Thesis or Dissertation Growth factors for uncultured bacteria and structural requirements of quercetin for inhibiting advanced glycation end products (AGEs) formation [an abstract of entire text]


The growth of strain ASN212 related to Leucobacter sp. was stimulated by thesupernatant of Sphingopyxis sp. strain GF9. In this study, novel porphyrin type growthfactors produced by strain GF9 were identified to induce vigorous proliferation of apreviously uncultured bacterial strain ASN212 at the picomolar to nanomolar levels.Even more surprisingly, the growth factors showed self-toxicities against the growthfactor producing bacterium, strain GF9 at the picomolar to nanomolar levels. Thesecond part of this study is the elucidation of structure-activity relationships for thedietary flavonoid, quercetin and its structural requirements for inhibition of advancedglycation end products (AGEs) formation. The study revealed that quercetin can rapidlytrap methylglyoxal (MGO) by forming mono- and di-MGO adducts under the oxygenfreeconditions. The positions C6 and C8 on the A ring of quercetin were the majoractive sites for trapping MGO that comprise the essential structural requirement forquercetin to inhibit AGEs formation.1. Identification of strain ASN212 growth factors produced by strain GF9Production of growth factors for strain ASN212 was achieved by a 72-h culture ofstrain GF9 in a 100 L stirred tank bioreactor (three batches). The active MeOH solublefraction (dried, 150 g) obtained from 210 l of culture broth was subjected to DiaionHP20 column chromatography to give a MeOH eluting fraction, which induced growthof strain ASN212 at equivalent concentration (EC). Further bioassay guidedfractionations by Sephadex LH-20 column chromatography led to the identification ofthe active fractions. Subsequent preparative ODS HPLC gave six different growthfactors A-F, which were identified as zinc coproporphyrin I (0.8 mg), coproporphyrin I(0.3 mg), zincphyrin (3.0 mg), coproporphyrin III (1.2 mg), structurally newzincmethylphyrin I (0.8 mg) and zincmethylphyrin III (2.0 mg). Minimun effective concentrations (MECs) were determined by measuring the dry weight of bacterial cellsfrom a 70-ml culture of strain ASN212 stimulated by each individual growth factor. Thegrowth stimulating-activity of growth factor C was most evident with a MEC value of14 pM followed by growth factor D (1.5 nM), F (4.8 nM), E (9.6 nM), A (20 nM), B(38 nM), respectively. A panel of commercial and synthetic porphyrins was tested toexplore the generality of porphyrin as growth factor for strain ASN212, whilecoproporphyrin I dihydrochloride, coproporphyrin III dihydrochloride, coproporphyrinIII tetramethyl ester, hemin and hematin showed significant growth stimulation of strainASN212. The structure of growth factors E and F named as zincmethylphyrin I andzincmethylphyrin III were determined by NMR analyses. It was also noteworthy that allsix growth factors completely inhibited the growth of strain GF9 itself, and the cocultureexperiments implied that strains GF9 and ASN212 are dependent on each otherfor their growth and survival in the environments. These results suggest thatcoproporphyrins function as global signal molecules that sustain the complex microbialcommunities as a network system. This research has broken new ground bydemonstrating that coproporphyrins produced by strain GF9 or commercially availableporphyrins could have an impact on the growth of previously uncultured bacterial strainin laboratory conditions.2. Structural requirements of quercetin for inhibiting AGEs formationStructure-activity relationship studies on fourteen commercial flavonoids indicatedthat quercetin was the most potent inhibitor against AGEs formation in glucose-, riboseorMGO-mediated bovine serum albumin (BSA) assay systems. To address thestructural requirement of quercetin as a potent inhibitor of AGEs formation, BSA wasincubated with MGO under both in conventional and oxygen-free conditions. Thereaction of quercetin with MGO in the presence of BSA yielded four different products.The structures of these compounds were determined to be quercetin-MGO diadduct, twodifferent quercetin-MGO monoadducts, and 2,3-dihydroxybenzoic acid. 2,3-Dihydroxybenzoic acid should be formed by an oxidative degradation of A and C ringsof quercetin, and no other degradation products were detected. Under the conventionalconditions, MGO accelerated the oxidative degradation of quercetin to form 2,3-dihydroxybenzoic acid. However, the diadduct formation of quercetin with MGO wasaccelerated under oxygen-free conditions, whereas the degradation of quercetin wassuppressed. The C6 and C8 positions of the A ring in quercetin were the major activesites for trapping MGO to form both mono- and diadducts. These results might providea new insight to prevent diabetes complications and aging caused by AGEs formation.
Hokkaido University(北海道大学). 博士(農学)

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