Koichiro Yamamoto

Interaction of dietary polyphenols with bovine milk proteins: Molecular structure–affinity relationship and influencing bioactivity aspects

SCOPE Dietary flavonoids and stilbenes are important polyphenols in foods, such as, e.g. fruits, vegetables, nuts, and tea as they are of great interest for their bioactivities, which are related to the anti-oxidative property. METHODS AND RESULTS The relationship between the structural properties of dietary polyphenols and their affinities for milk proteins (MP) was investigated. Methylation and methoxylation of flavonoids decreased (or hardly affected) the affinities for MP. Hydroxylation on the rings A and B of flavones and flavonols enhanced the interaction slightly. The hydroxylation on the ring A of flavanones significantly improved the affinities. Glycosylation of flavonoids weakened the affinities by 1-2 orders of magnitude. The hydrogenation of the C2==C3 double bond of flavonoids decreased the binding affinities by 7.24-75.86-fold. Galloylation of catechins significantly improved the binding affinities by about 100-1000-fold. Glycosylation of resveratrol decreased its affinity for MP. Esterification of gallic acid increased its binding affinity. MP significantly weakened the DPPH radical scavenging activity of polyphenols. The decreasing DPPH scavenging percentages of polyphenols increased with increasing affinities of MP-polyphenol complexes. CONCLUSION The binding affinities with MP were strongly influenced by the structural differences of dietary polyphenols. The MP-polyphenol interaction weakened with the DPPH free radical scavenging potential of polyphenols.

Advance in Dietary Polyphenols as α-Glucosidases Inhibitors: A Review on Structure-Activity Relationship Aspect

The dietary polyphenols as α-glucosidases inhibitors have attracted great interest among researchers. The aim of this review is to give an overview of the research reports on the structure-activity relationship of dietary polyphenols inhibiting α-glucosidases. The molecular structures that influence the inhibition are the following: (1) The hydroxylation and galloylation of flavonoids including catechins improve the inhibitory activity. (2) The glycosylation of hyroxyl group and hydrogenation of the C2=C3 double bond on flavonoids weaken the inhibition. (3) However, cyaniding glycosides show higher inhibition against than cyanidin. Proanthocyanidins oligomers exhibit a stronger inhibitory activity than their polymers. (4) The hydroxylation on B ring and the glycosylation of stilbenes reduce the inhibitory activity. (5) Caffeoylquinic acids display strong inhibition against α-glucosidases. However, hydroxycinnamic acid, ferulic acid, and gallic acid hardly inhibited α-glucosidases. (6) The coupled galloyl structures attached to C-3 and C-6 of the 4C1 glucose core of ellagitanin gave basic inhibitory activity. (7) The mono-glycosylation of chalcones slightly lowers the inhibition. However, the diglycosylation of chalcones significantly decreased the activity.

Structure-affinity relationship of flavones on binding to serum albumins: effect of hydroxyl groups on ring A.

Four flavones (flavone, 7-hydroxyflavone, chrysin, and baicalein) sharing the same B- and C-ring structure but a different numbers of hydroxyl groups on the A-ring were studied for their affinities for BSA and HSA. The hydroxylation on ring A of flavones increased the binding constants (K(a)) and the number of binding sites (n) between flavones and serum albumins. The affinities of 7-hydroxyflavone for BSA and HSA were about 800 times and 40 times higher than that of flavone, respectively. It appears that the optimal number of hydroxyl groups introduced to the ring A of flavones is one. As more hydroxyl groups were introduced to positions at C-5, C-6, and/or C-7 of flavones, the affinities for serum albumins decrease. The critical energy transfer distances (R(0)) between the hydroxylated flavones (1-3 OH on the ring A) and serum albumins decreased with the increasing affinities for serum albumins.

Molecular structure-affinity relationship of natural polyphenols for bovine γ-globulin

SCOPE The aim of this study was to investigate the interaction between polyphenols and bovine γ-globulin. METHODS AND RESULTS The relationship between the structural properties of natural polyphenols and their affinities for bovine γ-globulin were investigated by fluorescence titration analysis. Methylation of hydroxyl groups on flavonoids weakened the affinities for γ-globulin by 1.20-38.0 times. Hydroxylation on rings A, B, and C of flavonoids also significantly affected the affinity for γ-globulin. Glycosylation of flavonoids slightly affected the affinity depending on the conjugation site and the class of sugar moiety. Hydrogenation of the C2=C3 double bond on flavonoids decreased the binding affinities. Galloylated catechins and catechol-type catechins exhibited higher binding affinities for γ-globulin than non-galloylated and pyrogallol-type catechins. The glycosylation of resveratrol decreased its affinity for γ-globulin. CONCLUSION The binding process with γ-globulin was strongly influenced by the structural differences of polyphenols.

Bovine serum albumin significantly improves the DPPH free radical scavenging potential of dietary polyphenols and gallic acids.

Polyphenol-protein interaction (PPI) is reversible in that polyphenol-protein complex can be dissociated and release the free polyphenols. The aim of this study is to evaluate the contribution of polyphenol-protein interaction on improving the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging capacity of polyphenols. The DPPH radical scavenging potential of polyphenols was determined from 1 to 7 days under aerobic condition. The DPPH radical scavenging capacity of polyphenols depended on the structures of dietary polyphenols and gallic acids. The DPPH radical scavenging percentages of Group H polyphenols were weakened when kept in room temperature from 1 to 7 days. BSA rapid weakened the DPPH radical scavenging activity of polyphenols on the first day. However, the DPPH scavenging capacities of polyphenols in the presence of BSA overwhelmingly improved with increasing time. These results illustrated that BSA not only prolongs the effective time, but also improved the DPPH radical scavenging potential of polyphenols. The increasing DPPH scavenging percentages of polyphenols slightly decreased with increased affinities of BSA-polyphenol complexes.

Structural Requirements of Cholesterol for Binding to Vibrio cholerae Hemolysin

Cholesterol is necessary for the conversion of Vibrio cholerae hemolysin (VCH) monomers into oligomers in liposome membranes. Using different sterols, we determined the stereochemical structures of the VCH‐binding active groups present in cholesterol. The VCH monomers are bound to cholesterol, diosgenin, campesterol, and ergosterol, which have a hydroxyl group at position C‐3 (3βOH) in the A ring and a C–C double bond between positions C‐5 and C‐6 (C–C Δ5) in the B ring. They are not bound to epicholesterol and dihydrocholesterol, which form a covalent link with a 3αOH group and a C–C single bond between positions C‐5 and C‐6, respectively. This result suggests that the 3βOH group and the C–C Δ5 bond in cholesterol are required for VCH monomer binding. We further examined VCH oligomer binding to cholesterol. However, this oligomer did not bind to cholesterol, suggesting that the disappearance of the cholesterol‐binding potential of the VCH oligomer might be a result of the conformational change caused by the conversion of the monomer into the oligomer. VCH oligomer formation was observed in liposomes containing sterols with the 3βOH group and the C–C Δ5 bond, and it correlated with the binding affinity of the monomer to each sterol. Therefore, it seems likely that monomer binding to membrane sterol leads to the assembly of the monomer. However, since oligomer formation was induced by liposomes containing either epicholesterol or dihydrocholesterol, the 3βOH group and the C–C Δ5 bond were not essential for conversion into the oligomer.

The Gene Encoding the Prepilin Peptidase Involved in Biosynthesis of Pilus Colonization Factor Antigen III (CFA/III) of Human Enterotoxigenic Escherichia coli

The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5‐kDa preform pilin (prepilin) and then processed to 20.5‐kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly‐30‐Met‐31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. cofP consists of 819 bp encoding a 273‐amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram‐negative bacteria.

Signature-tagged mutagenesis of Vibrio vulnificus

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.