Interaction of different polyphenols with bovine serum albumin (BSA) and human salivary alpha-amylase (HSA) by fluorescence quenching

Phenolic compounds are responsible for major organoleptic characteristics of plant-derived food and beverages; these substances have received much attention, given that the major function of these compounds is their antioxidant ability. In the context of this study, our major aim was study the binding of several phenolic compounds such as (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, malvidin-3-glucoside, tannic acid, procyanidin B4, procyanidin B2 gallate, and procyanidin oligomers to different proteins (bovine serum albumin and human alpha-amylase) by fluorescence quenching of protein intrinsic fluorescence. From the spectra obtained, the Stern-Volmer, the apparent static, and the bimolecular quenching constants were calculated. The structure of polyphenols revealed to significantly affect the binding/quenching process; in general, the binding affinity increased with the molecular weight of polyphenol compounds and in the presence of galloyl groups. For catechin monomer and procyanidin dimer B4, the K(SV) was 14,100 and 13,800 M(-1), respectively, and for galloyl derivatives, the K(SV) was 19,500 and 21,900 M(-1), respectively. Tannic acid was shown to be the major quenching molecule for both proteins. However, comparing different proteins, the same polyphenol showed different quenching effects, which are suggested to be related to the three-dimensional structure of the proteins studied. For (+)-catechin and BSA, the K(SV) was 8700 M(-1), and with alpha-amylase, it was 14,100 M(-1); for tannic acid, the K(SV) was 10,0548 and 11,0674 M(-1), respectively. From the results obtained, besides the main binding analysis performed, we conclude that this technique is more sensitive than thought because we can detect several interactions that have not been proven by other methods, namely, nephelometry. Overall, fluorescence quenching has proven to be a very sensitive technique with many potentialities to analyze the interaction between polyphenols and proteins.     

Investigation on the interaction between ilaprazole and bovine serum albumin without or with different C-ring flavonoids from the viewpoint of food-drug interference

The interaction between ilaprazole and bovine serum albumin (BSA) has been investigated in the absence and presence of four popular flavonoids with different C-ring structures, quercetin, luteolin, taxifolin, and (+)-catechin, by means of fluorescence spectroscopy. The results indicated that ilaprazole had a strong ability to quench the intrinsic fluorescence of BSA, and site marker competitive experiments indicated that the binding of ilaprazole to BSA primarily took place in subdomain IIA. The quenching process of ilaprazole with BSA was easily affected by flavonoids,; however, they did not change the quchenching mechanism of ilaprazole with BSA, whereas all of the fluorescence quenching was initiated by a static quenching procedure combining with nonradiative energy transfer. The presence of flavonoids decreased the quenching constants of ilaprazole with BSA from 2.2 to 23.7% and decreased the binding constants from 73.7 to 98.3%, which depended on the different flavonoids' structures. The decreased binding constants and unchangeable spatial distance of ilaprazole with BSA by the introduction of quercetin, luteolin, and taxifolin may result from the competition of flavonoids and ilaprazole binding to BSA, whereas in the presence of (+)-catichin, decreased binding constants and increased spatial distance possibly resulted from the formation of a ternary ilaprazole-BSA-(+)-catechin complex. All of these results may have relevant consequences in rationalizing the interferences of common food to gastric ulcer treatments.     

Influence of B-ring hydroxylation on interactions of flavonols with bovine serum albumin

The B-ring substitution pattern of flavonols is a significant structural feature for their function as free radical scavengers and antioxidants. In this paper, four differently substituted B-ring hydroxylation flavonols (galangin, kaempferol, quercetin, and myricetin) and a flavonol glycoside (quercitrin) were studied for their ability to bind BSA by quenching the protein intrinsic fluorescence. From the spectra obtained, the biomolecular quenching constants, the apparent static binding constants, and the binding site values were calculated. The B-ring hydroxylation of flavonols significantly affected the binding/quenching process; in general, the binding affinity increased with the number of hydroxyl groups on the B-ring. The binding constants ( Ka) were determined as myricetin (4.90 x 10(8) L/mol) > quercetin (3.65 x 10(7) L/mol) > kaempferol (2.57 x 10(6) L/mol) > galangin (6.43 x 10(5) L/mol). The glycoside substitute at the C-ring position decreased the binding affinity. The chromatographic retention factor ( K') and logarithms of apparent partition coefficient (log Kow) were linear to the logarithms of apparent binding constants (log Ka) for flavonols with increasing hydroxyl groups on the B-ring. These results showed that the hydrogen bond force play an important role in binding flavonols to BSA. These results are also in agreement with the generally accepted structure-dependent free radical scavenger and antioxidant abilities of flavonols.     

Interaction of flavonoids with bovine serum albumin: a fluorescence quenching study

The interaction between four flavonoids (catechin, epicatechin, rutin, and quercetin) and bovine serum albumin (BSA) was investigated using tryptophan fluorescence quenching. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between the flavonoids and BSA. The binding affinity was strongest for quercetin and ranked in the order quercetin > rutin > epicatechin = catechin. The pH in the range of 5-7.4 does not affect significantly (p < 0.05) the association of rutin, epicatechin, and catechin with BSA, but quercetin exhibited a stronger affinity at pH 7.4 than at lower pH (p < 0.05). Quercetin has a total quenching effect on BSA tryptophan fluorescence at a molar ratio of 10:1 and rutin at approximately 25:1. However, epicatechin and catechin did not fully quench tryptophan fluorescence over the concentration range studied. Furthermore, the data suggested that the association between flavonoids and BSA did not change molecular conformation of BSA and that hydrogen bonding, ionic, and hydrophobic interaction are equally important driving forces for protein-flavonoid association.     

Optical spectroscopic exploration of binding of Cochineal Red A with two homologous serum albumins

Cochineal Red A is a negatively charged synthetic azo food colorant and a potential carcinogen. We present here the study of binding of Cochineal Red A with two homologous serum albumins, human (HSA) and bovine (BSA), in aqueous pH 7.4 buffer by optical spectroscopic techniques. Protein intrinsic fluorescence quenching by Cochineal Red A occurs through ground-state static interaction and its binding with BSA is stronger than with HSA. The magnitudes of thermodynamic parameters suggest that dye binding occurs principally via electrostatic complexation. Site-marker competitive binding shows that Cochineal Red A binds primarily to site I of serum albumins. Circular dichroic spectra indicate that dye binding results in some conformational modification of serum albumins. Increased ionic strength of the medium results in lowering of binding. This study provides an important insight into possible means of removal of dye toxicity.     

Bovine serum albumin nanoparticle promotes the stability of quercetin in simulated intestinal fluid

Quercetin (Que) is a flavonoid widely distributed in vegetables and fruits and exhibits strong antioxidant activity, but the poor stability of Que limits its function and application. The present study developed a nanoparticle (NP) using bovine serum albumin (BSA) as a matrix to encapsulate Que. The stability of encapsulated Que by BSA NP was tracked in a simulated intestinal fluid (SIF). The antioxidant activity of encapsulated Que was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays. Furthermore, the stabilizing mechanism of Que by BSA NP was investigated, using scanning transmisson electron microscopy (STEM), dynamic light scattering (DLS), UV-vis, fluorescence spectrometry, and circular dichroism (CD). The results revealed that Que was effectively encapsulated by BSA and formed spherical NP (<10 nm). BSA NP not only promoted the stability of encapsulated Que but also kept the antioxidant activity of encapsulated Que. The driving forces for BSA-Que association were hydrophobic interaction and hydrogen bond, and the latter was involved in the mechanism of Que stabilization. This suggested that BSA NP could be a good carrier to deliver hydrophobic flavonols.     

Production, characterization, and cross-reactivity studies of monoclonal antibodies against the coccidiostat nicarbazin

A cELISA was developed for the coccidiostat nicarbazin. On the basis of previous computer-assisted molecular modeling studies, p-nitrosuccinanilic acid (PNA-S) was selected as a hapten to produce antibodies to 4,4'-dinitrocarbanilide (DNC), the active component of the coccidiostat nicarbazin. Synthesis is described for the hapten [p-nitro-cis-1,2-cyclohexanedicarboxanilic acid (PNA-C)] used in a BSA conjugate as a plate coating antigen. Monoclonal antibodies (Mabs) were isolated that compete with nicarbazin, having IgM(kappa) isotype. Because of the lack of water solubility of nicarbazin, N,N-dimethylformamide (DMF) (3%, v/v) and acetonitrile (ACN) (10%, v/v) were added to the assay buffer to achieve solubility of nicarbazin and related compounds. The Nic 6 Mabs had an IC(35) value for nicarbazin of 0.92 nmol/mL, with a limit of detection of 0.33 nmol/mL. Nic 6 exhibited high cross-reactivity for PNA-S and PNA-C, and 3-nitrophenol, 4-nitrophenol, and 1-(4-chlorophenyl)-3-(4-nitrophenyl) urea. However, Nic 6 had little or no cross-reactivity with 15 other related compounds.     

Oxidation of skeletal muscle myofibrillar proteins in oil-in-water emulsions: interaction with lipids and effect of selected phenolic compounds

The effect of selected phenolic compounds, namely, gallic acid, cyanidin-3-glucoside, (+)-epicatechin, chlorogenic acid, genistein and rutin (50 and 200 microM), and alpha-tocopherol (50 microM) against the oxidation of oil-in-water emulsions (37 degrees C/10 days) containing 1% myofibrillar proteins (MPs), was investigated. Emulsions containing 1% bovine serum albumin (BSA) were also prepared for comparative purposes. Protein oxidation was assessed by measuring the loss of natural tryptophan fluorescence and the protein carbonyl gain by using fluorescence spectroscopy. Lipid oxidation was concurrently analyzed by measuring the increase of conjugated dienes (CDs) and hexanal. Proteins inhibited lipid oxidation in oil-in-water emulsions, and MPs showed a more intense antioxidant activity than BSA. MPs were also more resistant to oxidative deterioration than BSA. The different antioxidant capacity of MPs and BSA and their susceptibility to suffer oxidative reactions might be derived from their different amino acid composition and three-dimensional structures. The addition of the phenolic compounds resulted in a variety of effects, including both antioxidant and pro-oxidant effects. Gallic acid, cyanidin-3-glucoside, and genistein were the most efficient inhibitors of lipid and protein oxidation. The chemical structure of the phenolic compounds as well as the nature and conformation of the proteins were greatly influential on the overall effect against oxidative reactions.     

Postharvest variation in apple (Malus x domestica Borkh.) Flavonoids following harvest, storage, and 1-MCP treatment

The impact of 1-methylcyclopropene (1-MCP) on the synthesis and retention of flavonoid compounds during storage and ripening of red Delicious (Malus x domestica Borkh.) apples was investigated. Numerous anthocyanins, flavonols, flavan-3-ols, and a hydroxycinnamic acid from three different fruit harvest maturities were monitored after a 120 day storage and 1 week shelf life period using high-performance liquid chromatography/diode array detector analysis. The total flavonoid concentration was 5% greater in fruit treated with 1-MCP, whereas chlorogenic acid levels were 24% lower. All compounds analyzed increased in concentration during fruit harvest; however, the anthocyanins generally declined after storage, while chlorogenic acid levels increased. 1-MCP treatment resulted in the retention of anthocyanins in the latter stages of storage but did not affect the flavonols and flavan-3-ols. Chlorogenic acid biosynthesis from early and optimal fruit harvest maturities was greatly inhibited by 1-MCP during storage and the 1 week shelf life period. However, 1-MCP did not affect chlorogenic acid concentrations in late-harvested fruit. Results suggest that 1-MCP may inhibit the activity of phenylalanine ammonia-lyase and subsequent biosynthesis of flavonoid compounds. However, because very little postharvest biosynthesis of flavonoids occurs in apples, 1-MCP treatment may be useful for maintaining some of the intrinsic flavonoid levels of red Delicious apples, if applied at the proper harvest maturity.     

Pseudovitamin B(12) is the predominant cobamide of an algal health food, spirulina tablets.

The vitamin B(12) concentration of an algal health food, spirulina (Spirulina sp.) tablets, was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor chemiluminescence methods. The values determined with the microbiological method were approximately 6-9-fold greater in the spirulina tablets than the values determined with the chemiluminescence method. Although most of the vitamin B(12) determined with the microbiological method was derived from various vitamin B(12) substitutive compounds and/or inactive vitamin B(12) analogues, the spirulina contained a small amount of vitamin B(12) active in the binding of the intrinsic factor. Two intrinsic factor active vitamin B(12) analogues (major and minor) were purified from the spirulina tablets and partially characterized. The major (83%) and minor (17%) analogues were identified as pseudovitamin B(12) and vitamin B(12), respectively, as judged from data of TLC, reversed-phase HPLC, (1)H NMR spectroscopy, ultraviolet-visible spectroscopy, and biological activity using L. leichmannii as a test organism and the binding of vitamin B(12) to the intrinsic factor.     

Monitoring and estimation of the kinetics parameters in the binding process of tannic acid to bovine serum albumin with electrochemical quartz crystal impedance system

Combined measurements of piezoelectric quartz crystal impedance (PQCI) and electrochemical impedance (EI) were utilized in situ to monitor the adsorption of bovine serum albumin (BSA) onto the newly prepared Au colloid-modified electrode and study the binding process of tannic acid (TA) to BSA on the BSA-modified electrode surface. The time courses of the resonant frequency and the equivalent parameters of the sensor were simultaneously obtained during BSA adsorption and TA-BSA binding. Compared with the bare gold electrode, the Au colloid-modified gold electrode showed better biocompatibility, and the absorption capacity for BSA was increased by approximately 2.4 times. The observed frequency decrease was ascribed to the mass increase of the sensor surface resulting from the TA-BSA binding, which is believed to result mainly from the hydrogen bonding from FT-IR characterization. The maximal molar binding ratio of TA binding to immobilized BSA obtained from the frequency shift of the adsorbed BSA and TA was estimated to be 10.3:1. On the basis of the frequency decrease with time, the kinetics of the binding was quantitatively studied. By way of fitting the experimental data, the kinetics parameters, that is, binding and dissociation constant (k1, k(-1)), and the binding equilibrium constant (ka) were determined, giving values of 9.51 x 10(4) M(-1) s(-1), 3.15 s(-1), and 3.1 x 10(4) M(-1), respectively.     

Isolation and characterization of two flavonoids, engeletin and astilbin, from the leaves of Engelhardia roxburghiana and their potential anti-inflammatory properties

Engeletin, a flavonoid compound, was isolated from the leaves of Engelhardia roxburghiana for the first time, along with astilbin, another flavonoid. The chemical structures of engeletin and astilbin were confirmed by (1)H and (13)C nuclear magnetic resonance (NMR) and mass spectrometry (MS) spectra, and their anti-inflammatory activities were studied in lipopolysaccharide (LPS)-stimulated mouse J774A.1 macrophage cells. LPS induced the inflammatory state in macrophage cells and increased mRNA expressions of pro-inflammatory cytokines. Engeletin and astilbin exhibited remarkable inhibitory effects on interleukin (IL)-1β and IL-6 mRNA expression. Significant inhibition of LPS-mediated mRNA expressions were also seen in LPS binding toll-like receptor (TLR)-4, pro-inflammatory cytokine tumor necrosis factor (TNF)-α, IL-10, chemoattractant monocyte chemotactic protein (MCP)-1, and cyclooxygenase (COX)-2 genes. The reduced expression of these cytokines may alleviate immune response and reduce inflammatory activation, indicating that engeletin and astilbin may serve as potential anti-inflammatory agents.     

Conjugates of bovine serum albumin with chitin oligosaccharides prepared through the Maillard reaction

Chitin neoglycoconjugates (BSA-CO) were obtained by the conjugation of bovine serum albumin (BSA) with chitin oligosaccharides (CO) through the Maillard reaction (nonenzymatic glycation). CO produced by acid hydrolysis of chitin were fractionated using an ultrafiltration membrane system (1-3 kDa cutoff). The Maillard reaction was carried out by heating a freeze-dried mixture containing BSA and CO at 60 °C (under 43% relative humidity for 6 and 12 h). BSA-CO were characterized by available amino groups content, intrinsic tryptophan emission spectra, gel electrophoresis, and mass spectrometry. Biological assays included interaction with wheat germ agglutinin (WGA) and with bacterial adhesins of Escherichia coli K88+ and Salmonella choleraesuis. Glycation of BSA was revealed by reduction of available amino groups and fluorescence intensity and also retarded migration through SDS-PAGE. Conjugation of BSA with chitin oligomers appeared to be time dependent and was confirmed by mass spectrometry, by which molecular mass increase for monomers and dimers was observed. Monomers were estimated to contain either one or two glycation sites (at 6 and 12 h of treatment, respectively), with one or two tetrasaccharide units attached. Consequently, dimers showed two or four glycation sites. BSA-CO presented biological recognition by WGA and E. coli K88+ and S. cholerasuis adhesins. The strategy used in this work represents a simple method to obtain glycoconjugates to study applications involving protein-carbohydrate recognition.     

Investigation of flavonoids bearing different substituents on ring C and their cu2+ complex binding with bovine serum albumin: structure-affinity relationship aspects

The effects of 1:1 flavonoid-Cu(2+) complexes of four flavonoids with different C-ring substituents, quercetin (QU), luteolin (LU), taxifolin (TA), and (+)-catechin (CA), on bovine serum albumin (BSA) were investigated and compared with corresponding free flavonoids by spectroscopic analysis in an attempt to characterize the chemical association taking place. The results indicated that all of the quenching mechanisms were based on static quenching combined with nonradiative energy transfer. Cu(2+) chelation changed the binding constants for BSA depending on the structures of flavonoids and the detected concentrations. The reduced hydroxyl groups, increased steric hindrance, and hydrophilicity of Cu(2+) chelation may be the main reasons for the reduced binding constants, whereas the formation of stable flavonoid-Cu(2+) complexes and synergistic action could increase the binding constants. The changed trends of critical energy transfer distance (R(0)) for Cu(2+) chelation were contrary to those of binding constants.     

Antioxidative compounds from the outer scales of onion

Antioxidative compounds were isolated from the methanol extract of dry outer scales of onion (Allium cepa L.). Nine phenolic compounds (1-9) were finally obtained by reversed-phase high-performance liquid chromatography, and their structures were elucidated by NMR and mass spectrometry analyses. They were the six known compounds, protocatechuic acid (1), 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (2), quercetin 4'-O-beta-D-glucopyranoside (3), quercetin (5), 4'-O-beta-d-glucopyranoside of quercetin dimer (7), and quercetin dimer (8), and three novel compounds, condensation products of quercetin with protocatechuic acid (4), adduct of quercetin with quercetin 4'-O-beta-D-glucopyranoside (6), and quercetin trimer (9). These phenolic compounds were tested for their antioxidant properties using autoxidation of methyl linoleate in bulk phase or free radical initiated peroxidation of soybean phosphatidylcholine in liposomes. The flavonoid compounds having o-dihydroxy substituent in the B-ring were shown to be effective antioxidants against nonenzymic lipid peroxidation.     

Comparative studies on the interaction of genistein, 8-chlorogenistein, and 3',8-dichlorogenistein with bovine serum albumin

Chlorination can significantly enhance the antioxidant and antitumor activity of genistein. In this paper, genistein, 8-chlorogenistein, and 3',8-dichlorogenistein were selected to investigate the binding to bovine serum albumin (BSA) using fluorescence spectroscopy and circular dichroism (CD). The results showed that chlorination, especially at position 3', had significant effects on the binding constant value of chlorinated genistein derivatives to BSA; however, the binding site and the binding number were slightly affected. The thermodynamic parameters indicated that hydrophobic and electrostatic forces played important roles in the binding process and the enhanced binding affinity mainly associated with the increase of the hydrophobicity caused by the chlorine atom substitution. Furthermore, the CD data demonstrated that the conformation of BSA was slightly altered in the presence of genistein, 8-chlorogenistein, and 3',8-dichlorogenistein, with different reduced α-helix contents. The results obtained herein will be of biological significance in toxicology investigation and genistein derivative drug design.     

Targeted metabolite analysis and antioxidant potential of Rumex induratus

Targeted metabolite analysis of aqueous extract of Rumex induratus leaves, in terms of phenolic compounds and organic acids, and the study of its antioxidant activity against the DPPH(*) radical, a reactive oxygen species, hypochlorous acid, and a reactive nitrogen species, nitric oxide ((*)NO), were performed. The samples were collected in several locations, spontaneously occurring or from greenhouse culture, at different stages of development and seasons. The phenolic composition was achieved by high-performance liquid chromatography (HPLC)-diode array detection, and four hydroxycinnamic acid derivatives and 10 flavonoid glycosides (C- and O-heterosides) were determined. Organic acids composition was established by HPLC-UV, revealing five compounds. The total amount of phenolic compounds and organic acids were affected by growing conditions and developmental phase. The aqueous extract exhibited a dose-related activity against all tested reactive species.     

Phenolic antioxidants from the leaves of Corchorus olitorius L.

Six phenolic antioxidative compounds [5-caffeoylquinic acid (chlorogenic acid), 3,5-dicaffeoylquinic acid, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-(6-malonylglucoside), and quercetin 3-(6-malonylgalactoside) (tentative)] were identified from the leaves of Corchorus olitorius L. (moroheiya) by NMR and FAB-MS. The contents of these phenolic compounds, ascorbic acid, and alpha-tocopherol in C. olitorius leaves were determined, and their antioxidative activities were measured using the radical generator-initiated peroxidation of linoleic acid. The results obtained showed that 5-caffeoylquinic acid was a predominant phenolic antioxidant in C. olitorius leaves.     

Probing the binding of the flavonoid diosmetin to human serum albumin by multispectroscopic techniques

The binding mechanism of molecular interaction between diosmetin and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using atomic force microscopy (AFM) and various spectroscopic techniques including fluorescence, resonance light scattering (RLS), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by diosmetin was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The RLS spectra and AFM images showed that the dimension of the individual HSA molecules were larger after interaction with diosmetin. The thermodynamic parameters, ΔH° and ΔS° were calculated to be -24.56 kJ mol(-1) and 14.67 J mol(-1) K(-1), respectively, suggesting that the binding of diosmtin to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies and denaturation experiments in the presence of urea indicated site I as the main binding site for diosmetin on HSA. The binding distance between diosmetin and HSA was determined to be 3.54 nm based on the F?rster theory. Analysis of CD and FT-IR spectra demonstrated that HSA conformation was slightly altered in the presence of diosmetin.     

沙尘和遮阴复合胁迫对西梅叶片光合作用的影响

以5a生西梅为研究材料,对其叶片进行轻度沙尘+遮阴复合（1mg·cm-2沙尘+白色纱网1层）和重度沙尘+遮阴复合（5mg·cm-2沙尘+白色纱网3层）两种处理,分别在胁迫10、20、30和40d时测定其光合、叶绿素荧光等参数,探讨长时间沙尘和遮阴复合胁迫下西梅（Prunus domestica L．）叶片光合性能的变化机制。结果表明,两种处理下,随着胁迫时间的延长,总叶绿素（Chl）、叶绿素a（Chl-a）及叶绿素b（Chl-b）含量均表现为降低趋势。净光合速率（Pn）、蒸腾速率（Tr）和气孔导度（Gs）、水分利用效率（WUE）随处理时间的延长而下降,胞间CO2浓度（Ci）呈现先下降后上升的趋势,最大荧光（Fm）、PSII最大光化学效率（Fv/Fm）、潜在活性（Fv/F0）、光化学淬灭（qP）呈下降趋势,初始荧光（F0）、非光学淬灭（NPQ）呈上升趋势。说明沙尘和遮阴复合处理前期气孔因素是西梅光合作用的主要限制因素,处理后期复合胁迫使光合机构受到损伤,非气孔因素成为主要限制因素。复合胁迫影响了西梅叶片的光合参数,但并未对西梅叶片光反应系统造成不可逆的破坏。