Academic literature on the topic 'Antimutagenicita'

The antimutagenicity and fermentation pattern of three Bifidobacterium longum strains (B. longum, B. longum PS+, and B. longum PS-) in skim milk were studied. The increase in fermentation time significantly increased antimutagenicity with all strains tested against the mutagenicity of both 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in an Ames-like test using streptomycin-dependent strain SD510 of Salmonella typhimurium TA98. Bifidobacterium longum PS+, a polysaccharide-producing strain, had a longer lag phase but showed the highest inhibition percentage against both mutagens tested. The viability of B. longum PS+ cells was not affected by the low pH of 4.1, probably owing to the protection offered by the polysaccharide produced. The antimutagenicity of the fermented milk against Trp-P-1 was dose dependent. The strains were also able to bind with different amino acid pyrolysates, and B. longum showed the highest binding. Acetone extracts of fermented skim milk dissolved in water showed less antimutagenicity than extracts dissolved in dimethylsulfoxide. The isolated crude polysaccharide from B. longum PS+ showed a dose-dependent inhibition of the mutagenicity of Trp-P-1. Thus, we conclude that the polysaccharide of B. longum PS+ can be used as an antimutagen.Key words: Bifidobacterium longum, polysaccharides, fermented milk, heterocyclic amines.

The possible mechanisms of antimutagenicity against 4-nitroquinoline-N-oxide (4-NQO; a direct mutagen) and 3,2′-dimethyl-4-amino-biphenyl (DMAB; an indirect mutagen) were examined in fermented soymilk prepared with a coculture of Streptococcus thermophilus and Bifidobacterium infantis. The antimutagenicity in the fermented soymilk was not due to the bioantimutagenic effect of modulation of DNA repair processes. The mutagenicity of DMAB decreased with increased pre-incubation of fermented soymilk and the DMAB metabolite but not with intact DMAB or an S9 mixture. Mutagenicity of 4-NQO was not affected by preincubation of fermented soymilk with this mutagen. Mutagenicity of both 4-NQO and DMAB was reduced when Salmonella Typhimurium TA 100 was pretreated with fermented soymilk, indicating that fermented soymilk affected the function of the bacterial cell, which might also lead to reduced mutagenicity of the tested mutagens. Desmutagenic and blocking effects were the main mechanisms of antimutagenicity in the fermented soymilk against DMBA. In contrast, the antimutagenic effect of the fermented soymilk on 4-NQO was primarily due to a blocking effect.

Tato práce představuje pilotní studii testující souvislosti mezi biologickými vlastnostmi a strukturou huminových kyselin extrahovaných z původního a modifikovaného jihomoravského lignitu, důl Mír, Mikulčice. V první části práce byly testovány metody vhodné ke zvýšení výtěžku huminových kyselin extrahovaných z lignitu. Oxidace lignitu v plynné fázi nepřinesla uspokojivé zvýšení výtěžku a byla instrumentálně poměrně náročná. Dále proto byla zkoumána jen oxidace v kapalné fázi a modifikace nízkomolekulárními organickými kyselinami. Modifikace organickými kyselinami byla inspirována procesy podporujícími biologické funkce v rizosféře, t.j. kořenový systém vylučuje exudáty způsobující změny v supramolekulové struktuře okolní organické hmoty čímž zlepšuje její mobilitu a prostupnost buněčnými stěnami. Primární struktura huminových kyselin připravených v této práci byla zkoumána prostřednictvím elementární analýzy a spektrálních metod (13C CPMAS NMR, EPR a UV-VIS spektroskopie). Navzdory tomu, že primární struktura vykazovala jen malé rozdíly, měření biologické aktivity a genotoxického potenciálu prokázalo, že huminové kyseliny a jejich humáty získané z lignitu s rozdílnou předúpravou vykazují odlišnou bioaktivitu. Proto byla dále zkoumána supramolekulární struktura vzorků ve zředěných roztocích, a to prostřednictvím vysokoúčinné vylučovací chromatografie, měření ultrazvukové rychlosti a hustoty. Testovány byly dva různé protionty – draselný a amonný. Získané výsledky potvrdily předpoklad, že pozorované změny v kvalitě humátů jsou závislé na protiiontu, koncentraci humátu v roztoku a také na metodě předúpravy původního lignitu. Obě zvolené metody předúpravy lignitu prokázaly svůj potenciál produkovat huminové kyseliny s rozmanitými biologickými vlastnostmi, aplikovatelné v zemědělství, životním prostředí a potenciálně i ve farmakologii.

Orientador: Maria Edwiges Hoffmann
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A melatonina, honnônio produzido pela glândula pineal, apresenta um gr-ande interesse na atualidade, devido à demonstração de sua ação antioxidante tanto in vitro como in vivo. Neste trabalho, foi investigado o potencial de risco genético e toxicológico da melatonina em culturas de células, e sua atividade antimutagênica. A mutagenicidade da melatonina foi determinada pelo teste de Ames, na presença e na ausência de ativação metabólica (fração S9), nas linhagens de Salmonella typhimurium TA97, Tal00 e TA102. A exposição das culturas por 30 minutos, à diferentes concentrações de melatonina (29-1160 _g/placa) não induziu nenhum aumento significativo do número de colônias revertentes, tanto na presença como na ausência de ativação metabólica. A melatonina mostrou, também, uma reversão dose-dependente (29 ¿ 232 _g/placa) dos efeitos mutagênicos causados pelo H2O2 (0,5 mM), na linhagem T A1O2. A atividade citotóxica da melatonina para fibroblastos de hamster chinês (linhagem V79) foi determinada pela medida de seus efeitos sobre a viabilidade e proliferação celular. A melatonina não alterou a viabilidade celular, medida pela redução do MTT, na faixa de concentração entre 0,0001 a 1 mM. A exposição das células por 30 minutos a diferentes concentrações de melatonina (0,01 - 5 mM) não causou inibição significativa do crescimento celular, no período de 24 horas subsequente ao tratamento. Entretanto, a exposição das células por 24 horas a concentrações de melatonina entre 0,5 a 5 mM causou inibição significativa do crescimento celular, de fonna dependente da dose. A melatonina mostrou ser um antioxidante altamente eficiente em solução, inibindo a oxidação degradativa da desoxirribose, induzida pelo sistema H2O2/Fe+3/NTA, de forma dependente da dose. Este hormônio foi 10 vezes mais eficiente que a glutationa e 200 vezes mais que o manitol, ao sequestrar os radicais hidroxila. Porém, o trolox foi 15 vezes mais eficaz que a melatonina. Entretanto, ao avaliar crescimento celular em fibroblastos V79, a melatonina (0,001 mM) mostrou uma proteção apenas parcial (50+ 7%) do efeito inibitório induzido por H2_ (50 µM). Contudo, a melatonina não protegeu a membrana mitocondrial de fibroblastos V79 contra os ataques dos oxidantes H2O2 e HPC (2,5 mM), como demonstrado pela ausência de proteção no ensaio do MTT. Já em eritrócitos humanos, ela mostrou ser um antioxidante eficiente contra a peroxidação lipídica, induzida pelo H2O2 (5 mM), apresentando um efeito protetor dose-dependente. Estes resultados indicam que a melatonina não é mutagênica e, além disso, inibe a mutação induzida pela H2O2 . A análise da toxicidade da melatonina pelas células V79 mostrou que a melatonina é citotóxica somente quando presente em altas concentrações e por tempo prolongado. A melatonina mostrou ser um eficiente antioxidante em sistema livre de células, em cultura de fibroblastos V79 apresentou proteção apenas parcial contra os efeitos da H2O2 . Entretanto, em eritrócitos mostrou eficiente proteção contra danos de membrana induzidos por este oxidante
Abstract: The melatonin, hormone produced 1>y the pineal gland,presents _ great interest at the present time due to the demonstration of its antioxidant action as in vitro as in vivo. In this work, the potential of toxicological risk of the melatonin was investigated in bacterias and in mammal cells,and its antioxidant activity was examined in cell tree systems, in human fibroblast V79 and in erythrocyte. The mutagenicity of the melatonin was determined by Ames Test, in the presence and in the absence of metabolic activation (S9-traction), in the strains of Salmonella typhimurium TA97, TA100 and TA102. The exposition of cultures by 30 minutes, to different melatonin concentrations (29 to 1,160 µg/plate) didn't induce any significant increase of the number of reverted colonies, after 48 hours of cultivation, so much in the presence as in the absence of metabolic activation. The melatonin also showed, a dose dependent reversion (29 to 232 µg/plate) ofthe mutagenics effects caused by the hydrogen peroxide (0.5 mM), in the TA102 straip.. Theçytotmric activity of the melatonin to V79 Chinese hamster fibroblasts was determined by the measure of its effects on proliferation. The melatonin didn't chage the cellular viability mesured by the reduction of MTT, in the concentration range trom 0.0001 to 1 mM. The exposition of the cells for 30 minutes to different melatonin concentrations (0.01 -5 mM) didn't caus_significant iphibitionof the cellular growth, in the period of subsequent 24 hours of treatment. However, the exposition of the cells for 24 hours to increasing melatonin concentrations between 0.5 and 5 mM, caused significant inhibition in the cellular growth, in dependent way of the dose. The .melatonin,showed to be ahighly efficient antioxidant in solution, inhibiting thedegradative oxidation of the deoxyribose, induced by the system H2O2/Fe3+/NTA, in a dependent way to the dose. This honnone was more than 10 times efficient than the glutathione and 200 times more efficient than the manit04 when scaveng the hydroxil radical. Even so, the trolox was more than 15 times effective than the melatonin. However, when evaluating cellular growth in fibroblasts V79, the melatonin just showed a partial protection (50+ 7%) ofthe inhibition effect induced by H2O2 (50µM), in _low concentration (0.001 mM). However, the melatonin up to lmM didn't protect the mitochondrional membrane of fibroblasts V79 against the attacks of the hydrogen peroxide and cumene hydroperoxide (2.5 mM), as demonstrated by the absence of protection related to the inhibition of the MTT reduction induced by these oxidizers. Yet in human erythrocytes, it showed to be an efficient antioxidant against lipid peroxidation, induced in the membrane by hydrogen peroxide (5 mM), presenting a dose-dependent protecting effect. These results indicate that melatonin is not mutagenic; besides, it inhibits mutations induced by H2O2 . The analysis of melatonin toxicity through the cells V79 showed that melatonin is citotoxic, on1y when it is present in high concentrations, for a long time. Melatonin showed to be na efficient antioxidant in a cell ftee system, but in cultures of fibroblasts V79 it showed just a partial protection against the effects of H2O2 . However, in erythrocytes, it showed an efficient protection against membrane injuries caused by this oxidant
Mestrado
Bioquimica
Mestre em Ciências Biológicas

R. Staršelskytės magistro baigiamasis darbas/ mokslinė vadovė prof. N. Savickienė; Konsultantės: dr. Annabella Vitalone, prof. Gabriela Mazzanti, dr. Antonella Di Sotto; Lietuvos sveikatos mokslų universiteto, Farmacijos fakulteto, Farmakognozijos katedra. – Kaunas. Romos universiteto La Sapienza, Fiziologijos ir farmakologijos katedra. – Roma. Darbo tikslas: baltymų frakcijų, praturtintų lektinais, išskirtų iš Urtica doica L. žolės, antimutageninio, citotoksinio ir antioksidacinio aktyvumo įvertinimas. Darbo uždaviniai: 1. Įvertinti Urtica dioica L. baltymų frakcijų įtaką Salmonella typhimurium ir Escherichia coli kamienų mutageniškumui, naudojant AMES testo metodą. 2. Ištirti Urtica dioica L. baltymų frakcijų citotoksiškumą HepG2 ląstelių proliferacijai, naudojant MTT dažo redukcijos reakcijos metodą. 3. Ištirti Urtica dioica L. baltymų frakcijų antioksidacinį aktyvumą, naudojant modelinius ABTS (2,2-azino-bis-(3-etilbenztiazolin-6-sulfono rūgšties)) ir superoksido radikalus. Metodai: 1. Lektinais praturtintų baltymų frakcijų antimutageniškumas tiriamas AMES testo metodu (grįžtamosios mutacijos modeliu in vitro) su S9 metabolinės aktyvacijos sistema (supernatantu iš žiurkių (paveiktų fenobarbitalio/β-naftoflavono mišiniu) kepenų lątelių mitochondrijų) ir be S9 metabolinės aktyvacijos sistemos. Eksperimentui naudojami trys bakterijų kamienai: S. typhimurium TA98, S. typhimurium TA100 ir E. coli WP2uvrA. 2. Citotoksiškumas nustatomas MTT dažo redukcijos reakcijos metodu... [toliau žr. visą tekstą]
Rasa Staršelskytė master thesis/ Supervisor of the research paper: prof. Nijolė Savickienė1 Consultants: PhD Annabella Vitalone, prof. Gabriela Mazzanti, PhD Antonella Di Sotto2 1Department of Pharmacognosy, Faculty of pharmacy, Lithuanian University of Health Sciences, Lithuania 2Department of Physiology and Pharmacology, Sapienza University of Rome, Italy Objective of work: evaluation of antimutagenicity, cytotoxicity and antioxidant activity of lectin-enriched protein fractions from herb of Urtica dioica L. Main tasks: 1. To evaluate antimutagenic activity of lectin-enriched protein fractions by bacterial reverse mutation assay. 2. To determine cytotoxicity of lectin-enriched protein fraction by the tetrazolium dye (MTT) colorimetric assay. 3. To evaluate antioxidant activity of lectin-enriched protein fraction against ABTS-free radical and superoxide-radical. Methods: 1. The antimutagenicity was studied in a bacterial reverse mutation assay (Ames test), both in the absence and presence of an exogenous metabolic activator S9 (the liver postmitochondrial supernatant of rats treated with the mixture phenobarbital/β-naphthoflavone to induce the hepatic microsomal enzymes). A set of three strains, S. typhimurium TA98, S. typhimurium TA100 and E. coli WP2uvrA, was used. 2. Cytotoxicity was determined by the tetrazolium dye (MTT) colorimetric assay in HepG2 human hepatoblastoma cell line. 3. The antioxidant activity was evaluated by ABTS-free radical scavenging activity test... [to full text]

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Silymarin (SM) is a standardized extract from the seeds and leaves of milk thistle Silybum marianum (L.) Gaertn. It is composed mainly of flavonolignans, with silibinin (SB) being its principal active constituent. Known mainly as antioxidant and hepatoprotector, SM and SB were found to be clinically effective in the treatment of a variety of liver disorders, including acute and chronic viral hepatitis, toxin and drug-induced hepatitis and cirrhosis. Due to the wide biological activities presented by SM and SB, the present study aimed to evaluate their antimutagenic activities using the Ames mutagenicity test in Salmonella typhimurium, their antigenotoxic activities using the mouse bone marrow micronucleous test and the alkaline comet assay, and to assess their effect on the gene expression pattern of some genes associated with the process of carcinogenesis and chemoprevention. To assess antimutagenicity, bacterial suspensions of Salmonella typhimurium (TA98 and TA100 strains) were treated with different concentrations of SM or SB simultaneously with the appropriate positive controls for each strain. To assess antigenotoxicity, Swiss mice were orally treated with different concentrations of SM or SB simultaneously with a single intraperitoneal dose of mitomycin C (MMC) for the micronucleus test, and human blood lymphocytes were cotreated with SM or SB and methyl methanesulfonate (MMS) for the alcaline comet assay. To investigate the role of SM and SB in modulating gene expression, we conducted microarray analysis. The results showed that SM was not significantly effective in reducing the number of frameshift mutations in strain TA98, while SB demonstrated significant protection at higher doses (p < 0.05). Regarding strain TA 100, SM and SB significantly decreased mutagenicity (point mutations) (p < 0.05). The results of the antigenotoxic evaluation demonstrated that SM and SB significantly reduced the frequency of micronucleated polychromatic erythrocytes (MNPCE) (p < 0.05). The results also indicated that SM and SB significantly attenuated MMC induced cytotoxicity (p < 0.05). In the comet assay, SM and SB significantly reduced the genotoxicity of MMS (p < 0.05), with a stronger antigenotoxic activity exerted by the extract complex (SM) than the one exerted by the isolated main active constituent (SB). The expression array analysis of five genes related to DNA damage, carcinogenesis and/or chemoprevention mechanisms demonstrated an up-regulation of PTEN and BCL2, down-regulation of BAX and ABL1 and no significant change in ETV6 expression levels.In conclusion, our results demonstrated that both SM and SB presented antimutagenic and antigenotoxic actions, as well as modulated the expression levels of genes analysed under the experimental conditions of this study.
A silimarina (SM) é um extrato padronizado obtido a partir das sementes e folhas de Silybum marianum (L.) Gaertn. SM é composta principalmente de flavonóides, sendo a silibinina (SB) seu principal componente ativo. Conhecidas principalmente como antioxidantes e hepatoprotetoras, SM e SB foram consideradas clinicamente eficazes no tratamento de uma variedade de doenças do fígado, incluindo hepatites virais agudas e crônicas, hepatites induzidas por toxinas e/ou drogas e cirrose. Assim, devido à ampla gama de atividades biológicas apresentadas pela SM e SB, o presente estudo teve como objetivo avaliar suas atividades antimutagênicas utilizando o teste de Ames em Salmonella typhimurium, suas atividades antigenotóxicas pelo teste do micronúcleo em medula óssea de camundongos e pelo teste do cometa em linfócitos humanos e avaliar seus efeitos nos perfis de expressão gênica de alguns genes associados ao processo de carcinogênese e quimioprevenção. Para a avaliação da antimutagenicidade, suspensões bacterianas de Salmonella typhimurium (cepas TA98 e TA100) foram co-tratadas com diferentes concentrações de SM ou SB e os controles positivos adequados para cada cepa. Para a avaliação de antigenotoxidade, camundongos Swiss foram tratados oralmente com diferentes concentrações de SM ou SB concomitantemente a uma única dose intraperitoneal de mitomicina C (MMC) para o teste do micronúcleo, e linfócitos humanos foram tratados simultaneamente com SM ou SB e metil-metanossulfonato (MMS) para o ensaio do Cometa. Os resultados mostraram que a SM não foi significativamente efetiva em reduzir o número de mutações com deslocamento de quadro de leitura na cepa TA 98, enquanto que a SB apresentou uma proteção significativa nas doses maiores (p < 0.05). Em relação à cepa TA100, SM e SB reduziram significativamente a mutagenicidade (mudanças de pares de bases) (p < 0.05). Na avaliação de antigenotoxidade, SM e SB reduziram significativamente a frequência de eritrócitos policromáticos micronucleados (EPCMN) (p<0,05). Os resultados também mostraram que a citotoxicidade causada pela MMC foi significativamente atenuada pela SM e SB (p<0,05). No ensaio do cometa, SM e SB reduziram significativamente a genotoxicidade provocada pelo MMS (p<0.05), com uma atividade antigenotóxica maior exercida pelo extrato complexo (SM) do que pelo principal componente ativo isolado (SB). A análise dos níveis de expressão de cinco genes relacionados ao dano no DNA, mecanismos de carcinogênese e/ou quimioprevenção demonstrou um aumento na expressão de PTEN e BCL2, diminuição na expressão de BAX e ABL1 e ausência de mudança significativa nos níveis de expressão do ETV6. Com base nesses resultados, conclui-se que a SM e a SB apresentaram ações antimutagênicas e antigenotóxicas, e também modularam os níveis de expressão dos genes analisados sob as condições experimentais deste estudo.

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Ipriflavone is a synthetic isoflavone derivative from daidzein and clinically prescribed for treating and preventing osteoporosis in postmenopausal women. We investigated the potential of this drug against the cytotoxic and mutagenic effects induced by cyclophosphamide (CPA) chemotherapy, using the micronucleus assay in bone marrow erythrocytes of Swiss albino mice (Mus musculus) in vivo. To evaluate their possible mechanisms of action, performed the evaluation of antioxidant activity by DPPH assay. For in vivo testing was carried out three protocols: pretreatment, simultaneous treatment and post treatment. The ipriflavone was evaluated in three different concentrations dissolved in DMSO (1,71; 8,57 e 42,85mg.kg-1 m.c) and administered by oral via. The bone marrow was collected for the evaluation of polycromatic erythrocytes (PCE) and the ratio PCE/(PCE+NCE) (polychromatic erythrocytes / polychromatic erythrocytes + normochromatic erythrocytes). For the DPPH test were assessed five concentrations of ipriflavone (500, 250, 150, 50 e 10μg.mLˉ¹) using DPPH solution (60μM). The results of in vivo tests show that the three concentrations of ipriflavone studied significantly reduced the frequency of MNPCEs induced by CPA, in the pre-treatment protocol and demonstrated the same effect at the concentrations of 1,71 e 42,85mg.kg-1 m.c in the post-treatment. However, simultaneous treatment did not reduce the frequency of MNPCE in any of the concentrations tested. In all protocols performed, the ratio PCE/(PCE+NCE) increased. There was variation between the genders in some of the experimental groups and the evaluation of antioxidant activity of ipriflavone showed no ability to donate hydrogens, suggesting that it acts through other mechanisms, such as inactivation of the enzyme activity of cytochrome P-450
Ipriflavona é uma isoflavona sintética derivada da daidzeína e utilizada no tratamento e prevenção da osteoporose em mulheres pós-menopausadas. Investigamos o potencial dessa droga contra os efeitos citotóxico e mutagênico induzidos pelo quimioterápico ciclofosfamida (CPA), por meio do ensaio do micronúcleo em eritrócitos de medula óssea de camundongos albinos Swiss (Mus musculus) in vivo. Para avaliar um de seus possíveis mecanismos de ação realizamos a avaliação de sua atividade antioxidante pelo método de DPPH. Para os testes in vivo foram realizados três protocolos: pré-tratamento, tratamento simultâneo e pós-tratamento. A ipriflavona foi avaliada em três concentrações dissolvidas em DMSO (1,71; 8,57 e 42,85mg.kg-1 m.c) e administrada via oral. A medula óssea foi coletada para a avaliação dos eritrócitos policromáticos micronucleados (MNPCEs) e da razão PCE/(PCE+NCE) (eritrócitos policromáticos/eritrócitos policromáticos + eritrócitos normocromáticos). Para o teste de DPPH foram avaliadas 5 concentrações de ipriflavona (500, 250, 150, 50 e 10μg.mLˉ¹) utilizando solução de DPPH 60μM. Os resultados obtidos nos testes in vivo demonstram que a ipriflavona nas três concentrações pesquisadas reduziu significativamente a frequência de MNPCEs induzidos pela CPA no protocolo de pré-tratamento e demonstrou o mesmo efeito nas concentrações de 1,71 e 42,85mg.kg-1 m.c, no pós-tratamento. Entretanto, no tratamento simultâneo, ela não reduziu a frequência de MNPCE em nenhuma das concentrações testadas. Em todos os protocolos realizados houve o aumento da razão PCE/(PCE+NCE), demonstrando sua eficácia na redução da citotoxicidade induzida pela CPA. Houve variação entre os gêneros em alguns dos grupos experimentais. A avaliação da atividade antioxidante da ipriflavona revelou sua ausência de capacidade em doar hidrogênios para o radical DPPH, sugerindo que a mesma atua por meio de outros mecanismos, como por exemplo, inativação da atividade enzimática das isoenzimas do citocromo P-450

The purpose of this study was to identify antimutagens in yogurt active against the experimental colon carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Our initial experiments showed that acetone extracts of yogurt, or milk fermented by various lactic acid bacteria were antimutagenic against MNNG and 3,2'-dimethyl-4-aminobiphenyl (DMAB) in the Ames test (Salmonella typhimurium TA 100). Further experiments carried out with milk fermented by Lactobacillus delbrueckii ssp. bulgaricus 191R showed that the putative compounds were more soluble in DMSO than in water, and that extractability of activity against MNNG and DMAB varied with pH, suggesting the presence of ionizable groups. Subsequent experiments demonstrated the antimutagenicity of yogurt. An acetone extract of yogurt was found to be active against a range of mutagens and promutagens in the Ames test. Simulation of fermentation by addition of lactic acid, lactic acid bacteria, or both to milk did not increase antimutagenicity, suggesting that compounds responsible for the activity may be formed during fermentation. Conjugated linoleic acid (CLA), a known dairy anticarcinogen, did not inhibit MNNG or DMAB indicating that other antimutagens may be present in yogurt. Fractionation of the acetone extract by HPLC showed that anti- MNNG and anti-DMAB activities did not co-elute, indicating that different compounds were responsible for the two activities. Using the Ames test to direct purification, isolation of an anti-MNNG active compound was accomplished using silica gel, Sephadex LH-20 and C18 reversed phase medium pressure chromatographies. The antimutagen was identified as palmitic acid by: a) co-elution with authentic palmitic acid on GC and HPLC columns, and b) by comparison of mass and ¹³C-NMR spectra. Minor components of milk fat such as iso methyl branched fatty acids (isopalmitic acid, isomargaric acid, isomyrsitic acid, and isostearic acid) were found to be more active than their straight chain counterparts. Isopalmitic acid also inhibited 4-nitroquinoline-N-oxide (4NQO) and the P450-mediated activation of 7,12-dimethylbenz[a]anthracene (DMBA). The mechanism of antimutagenesis against MNNG has not been established.
Graduation date: 1996

碩士
中國文化大學
生活應用科學研究所
92
Abstract The aim of this study was to evaluate the antioxidative capacity and antimutagenicity of three phytochemicals, including chlorophyll and their derivatives, flavonols and anthocyanins and acid hydrolysates of leafy sweet potato. The antioxidantive substances included porphyrins, flavonols and anthocyanins were determinated. The antioxidative substances of acid hydrolysates of leafy sweet potato were also determined. The flavonols such as myricetin, quercetin, morin and kaempferol, and anthocyanins such as cyanidin and malvidin were determined. The antioxidative capacity was evaluated by the Trolox Equivalent Antioxidant Capacity (TEAC) assay and by the inhibition percentage of conjugated diene formation in linolenic acid emulsion autoxidation system. The substances, such as chlorophyllin (CHL), pheophytin a (Phe a), pheophytin b (Phe b), quercetin, cyanidin and acid hydrolysates of leafy sweet potato were evaluated their antimutagenicity by Ames test. In results, the porphyrins contents were significantly rich in Taoyuan 2. The flavonols, such as morin and quercetin were significantly rich in Ipomoea batatas (L.)(red). The anthocyanins only existed in Ipomoea batatas (L.) (red). In TEAC assary, at 7.5μM and 100μg/ mL, the antioxidative capacity of flavonols, anthocyanins and acid hydrolysates of leafy sweet potato were more than 90%. Excepting CHL, the antioxidative capacity of chlorophyll and their derivatives were less than 50%. In general, the antioxidative capacity of chlorophyll and their derivatives were much less than that of flavonols and anthocyanins. In Ames test, CHL, Phe a, Phe b, cyanidin, quercetin and acid hydrolysates of leafy sweet potato had antimutagenicity effect. In S. typhimurium TA98 system, Phe a, Phe b and quercetin had the best inhibition percentage. In TA100 system, Phe a and cyanidin had the best inhibition percentage. In TA98 and TA100 systems, the various acid hydrolysates of leafy sweet potato had antimutagenicity effect, and the inhibition percentage of Taoyuan 2 was better than that of Ipomoea batatas (L.)(red). The TEAC percentage of samples treated with acid hydrolysates of leafy sweet potato was negative correlated with porphyrins but was positive correlated with flavonols and anthocyanins. In TA98 and TA100 systems, the antimutagenicity of samples treated with acid hydrolysates of Ipomoea batatas (L.)(red) was positive correlated with cyanidin while the antimutagenicity of samples treated with acid hydrolysates of Taoyuan 2 was positive correlated with Chl a. The TEAC percentage of CRCs and cyanidin were negative correlated with the antimutagenicity of TA98 while the TEAC percentage of CRCs, cyanidin and acid hydrolysates of Ipomoea batatas (L.)(red) were positive correlated with the antimutagenicity of TA100. In conclusion, the phytochemicals including chlorophyll and their derivatives, flavonols and anthocyanins, and acid hydrolysates of leafy sweet potato had antioxidative capacity and antimutagenicity, but there was no identical correlation. Keywords: Chlorophyll, Flavonols, Anthocyanins, Phytochemicals, Antioxidative, TEAC, Ames test

博士
國立臺灣大學
食品科技研究所
91
Antimutagenic activities of MRS cultures of several probiotic bifidobacteria against a potent mutagen, benzo[a]pyrene (B[a]P), were examined by the Ames test using Salmonella typhimurium TA100. These MRS cultures of bifidobacteria were neither toxic nor mutagenic. Most bifidobacterial cultures showed more than 50% inhibitory effect on B[a]P. B. bifidum CCRC14615, B. lactis Bb-12 and B. longum CCRC14634 showed significantly higher antimutagenicity than B. adolescentis CCRC14606, B. breve CCRC11846, and B. infantis CCRC14633 against B[a]P; however, the bioantimutagenic activities were lower. The cultures preincubated with mutagenic factors such as B[a]P and S9 mix displayed characteristic antimutagenic activities. Among these cultures, B. lactis exhibited the highest antimutagenicity. The cells of B. lactis and B. longum showed higher antimutagenic activities than their supernatants. The mutagenicity of B[a]P decreased as the reaction time of cells with B[a]P, S9 mix and B[a]P metabolites increased. According to this time-dependent inhibition study, the antimutagenicity of cells toward B[a]P was chiefly attributed to an interaction of cells with B[a]P and B[a]P metabolites. Crude cell walls of B. lactis and B. longum showed higher antimutagenic activities than heat-treated cells and cell extracts. Sequential preincubation studies showed that the main mechanism of antimutagenicity is action of desmutagenicity, involving the formation of chemical complexes between bifidobacteria, B[a]P, and B[a]P metabolites, and the inactivation of P450-mediated metabolism. The antimutagenic activities of bifidobacterial cells against B[a]P were also affected by the acidic and bile treatment mimicking gastrointestinal conditions. When bifidobacterial cells were treated at pH 2.0 for 3 h or 1% bile for 6 h, their antimutagenic activities against B[a]P were increased as compared to controls at pH 7.0 for 0 h. The viable counts substantially reduced to < 2.0 log cfu/ml after 3 h of incubation at pH 2.0, but the cells number at 1% bile for 6 h remained almost the same as original levels. After sequential acidic pH and bile treatments, B. lactis displayed the highest antimutagenic activity although its viable cells number was less than 2.0 log cfu/ml. B. infantis showed the highest survival counts (4.0 log cfu/ml), however its antimutagenic activity was less than B. lactis and B. longum. The antimutagenic activity of B. lactis against B[a]P was increased as pH values were increased from 2.0 to 7.0 and reaction time was extended from 1 to 3 h. However, antimutagenic activity was decreased as bile salts concentration was increased from 0.5 to 2.0%. The antimutagenic activity of B. lactis against B[a]P was increased in the presence of whole milk, semi-skimmed milk and skimmed milk. When B. lactis was preincubated with B[a]P and milk substrates in 1% bile for 6 h, its antimutagenic activity was increased to 99∼100%.

碩士
國立臺灣大學
食品科技研究所
91
The lactic acid bacteria (LABs) can improve nutrition bioavailability, stimulate the immune system, prevent diarrhea, decrease cholesterol level, maintain the mucosal integrity, and have the ability of antimutagenity/ antitumor. The LABs promote and support a beneficial balance of the autochthonous microbial population of GIT. Studies show probiotics can bind with mutagens-carcinogens; therefore, they can decrease the interaction of the mutagens and carcinogens when assimilating the products of probiotics. This study selected 9 strains from 15 strains LABs according to the antimutagenicity of cell suspensions. The 9 strains are Bifidobacteria bifidum CCRC 14615, B. infantis CCRC 14602, B. breve CCRC 11846, B. lactis Bb-12, Lactobacillus rhamnosus GG ATCC 53103, L. casei, L. acidophilus, Lactococcus lactis and Streptococcus salivarius subsp. thermophilus CCRC 14085. The LABs were treated against 4-nitroquinoline-N-oxide (4NQO) in different temp, pH, concentration and reaction time to understand the effect of various physical factors on the antimutagenicity and in advanced we isolated the crude cell walls and cell extracts to teat the two parts on the antimutagenicity against 4NQO. The antimutagenicities of 9 strains under heat shock treatment (42℃ for 15 min) and cold shock treatment (10℃ for 4 h) were 88∼96% and 66∼97% . After thermal death treatment (100℃ for 15 min), the antimutagenicities of probiotics were significanty less than 50% . The antimutagenicities of 9 strains under the pH 3.0 treatment were 54∼93% and the results showed this treatment increased the antimutagenicity of LABs. The concentration of LABs was 9 log CFU/ mL and it showed 2 to 25-fold of antimutageniciity against 4NQO to the 8 log CFU/ mL. When the reaction time of LABs and 4NQO were extened 20∼40 min, the antimutagenicities increased 14∼43% . Finally, the crude cell walls and cell extracts did not have the antimutagenicity and they did not bind with 4NQO, but the whole cell had 90∼94% antimutagenicity and bind with 80∼90% 4NQO.

Marigold flower (Tagetes erecta L.) produces lutein compounds which present biological activities such as antioxidant, antiinflammatory, antimutagenicity, and immunomodulatory effects. The study was to investigate the antioxidant activity of the lutein of T. erecta L. and the effect of lutein on the activity and phagocytic capacity of macrophage cells. The antioxidant screening was carried out using diphenyl picrylhydrazyl (DPPH), 2,2′-and-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging assay with serial concentrations and ferric-reducing antioxidant power (FRAP) method. For the observation of activity and phagocytic capacity of peritoneal macrophages, twenty-eight mice were used and divided into seven groups each comprising four replicates, i.e., Group (I) normal controls, mice were untreated (II) a negative control, mice were induced by Staphylococcus aureus (III) positive control, mice were induced by S. aureus and treatment of meniran extract (Phyllanthus niruri). The treatment group (IV-VII) mice were induced by S. aureus and treated crude lutein, respectively: 0.15 mg, 0.30 mg, 0.60 mg, and 0.90 mg. 20 g−1 of body weight. The lutein extracted from T. erecta shows an antioxidant activity against DPPH radical with an IC50 value of 53.58 μg.ml−1, while the antioxidant activity against ABTS has an IC50 value of 72.91 μg.ml−1. The antioxidant activity test results by the FRAP method at each lutein concentrations of 10, 25, 50, and 75 ppm were obtained respectively of 33, 88, 185.5, and 288.5 μmol Fe2+/g extract. The data were analyzed using one-way ANOVA and Duncan’s multiple range test (DMRT) after. The phagocytic activity was 45.5%; 54.75%; 57.50% and 67.0%, respectively, while the phagocytic capacity values ​​were 355; 519; 611 and 767 S. aureus bacterial cells per 50 macrophage cells. The lutein from marigolds (T. erecta L.) is capable of scavenging free radicals and reducing oxidants. Lutein can increase the activity and capacity of phagocytic of peritoneum macrophage cells in mice.