Flavonoid content in leaf extracts of the fig (Ficus carica L.), carob (Ceratonia siliqua L.) and pistachio (Pistacia lentiscus L.)

The total flavonoid content of leaf extracts (70% ethanol) from fig (Ficus carica L.), carob (Ceratonia siliqua L.) and pistachio (Pistacia lentiscus L.) plants were determined by using reverse phase high-performance liquid chromatography (HPLC)-and analyzed by UV/VIS array and electrospray ionization (ESI)-mass spectrometry (MS) detectors. As a base for comparison, flavonoid type and level were also determined in extracts from soybeans and grape seeds. It was found that the major flavonoids in Ficus are quercetin and luteolin, with a total of 631 and 681 mg/kg extract, respectively. In Ceratonia leaves, nine different flavonoids were detected. The major one was myricetin (1486 mg/kg extract), with a similar level in Pistacia (1331 mg/kg extract, myricetin). The present study is the first to report the presence of the isoflavone genistein in the Pistacia leaf, which was discovered to consist of about a third of the genistein level detected in soybean.     

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.     

Salvage synthesis of purine nucleotides by Helicobacter pylori

G.L. MENDZ, B.M. JIMENEZ, S.L. HAZELL, A.M. GERO AND W.J. O'SULLIVAN. 1994. The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy. The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine. Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed. The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established. The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected. Phosphorylase activities were not observed with any of the nucleosides. Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine. No nucleoside kinase activities were observed with any of the nucleosides. The presence of adenylate kinase was established, but no guanylate kinase activity was observed. The study provided evidence for the presence in H. pylori of salvage pathways for the biosynthesis of purine nucleotides.     

Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium.

Plasma membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine.     

Labeling of nucleosides with fluorescamine and detection by spectrofluorometer for End Stage Renal Disease

Nucleosides are characterized as biomarkers in AIDS, Alzheimer, tumor, breast cancer and various malignant diseases. In the present work a direct method for the detection of nucleosides (adenosine, cytidine, uridine and guanosine) from urine samples has been developed. Nucleosides represent the extent of damage in genetic material, analysis of nucleosides by ultrasonic assisted microextraction effectively eliminates the interfering constituent of urine. This has made it a highly selective and sensitive method to analyze the nucleosides with a lower limit of detection 0.220 μmol/L and Limit of quantitation 0.660 μmol/L. The method has been validated with good linearity and correlation of coefficients of the calibration curves was higher than 0.997. The coefficients were in the range of 0.11–16.92% (inter-day) and 0.38–16.43% (intra-day), respectively.     

HEARTWOOD FLAVONOIDS AND THE INFRAGENERIC RELATIONSHIPS OF RHUS (ANACARDIACEAE)

Heartwood flavonoids of 23 taxa of Rhus L. were surveyed in order to assess infrageneric relationships and classification. Fourteen flavonoids and two coumarins were detected in the heartwood extracts. All taxa were characterized by a flavonoid complement consisting of eight 5-deoxyflavonoids involving several aglycone classes (e.g., flavonols, flavones, aurones, chalcones and dihydroflavonols) and the aurone rengasin. None of the 5-hydroxyl analogs of the 5-deoxyflavonoids were detected in the heartwood extracts. Infraspecific flavonoid patterns were uniform in different populations, although the presence of 3′,4′-dihydroxyflavone 4′-O-β-glucoside varied in some taxa. Taxa of Rhus subgenus Rhus consistently differed from all taxa of Rhus subgenus Lobadium in lacking glycosides of fisetin, butein and 3′,4′-dihydroxyflavone. The major evolutionary trend in the heartwood flavonoids of Rhus appears to be accumulation of simple mono- or diglucosides. Data from heartwood flavonoids suggest that Rhus be treated as consisting of two subgenera (Rhus and Lobadium) and that subgenus Lobadium be divided into three sections.     

人为扰动对乌拉尔甘草不同部位甘草酸与总黄酮含量的影响

人为扰动对甘草不同部位甘草酸含量和总黄酮含量均有较大的影响。即使是轻度的人为扰动 (土壤翻松 1次 )也会导致野生甘草的甘草酸含量明显下降 ,尤其对地下根茎 (兼有运输和储存功能 )中的甘草酸的积累影响最大。重度扰动栽培甘草各部位的甘草酸含量均较低 ,相对而言黄酮类物质的积累速率高于甘草酸 ,表明土壤扰动因素对甘草酸的形成与积累的影响大于总黄酮的形成与积累。无扰动野生甘草和轻度扰动野生甘草总黄酮含量从地上到地下呈下降趋势 ,而重度扰动栽培甘草的叶和不定根各有一个含量较高的部位 ,据此推断叶和不定根 (含毛状根 )是黄酮类物质的主要产生部位 ;具有输导功能的地上茎、复叶柄中总黄酮含量较低且波动较大。人为扰动对不同土壤深度甘草主根中甘草酸含量和甘草总黄酮含量的变化规律影响较小。无扰动野生甘草和轻度扰动野生甘草主根在 1.0～ 2 .0 m深度甘草酸含量分布较高是对该生境不同土壤深度长期适应的结果 ,而重度扰动栽培甘草主根可能尚未达到相应的土壤深度 ,因而表现为 1.0 m以下深层土壤中甘草酸含量较高。总之 ,旨在改善甘草生长条件的人为扰动对甘草酸和总黄酮的积累具有消极影响 ,适当的胁迫环境条件对提高药用植物的品质有益     

Behaviour of the pathogen surrogates Listeria innocua and Clostridium sporogenes during production of parsley in fields fertilized with contaminated amendments

The survival and transfer of Listeria innocua and Clostridium sporogenes, used as surrogates of the food borne pathogens Listeria monocytogenes and Clostridium botulinum, were quantitatively assessed under field conditions. In the soil, spores of C. sporogenes declined by less than 0.7 log cycles within 16 months and were detected on parsley leaves throughout the experiment. In contrast, L. innocua in the soil declined by 7 log cycles in 90 days and was detected on leaves in low numbers (>0.04 MPN g(-1)) during the first 30 days. Rates of decline in soil were similar in the laboratory at 20 degrees C for two strains of L. innocua and L. monocytogenes ; and in the field for L. innocua over two different years. L. innocua survived better in winter, indicating an important influence of temperature. The major cause of transfer of L. innocua from soil to parsley leaves was splashing due to rain and irrigation. As few as 1 CFU g(-1) Listeria in soil led to contamination of parsley leaves. Internalisation of Listeria through parsley roots was not observed. Under the conditions of soil and climate studied, a delay of 90 days between application of potentially contaminated fertilizer and harvest should be sufficient to eliminate L. monocytogenes.     

Prevalence of Listeria in soil

One hundred thirty soil samples from agricultural fields and animal-inhabited areas were examined for the presence of Listeria. The microorganism was identified in 23 (17.7%) samples. L. monocytogenes was detected in 7 samples (5.4%), L. ivanovii in 2 (1.5%), L. innocua in 10 (7.7%) and L. welshimeri in 4 samples (3.1%). Prevalence of Listeria in soil from agricultural fields (17.5%) did not differ considerably from that in the soil and animal-inhabited area (18.0%), but L. ivanovii was isolated only from the latter source. Frequency of occurrence of different species of Listeria differed from place to place.     

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography–electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC™) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 μmol/L (UMP and AMP) to 1.3 μmol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.     

Negative allelopathic effects of rutin and quercetin on fourteen soil and enteric microbes

The flavonoids rutin and quercetin were assayed separately and in combination for antimicrobial activity againts 14 soil and enteric microbes. Microbial inhibition was observed when rutin and quercetin (pH 5.7 and 5.8 respectively) solutions were not adjusted to pH 7.0. At neutral pH no antimicrobial activity was observed. The addition of flavonoids at neutral pH did not effect the bacterial growth curve of Agrobacterium tumefaciens or the exponential growth of Escherichia coli. Both bacteria grew to turbidity from a loop-inoculum in medium containing rutin or quercetin as the sole carbon source. Furthermore, medium containing rutin and/or quercetin did not decrease either the number or relative frequency of several microbes from open field and forest floor soil samples. It is concluded that microbial inhibition has not played a major evolutionary role in the production of flavonoids in plants.     

基于灰色关联的苗药红禾麻总黄酮含量影响因子分析

采用Al Cl3比色法对苗药红禾麻42个居群中的总黄酮含量进行分析测定,并用土壤养分测定仪测定对应产地土壤中的铵态氮、速效磷、有效钾、p H值和水分,通过向距产地较近的气象部门查询和以全球卫星定位系统GPS、海拔表等测定地理气候因子,结合42个居群红禾麻ISSR遗传多样性分析结果,运用灰色关联度分析法对红禾麻不同种质资源药材中总黄酮的含量与各影响因子进行相关性分析。结果表明:42个红禾麻不同种质资源药材总黄酮含量为0.42%~2.16%,平均加样回收率为97.86%,RSD为1.5%;环境因子和遗传因子中与总黄酮含量关联度较大的因素分别为无霜期和Shannon信息指数I,而各影响因子与总黄酮含量的关联度中,以Shannon信息指数I(r=1.03)最大,土壤p H(r=0.49)最小,表明遗传因子对红禾麻药材总黄酮含量的影响大于环境因子。该研究结果为红禾麻药材优良种质资源筛选及野生变家种研究提供了依据。     

NO对银杏悬浮细胞生长及黄酮类物质合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(NO)的供体,向银杏悬浮细胞培养液中加入不同浓度的SNP,研究外源NO对银杏悬浮细胞生长状况、过氧化氢酶(CAT)活性、苯丙氨酸解氨酶(PAL)活性和黄酮类物质生物合成的影响.结果表明,低浓度SNP有利于银杏悬浮细胞生长,而高浓度SNP可以促进黄酮类物质的合成.银杏悬浮细胞在添加0.5和10 mmol/L SNP的培养基中培养16 d时,细胞干重分别为对照组的134%和73%;在添加10 mmol/L SNP的培养基中培养20 d时,细胞中黄酮类物质的含量为对照组的136%.同时,10 mmol/L SNP促进银杏悬浮细胞PAL和CAT活性显著升高.NO专一性淬灭剂c-PITO(carboxyl phenyltetramethylimidazoleoxide)抑制SNP对银杏悬浮细胞生长、CAT活性、PAL活性和黄酮类物质含量的促进作用,说明SNP是通过其分解产物NO影响细胞生长和黄酮类物质的合成.根据这些结果推测,NO可能通过触发银杏悬浮细胞的防卫反应,激活了细胞中黄酮类物质的生物合成途径.     

不同群落类型柔毛淫羊藿总黄酮和淫羊藿苷含量及其与土壤因子的关系

2009年8月,采用高效液相色谱法和紫外分光光度法测定了唐家河自然保护区次生落叶阔叶林红桦群落(群落Ⅰ)、常绿落叶阔叶混交林细叶青冈群落(群落Ⅱ)和常绿阔叶林油樟群落(群落Ⅲ)下生长的柔毛淫羊藿各器官的总黄酮和淫羊藿苷含量,分析其与土壤因子的关系结果表明：柔毛淫羊藿叶片中总黄酮和淫羊藿苷含量最高、茎中最低;群落Ⅰ的柔毛淫羊藿总黄酮和淫羊藿苷含量［(5.32±0.23)%和(0.47±0.05)%］均显著高于群落Ⅱ［(4.06±0.03)%和(0.32±0.01)%］和群落Ⅲ［(4.15±0.07)%和(0.28±0.09)%］;土壤氮含量和pH值对柔毛淫羊藿的总黄酮和淫羊藿苷含量影响较大,柔毛淫羊藿总黄酮和淫羊藿苷含量与土壤全氮和碱解氮呈显著负相关(P<0.05),与土壤pH值呈极显著正相关(P<0.01).土壤较低的氮供应水平和较高的土壤酸度可能使群落Ⅰ柔毛淫羊藿的总黄酮和淫羊藿苷含量增加.     

Antioxidant properties of complexes of flavonoids with metal ions

The formation of complexes of metal ions with the flavonoids quercetin (L1), rutin (L2), galangin (L3) and catechin (L4) has been investigated by UV-visible spectroscopy. The antioxidant activities of the compounds were evaluated by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicalscavenging method. In this work, we have shown that the complexed flavonoids are much more effective free radical scavengers than the free flavonoids. We suggest that the higher antioxidant activity of the complexes is due to the acquisition of additional superoxide dismutating centers. Radical scavenging activities of the compounds were also investigated from an electrochemical point of view. There is a relationship between the logarithm of the antioxidant activity (represented by EC50) and the oxidation potential. The synergic effect of the complexes and ascorbic acid were studied by [13C]-NMR analyses. The results show that ascorbic acid can protect flavonoids from oxidative degradation, and reveal antioxidant synergies between ascorbic acid and the compounds.     

灵芝孢子粉中核苷类成分分析

本文利用高效液相色谱方法（HPLC）同时对灵芝孢子粉中的15种核苷类成分的含量进行测定。采用Ultimate AQ-C₁₈（4.6mm×250mm,5μm）色谱柱,以甲醇和水为流动相进行梯度洗脱,流速1.0mL/min,检测波长259nm,柱温30℃,进样量10μL。方法学考察结果表明,该方法准确度高,稳定性、精密度、重现性好,适用于灵芝孢子粉中核苷类成分的测定分析。运用建立的方法对不同破壁时间、不同采收时期龙泉、奉化、大别山、黄山4个产区的灵芝孢子粉中的15种核苷类成分的含量进行测定。结果表明破壁处理对灵芝孢子粉中核苷类成分提取率的影响不大,不同产地的灵芝孢子粉中核苷类成分的组成和含量具有显著差异,且孢子粉中的核苷含量随着产粉时间的延长有所增加。各待测样品中均含有胞嘧啶、尿苷、腺嘌呤、鸟苷、腺苷等成分,其中尿苷、鸟苷、腺苷3种核苷的含量占总量的比例在待测样品中均达到70%以上,为灵芝孢子粉中的主要核苷类成分。     

提取工艺和植物生长调节剂对罗布麻愈伤组织中黄酮含量的影响

以罗布麻愈伤组织粉末为材,在单因素实验的基础上,利用响应曲面法对罗布麻愈伤组织中黄酮的提取工艺进行优化。响应曲面分析结果表明,提取试剂和提取温度对提取的黄酮含量存在显著影响。通过响应曲面分析得到罗布麻愈伤组织中黄酮提取的最佳条件为:提取试剂为70%甲醇,物料比1∶40,提取时间为4 h,提取温度为70℃。培养并比较了30种不同植物生长调节剂浓度与配比诱导100 d生长的愈伤组织中黄酮的含量,结果得出MB+KT(1.0 mg/L)+NAA(0.2 mg/L)上培养约100 d的愈伤组织中黄酮含量最高,为73.90mg/g。测定愈伤组织培养30 d内黄酮的积累动态,探明从培养的愈伤组织提取黄酮的最佳时段。通过优化提取工艺和筛选最佳植物生长调节剂浓度与配比,运用组织培养技术提高了罗布麻愈伤组织中黄酮含量。     

Deoxynucleoside composition of DNAs and modified nucleoside composition of tRNAs determined at nanomole sensitivity by reversed-phase liquid chromatography

Selected purified tRNA and DNA samples were digested by standard enzymatic methods, and the nucleosides were separated by high-pressure liquid chromatography on reversedphase columns. Nanomole sensitivity was obtained by use of an integrator. The nucleosides were detected at wavelengths near their uv-absorption maxima, including 220 nm for dihydrouridine, and 330 nm for 4-thiouridine. Recovery values for the individual nucleosides were in the range of 94–100%. The nucleoside composition of the DNA and tRNA digests were in accurate and precise agreement with published values.