RESUMEN
Sesamol, the phenolic degradation product of sesamolin, although recognised for its anti-inflammatory effects, has low bioavailability. In this manuscript, we attempted to improve its bioavailability by encapsulation in mixed phosphatidylcholine micelles. Sesamol could be solubilised and entrapped in phosphatidylcholine mixed micelles (PCS) with 96.8% efficiency (particle size 3.0±0.06nm). Fluorescence spectra of PCS revealed lower relative fluorescence intensity (RFI 112) compared to 'free' sesamol (FS) (RFI 271). The bioaccessibility, transport across a monolayer of cells and cellular uptake of PCS was 8.58%, 1.5-fold and 1.2-fold better, respectively, compared to FS. The anti-inflammatory effects of FS and PCS were compared using LPS treated RAW 264.7 cell line and lipoxygenase inhibition. PCS effected downregulation of iNOS protein expression (27%), NO production (20%), ROS (32%) and lipoxygenase inhibition (IC50=31.24µM) compared to FS.
Asunto(s)
Antiinflamatorios/uso terapéutico , Benzodioxoles/química , Fenoles/química , Disponibilidad Biológica , Células CACO-2 , Humanos , MicelasRESUMEN
Reactive oxygen species, the byproducts of oxygenases reaction, when in excess, promote degenerative diseases like cardiovascular, cancer and arthritis. Sesame lignans- sesamin, sesamolin and the phenolic degradation product of sesamolin, sesamol, are empirically known for their health promoting properties like antioxidant, antimutagenic, antiaging and antiinflammatory activities. In the current study, the effect of sesamol on the inflammatory oxygenase - lipoxygenase (LOX) was investigated. Enzyme kinetics and spectroscopic techniques were used to understand the inhibition mechanism. Sesamol was a potent inhibitor of soy LOX-1. It inhibited soy LOX-1 in a dose dependent manner with IC50 value of 51.84µM and Ki of 4.9µM. Binding studies using circular dichroism and corroborated by surface plasmon resonance, revealed that sesamol does not bind or change the conformation of LOX. Further, sesamol prevented the conversion of inactive LOX (Fe2+) to active LOX (Fe3+) by arresting the oxidation state of iron and prolonging the lag phase by virtue of its ability to scavenge hydroperoxides. Understanding the mechanism of action of such molecules will help in their application and promotion as nutraceuticals.
Asunto(s)
Antioxidantes/química , Benzodioxoles/química , Inhibidores Enzimáticos/química , Quelantes del Hierro/química , Lipooxigenasa/química , Fenoles/química , Proteínas de Plantas/antagonistas & inhibidores , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Antioxidantes/aislamiento & purificación , Benzodioxoles/aislamiento & purificación , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Hierro/química , Quelantes del Hierro/aislamiento & purificación , Cinética , Oxidación-Reducción , Fenoles/aislamiento & purificación , Proteínas de Plantas/química , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Sesamum/química , Soluciones , Glycine max/química , Glycine max/enzimologíaRESUMEN
Sesamin, sesamolin (lignans) and sesamol--from sesame seed (Sesamum indicum L.)--are known for their health promoting properties. We examined the inhibition effect of sesamol, a phenolic degradation product of sesamolin, on the key enzyme in melanin synthesis, viz. tyrosinase, in vitro. Sesamol inhibits both diphenolase and monophenolase activities with midpoint concentrations of 1.9 µM and 3.2 µM, respectively. It is a competitive inhibitor of diphenolase activity with a K(i) of 0.57 µM and a non-competitive inhibitor of monophenolase activity with a K(i) of 1.4 µM. Sesamol inhibits melanin synthesis in mouse melanoma B16F10 cells in a concentration dependant manner with 63% decrease in cells exposed to 100 µg/mL sesamol. Apoptosis is induced by sesamol, limiting proliferation. This study of the chemistry and biology of lignans, in relation to the mode of action of bioactive components, may open the door for drug applications targeting enzymes.
Asunto(s)
Benzodioxoles/metabolismo , Benzodioxoles/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Melaninas/biosíntesis , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Fenoles/metabolismo , Fenoles/farmacología , Agaricales/enzimología , Animales , Apoptosis/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Línea Celular Tumoral , Culinaria , Cobre/metabolismo , Lignanos/farmacología , Ratones , Monofenol Monooxigenasa/química , Oxidorreductasas/antagonistas & inhibidores , Unión Proteica , Aceite de Sésamo/química , Análisis EspectralRESUMEN
Porcine pancreatic alpha-amylase (PPA) and its isoforms (PPA-I and PPA-II) were deglycosylated by peptide-N-glycosidase F (PNGase F) to investigate the role of bound carbohydrate. On deglycosylation, the effect on thermal stability was less pronounced. Deglycosylation resulted in a shift of the mid-point of thermal transition by 1-2 degrees C towards lower temperature. The fluorescence emission maxima of PPA, PPA-I and PPA-II were found to be 340nm indicating the presence of tryptophan residues in a fairly hydrophilic environment. A red shift in emission spectra accompanied by an increase in fluorescence intensity was observed upon deglycosylation.
Asunto(s)
Páncreas/enzimología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Sus scrofa , alfa-Amilasas/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Isoenzimas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
The napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits - one small (29 amino acid residues) and one large (86 amino acids residues) - held together by disulfide bonds. The thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with T(M1) and T(M2) at 323.5 K and 335.8 K, respectively; DeltaC(P1) and DeltaC(P2) are 2.05 kcal mol(-1) K(-1) and 1.40 kcal mol(-1) K(-1), respectively. In the temperature range 310-318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N <_-_-> I <_-_-> U with DeltaG(1H2O) and DeltaG(2H2O) values of 5.2 kcal mol(-1) and 5.1 kcal mol(-1) at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from 20 A to 35.0 A due to unfolding by guanidinium hydrochloride. In silico alignment of sequences of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition.
Asunto(s)
Planta de la Mostaza/metabolismo , Proteínas de Plantas/química , Albuminas 2S de Plantas , Secuencia de Aminoácidos , Rastreo Diferencial de Calorimetría , Cromatografía , Cromatografía en Gel , Dicroismo Circular , Guanidina/química , Datos de Secuencia Molecular , Desnaturalización Proteica , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Temperatura , Termodinámica , Factores de TiempoRESUMEN
Napin from mustard (Brassica juncea L.) is a seed storage protein consisting of two subunits linked through disulfide bonds and is predominantly helical in nature. Resistance to trypsin digestion and allergenicity limit its food applications. The role of disulfide linkages, electrostatic as well as hydrophobic interactions, in napin stability have been investigated through spectroscopic methods, employing different fluorescent probes and additives. The subunits are hydrophilic in nature and possess extended structure. With the addition of 0.5 M NaCl, the surface hydrophobicity of napin decreases, whereas the helical content increases by 25%. In the presence of NaCl, emission maximum shifts toward shorter wavelength and the Stern-Volmer constant decreases from 6.5 to 3.4 M-1, indicating compaction of napin. Na2SO4 has no significant effect on the structure due to the lack of a hydrophobic core. In the presence of monohydric alcohols and trifluoroethanol, there is an increase in ordered structure. These studies indicate that the structure of napin, which is hydrophilic in nature, is stabilized by electrostatic interactions, in addition to disulfide linkages.
Asunto(s)
Alcoholes , Planta de la Mostaza/química , Proteínas de Plantas/química , Disulfuros/química , Interacciones Hidrofóbicas e Hidrofílicas , Sales (Química) , Semillas/química , Electricidad EstáticaRESUMEN
Genistein and daidzein, the major isoflavones present in soybeans, possess a wide spectrum of physiological and pharmacological functions. The binding of genistein to human serum albumin (HSA) has been investigated by equilibrium dialysis, fluorescence measurements, CD and molecular visualization. One mole of genistein is bound per mole of HSA with a binding constant of 1.5 +/- 0.2 x 10(5) m(-1). Binding of genistein to HSA precludes the attachment of daidzein. The ability of HSA to bind genistein is found to be lost when the tryptophan residue of albumin is modified with N-bromosuccinimide. At 27 degrees C (pH 7.4), van't Hoff's enthalpy, entropy and free energy changes that accompany the binding are found to be -13.16 kcal x mol(-1), -21 cal x mol(-1) K(-1) and -6.86 kcal x mol(-1), respectively. Temperature and ionic strength dependence and competitive binding measurements of genistein with HSA in the presence of fatty acids and 8-anilino-1-naphthalene sulfonic acid have suggested the involvement of both hydrophobic and ionic interactions in the genistein-HSA binding. Binding measurements of genistein with BSA and HSA, and those in the presence of warfarin and 2,3,5-tri-iodobenzoic acid and Förster energy transfer measurements have been used for deducing the binding pocket on HSA. Fluorescence anisotropy measurements of daidzein bound and then displaced with warfarin, 2,3,5-tri-iodobenzoic acid or diazepam confirm the binding of daidzein and genistein to subdomain IIA of HSA. The ability of HSA to form ternery complexes with other neutral molecules such as warfarin, which also binds within the subdomain IIA pocket, increases our understanding of the binding dynamics of exogenous drugs to HSA.