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]

BHUIYAN, MOHAMMAD NAZRUL ISLAM

2015-09-25
Description
The growth of strain ASN212 related to Leucobacter sp. was stimulated by the supernatant of Sphingopyxis sp. strain GF9. In this study, novel porphyrin type growth factors produced by strain GF9 were identified to induce vigorous proliferation of a previously uncultured bacterial strain ASN212 at the picomolar to nanomolar levels. Even more surprisingly, the growth factors showed self-toxicities against the growth factor producing bacterium, strain GF9 at the picomolar to nanomolar levels. The second part of this study is the elucidation of structure-activity relationships for the dietary flavonoid, quercetin and its structural requirements for inhibition of advanced glycation end products (AGEs) formation. The study revealed that quercetin can rapidly trap methylglyoxal (MGO) by forming mono- and di-MGO adducts under the oxygenfree conditions. The positions C6 and C8 on the A ring of quercetin were the major active sites for trapping MGO that comprise the essential structural requirement for quercetin to inhibit AGEs formation. 1. Identification of strain ASN212 growth factors produced by strain GF9 Production of growth factors for strain ASN212 was achieved by a 72-h culture of strain GF9 in a 100 L stirred tank bioreactor (three batches). The active MeOH soluble fraction (dried, 150 g) obtained from 210 l of culture broth was subjected to Diaion HP20 column chromatography to give a MeOH eluting fraction, which induced growth of strain ASN212 at equivalent concentration (EC). Further bioassay guided fractionations by Sephadex LH-20 column chromatography led to the identification of the active fractions. Subsequent preparative ODS HPLC gave six different growth factors 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 new zincmethylphyrin I (0.8 mg) and zincmethylphyrin III (2.0 mg). Minimun effective concentrations (MECs) were determined by measuring the dry weight of bacterial cells from a 70-ml culture of strain ASN212 stimulated by each individual growth factor. The growth stimulating-activity of growth factor C was most evident with a MEC value of 14 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 to explore the generality of porphyrin as growth factor for strain ASN212, while coproporphyrin I dihydrochloride, coproporphyrin III dihydrochloride, coproporphyrin III tetramethyl ester, hemin and hematin showed significant growth stimulation of strain ASN212. The structure of growth factors E and F named as zincmethylphyrin I and zincmethylphyrin III were determined by NMR analyses. It was also noteworthy that all six growth factors completely inhibited the growth of strain GF9 itself, and the coculture experiments implied that strains GF9 and ASN212 are dependent on each other for their growth and survival in the environments. These results suggest that coproporphyrins function as global signal molecules that sustain the complex microbial communities as a network system. This research has broken new ground by demonstrating that coproporphyrins produced by strain GF9 or commercially available porphyrins could have an impact on the growth of previously uncultured bacterial strain in laboratory conditions. 2. Structural requirements of quercetin for inhibiting AGEs formation Structure-activity relationship studies on fourteen commercial flavonoids indicated that quercetin was the most potent inhibitor against AGEs formation in glucose-, riboseor MGO-mediated bovine serum albumin (BSA) assay systems. To address the structural requirement of quercetin as a potent inhibitor of AGEs formation, BSA was incubated with MGO under both in conventional and oxygen-free conditions. The reaction 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, two different quercetin-MGO monoadducts, and 2,3-dihydroxybenzoic acid. 2,3- Dihydroxybenzoic acid should be formed by an oxidative degradation of A and C rings of quercetin, and no other degradation products were detected. Under the conventional conditions, MGO accelerated the oxidative degradation of quercetin to form 2,3- dihydroxybenzoic acid. However, the diadduct formation of quercetin with MGO was accelerated under oxygen-free conditions, whereas the degradation of quercetin was suppressed. The C6 and C8 positions of the A ring in quercetin were the major active sites for trapping MGO to form both mono- and diadducts. These results might provide a 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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