Antioxidative and antihepatotoxic principles of Tuocha

沱茶は中国雲南省のアッサム茶の葉を蒸して作られる緊圧茶で,抗老化,コレステロール低下,免疫増強,血圧降下等の作用があると言われている。我々は,DPPHラジカル捕捉活性を指標として成分検索を行ない,得られた成分についてDPPHラジカル捕捉活性と共にD-GalN/TNF-α誘発マウス肝細胞死阻害活性を測定した。その結果,沱茶がフェノール類,リグナン,フラボノイド,フラバン3オール類などの抗酸化成分を多く含む事を明らかにし,それら成分の構造と活性との関係について考察した。 Tuocha is one of the special varieties of fermented compressed tea leaves, praised for its important health benefits, such as anti-aging, lowering cholesterol, enhancing immune function, lowering of blood pressure, reducing heart attacks etc. In the present study, we carried out fractionation and isolation of the active constituents, guided by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. In addition, antioxidative and antihepatotoxic potency on D-galactosamine (D-GalN)/tumor necrosis factor-alpha (TNF-α)-induced cell death in primary cultured mouse hepatocytes was also examined. Our study revealed that, tuocha is rich in antioxidants such as phenolics, lignans, flavanoides and flavan-3-ols, and showed a good understanding between structure and activity relationship

Division of Natural Products Chemistry

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されまし

Division of Natural Products Chemistry

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されまし

Analysis of Two Maize Mutations that Arose After Exposure to Radiation from a Plutonium-Beryllium Source and Developing Molecular Markers for Antimorph AEI1-5180, An Alternative to AEI to Increase Amylose Content in Maize Endosperm

This thesis consists of two independent projects dealing with two different aspects of maize. The first project is about the maize reproduction, where we try to uncover the candidate genes of two mutations that affect pollen viability and successful fertilization. The second project is about maize endosperm starch, where we developed molecular markers to characterize a mutant allele that knocks a major starch branching enzyme in maize. Analysis of two maize mutations that arose after exposure to radiation from a plutonium-beryllium source Maize is a monoecious and diclinous plant. Sexual reproduction takes place by the fusion of haploid male and female gametes produced in the anther and ovule, respectively. The male gametophyte is pollen, a trinucleate structure, that consists of two sperm cells and a vegetative cell. The female gametophyte is embryo sac, that consists of an egg cell, two synergids, two central cells and a variable number of antipodal cells. After pollination, the vegetative cell germinates and forms a pollen tube. The pollen tube elongates through the style, penetrates the ovule, and finally burst after reaching embryo sac to release two sperm cells. One sperm cell fuse with the haploid egg cell in embryo sac and forms a diploid embryo, whereas the other fuses with two central cells to form a triploid endosperm. In the first project, we developed two mutant lines, PB1 and PB2 by exposing kernels heterozygous for r1 (colored 1) and c1 (colored-aleurone 1) color factors to a fast neutron source for three and six months, respectively. Two mutagenized plants when crossed with r1 and c1 testers, showed reduced transmission for the R1 marker through pollen, but normal transmission through female. Instead of 50%, the R1 transmission rate through pollen was 23.6% ±0.61% (mean ±se) for PB1, and 1.48% ±0.44% (mean ±se) for PB2. Comparison of whole genome sequences of PB1, PB2 and the unmutated parental line PB1, shows that 32 genes are affected by the deletions in PB1, whereas in PB2, 45 genes are affected. In PB2, genes that code for mechanosensitive ion channel protein, C2H2 type Zinc finger family protein and β-expansin have been previously reported to affect pollen tube germination and elongation, which makes them good candidates. Developing molecular markers for antimorph Ae1-5180, an alternative to ae1 to increase amylose content in maize endospermMaize endosperm consists of mostly starch, which is made of unbranched amylose and branched amylopectin residues. In most Midwestern dents, the endosperm starch is about 75% amylopectin and 25% amylose. Branch formation is catalyzed by starch branching enzymes. Starch branching enzyme IIB (SBEIIb) encoded by ae1 (amylose extender 1), is the primary starch branching enzyme that forms amylopectin. The amylose content increases up to 50% when ae1 is homozygous recessive. An antimorph allele (Ae1-5180), was identified that acts in a dominant fashion to eliminate SBEIIb (Stinard et al., 1993). The essential portion of this mutant allele is a 3kb XhoI fragment that includes Mutator1 (Mu1) element flanked by an inverted duplication of the ae1 gene. Based on the restriction map (Stinard et al., 1993) we developed an approximate predicted sequence for Ae1-5180 using the known sequences of the wildtype ae1 gene and Mu1 transposon. Using this, we designed PCR primers targeted to amplify regions within a 3 kb XhoI restriction fragment that appears crucial for the dominant action of Ae1-5180. The primers amplify two DNA bands from samples expected to have Ae1-5180, but none from B73. The sequence of the lower molecular weight band matches with 181 bp of Mu1 and 364 bp of Ae1 (545 bp). The higher molecular weight band also possesses 181 bp of Mu1 but includes 438 bp of Ae1 (619 bp). The additional 74 bp includes a restriction site for NotI, which was predicted from the original restriction map. Sequencing results indicate that these primers reliably detect the presence or absence of the Ae1-5180 allele. Also, the insertion site of Mu1 in Ae1-5180 is now exactly defined

Quantitative Trait Loci Analysis of Low Temperature Responses in Grapevine F2 Population

Freezing injury, caused by freezing temperatures in the late fall, midwinter, or early spring, can result in significant loss to grape growers. The damage may range from the partial damage of parts of the plants to the total death of the plant, and may vary between years. Freezing tolerance is a multi-genetic, complex quantitative trait that involves many related traits like dormancy induction, growth cessation, acclimation, deacclimation and bud break. Developing an understanding of the genetics behind the complex trait requires connecting the phenotype with the genotype to enable discovering the underlying genes that can contribute to quantifiable differences between individuals. The objective of this thesis is to detect the genomic location(s) underlying genetic variation in low-temperature response traits like freezing tolerance and bud break using quantitative trait loci analysis (QTL). An F2 population developed by selfing a single F1 from a cross between American species Vitis riparia and a hybrid grapevine Seyval, was used to conduct QTL analysis for subzero temperature tolerance. The low temperature exotherms (LTE) obtained from differential thermal analysis (DTA) were used as phenotype data for the QTL analysis. The phenotyping was conducted in multiple months and dormant seasons. LTE results when the supercooled water inside the bud freezes and identifies the temperature at which buds are killed. Best linear unbiased predictors (BLUP) was also calculated using the LTEs to calculate random genetic effects. Composite interval mapping (CIM) was conducted using either the average LTEs or BLUP values in R/QTL with 1000 permutations and error rate of 5%. The LTEs for individual genotypes varied in the different months. QTLs were identified using either average LTEs or BLUP values for all the months and dormant seasons. QTLs appeared on Chromosome 1, 5, 9, 13 and 16 in different months or dormant seasons. Many potential candidate genes associated with calcium signaling, ethylene signaling, ABA signaling, cellular metabolism and dehydration response were found underlying the 1.5 LOD interval of these QTLs. The bud break phenology was studied at three chilling periods to identify bud break related QTL. Bud break occurs upon the exposure to optimal growth conditions, after the chilling requirement fulfillment transitions the bud from endodormancy to ecodormancy. Three one node cuttings of the grapevines were forced at 13 hours of daylength and 25⁰C/20⁰C thermoperiod in the growth chamber for four weeks. Each week the growth stage of the cuttings was scored using Modified Eichhorn-Lorenz (Modified E-L) phenology scale. This was repeated in multiple dormant seasons and months. The bud break data was ordinal, monotonic and based on repeated measurements, thus similar to data in disease epidemiological studies in which disease severity is scored repeatedly over a time period. Therefore, the concept of area under the curve (AUC) was applied to the bud break data. The area under the bud break progression curve (AUBPC) was calculated for the bud break data of each individual for an individual month. CIM was conducted with the average week 3 phenology score data or AUBPC data in R/qtl using 1000 permutations. Both data processing approaches provided similar QTL results on chromosome 3, 7, 8, 9, 13, 18 and 19. These QTLs encompassed candidate genes involved in calcium signaling, auxin signaling, ethylene signaling, circadian clock signaling, cellular metabolism, primary and secondary metabolism, dehydration stress response, drought stress response, fruit ripening and many transcription factors. The results from this study can be useful in developing low temperature response trait markers that can be applied in marker-assisted breeding and selection to develop the grapevine cultivars suitable for northern continental climates where freezing temperatures are common. This will enhance the sustainability of cold climate grape production and promote expansion of grape growing regions