amylases with certain commercial detergents was also shown to be good as enzymes retained up to 98% of their activities after 30 min of incubation at 80°C. DISCUSSION AND CONCLUSION: Analysis of amylolytic reaction products by TLC and HPLC suggested that Kahli amylase was an amyloglucosidase and Bidhi amylase was ß-fructose, α(1-4) glucose. Bidhi amylase is a good choice for application in starch, food, detergents and medical industries.

A new chymotrypsin-like serine protease involved in dietary protein digestion in a primitive animal, Scorpio maurus: purification and biochemical characterization.

BACKGROUND: Most recent works on chymotrypsins have been focused on marine animals and insects. However, no study was reported in chelicerate. RESULTS: Scorpion chymotrypsin-like protease (SCP) was purified to homogeneity from delipidated hepatopancreases. The protease NH2-terminal sequence exhibited more than 60% monoacids identity with those of insect putative peptidases. The protease displayed no sequence homology with classical proteases. From this point of view, the protease recalls the case of the scorpion lipase which displayed no sequence homology with known lipases. The scorpion amylase purified and characterized by our time, has an amino-acids sequence similar to those of mammalian amylases. The enzyme was characterized with respect its biochemical properties: it was active on a chymotrypsin substrate and had an apparent molecular mass of 25 kDa, like the classically known chymotrypsins. The dependence of the SCP activity and stability on pH and temperature was similar to that of mammalian chymotrypsin proteases. However, the SCP displayed a lower specific activity and a boarder pH activity range (from 6 to 9). CONCLUSION: Lower animal have a less evaluated digestive organ: a hepatopancreas, whereas, higher ones possess individualized pancreas and liver. A new chymotrypsin-like protease was purified for the first time from the scorpion hepatopancreas. Its biochemical characterization showed new features as compared to classical chymotrypsin-higher-animals proteases.

Digestive amylase of a primitive animal, the scorpion: purification and biochemical characterization.

Scorpion, one of the most ancient invertebrates was chosen, as a model of a primitive animal, to purify and characterize an amylase located in the hepatopancreas. The scorpion digestive amylase (SDA) was purified. Pure SDA was obtained after heat treatment followed by ammonium sulfate fractionation and three steps of chromatography. The pure amylase is not glycosylated and has a molecular mass of 59,101 Da determined by MALDI-TOF MS analysis. The maximal amylase activity was measured at pH 7.0 and 50 degrees C, in the presence of Ca2+ and using potato starch as substrate. The enzyme was able to hydrolyze also, glycogen and amylose. The 23 NH2-terminal amino acid SDA residues were sequenced. The sequence obtained is similar to those of mammalian and avian pancreatic amylases. Nevertheless, polyclonal antibodies directed against SDA failed to recognize classical digestive amylases like the porcine pancreatic one.