酶法辅助提取柑桔皮总黄酮的工艺优化研究

为了充分利用农业废弃物柑桔皮,进一步提高柑桔皮中总黄酮的提取效率,在单因素试验的基础上,采用中心组合设计对柑桔皮总黄酮纤维素酶法辅助提取工艺中的提取时间、提取温度、pH值和酶用量4因子的最优化组合进行了定量研究,建立并分析了各因子与黄酮得率关系的数学模型.结果表明:最佳的工艺条件为酶用量0.55%、提取时间123 min、pH值4.4、温度48℃.经试验验证,在此条件下,黄酮得率为(3.51±0.13)%,与理论计算值3.62%基本一致.说明回归模型能较好地预测柑桔皮中总黄酮的提取得率.     

Structure-activity relationships of flavonoids in the cellular antioxidant activity assay

Chemical antioxidant activity assays are used extensively to evaluate the potential bioactivity of plant foods and their phytochemical constituents, but they do not mimic the complexity of biological systems. The cellular antioxidant activity (CAA) activity assay was developed to be a more biologically relevant model to measure antioxidant activity. Structure-activity relationships of flavonoids have been determined in many chemistry antioxidant activity assays, and they vary with the protocols. The objective of this study was to determine structure-activity relationships of selected flavonoids in the CAA assay. The structures that conferred flavonoids with the most antioxidant activity in the CAA assay were a 3',4'- o-dihydroxyl group in the B-ring, a 2,3-double bond combined with a 4-keto group in the C-ring, and a 3-hydroxyl group. Isoflavones had no cellular antioxidant activity. Flavanols with a galloyl moiety had higher antioxidant activity than those without, and a B-ring 3',4',5'-trihydroxyl group further improved their efficacy. ORAC values for flavonoids were not related to their CAA values. Knowledge of structure-activity relationships in the CAA assay may be helpful in assessing potential in vivo antioxidant activity of flavonoids.     

Isolation and Caenorhabditis elegans lifespan assay of flavonoids from onion

The main flavonoids were isolated from three selected onion cultivars. Three phenolic compounds were obtained by reverse-phase HPLC, and their structures were elucidated by multiple NMR measurements. There were two known compounds, quercetin and quercetin 3'-O-β-D-glucopyranoside (Q3'G), and one novel compound, quercetin 3-O-β-D-glucopyranoside-(4→1)-β-d-glucopyranoside (Q3M), which was identified in onion for the first time. These flavonoids were found to be more abundant in the onion peel than in the flesh or core. Their antioxidative activities were tested using the DPPH method, and their antiaging activities were evaluated using a Caenorhabditis elegans lifespan assay. No direct correlation was found between antioxidative activity and antiaging activity. Quercetin showed the highest antioxidative activity, whereas Q3M showed the strongest antiaging activity among these flavonoids, which might be related to its high hydrophilicity.     

The biogeochemical cycling of Hg in forests: Alternative methods for quantifying total deposition and soil emission

A biogeochemical cycling approach to the Hg cycle is explored using existing data from the literature in order to begin to identify important processes that might be studied in detail and extrapolated to a regional scale. If it is assumed that all foliar Hg is taken up from the atmosphere, then estimates of total Hg deposition are rather easily made from measurements of leaf litterfall and throughfall. This assumption needs absolute verification before such an extrapolation can be made, however, because litterfall is a major Hg flux to the forest floor. Hg⁰ evolution from soils can be an important process and needs to be measured in more ecosystems. The diffusion model for soil gaseous efflux may be useful in this regard and deserves testing. There is a critical need for a systematic analysis of Hg cycles using common protocols so as to minimize artifacts associated with sampling methodology (i.e., soil Hg efflux). This should be done in several soil and forest types, given the high degree to which Hg transformations in and emissions from soils are dependent upon soil organic matter content, redox potential, and temperature.     

Interactions between flavonoids and proteins: effect on the total antioxidant capacity

Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect of the interaction between tea flavonoids and proteins on the antioxidant capacity was examined. Their separate and combined antioxidant capacities were measured with the Trolox equivalent antioxidant capacity (TEAC) assay. It was observed that the antioxidant capacity of several components of green and black tea with alpha-, beta-, and kappa-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction. This masking depends on both the protein and the flavonoid used. Components in green and black tea, which show the highest masking in combination with beta-casein, are epigallocatechin gallate and gallic acid. The results demonstrate that the matrix influences the efficacy of an antioxidant.     

Novel sediment source fingerprinting quantifying erosion-induced total nitrogen and total phosphorus outputs from an intensive agricultural catchment,North China

Intensive farming is a primary cause of increased sediment and associated nitrogen (N) and phosphorus (P) loads in surface water systems. Determining their contributing sources, pathways and loads present major challenges in the high-intensity agricultural catchments. Herein, we quantify the sediment sources and magnitude of sediment total N and total P from different sources using a novel application of compound-specific stable isotope (CSSI) and fallout radionuclides (FRNs) of ¹³⁷Cs and ²¹⁰Pbex in an intensive agricultural catchment in North China. Sediment sources from surface and sub-surface soils were estimated from FRNs fingerprint and accounted for 62 ± 7% and 38 ± 7% respectively, while surface soil from land uses that originated from hillslope were identified by CSSI fingerprint. Using a novel application of FRNs and CSSI sediment fingerprinting techniques, the dominant sediment source was derived from maize farmland (44 ± 0.1%), followed by channel bank (38 ± 7%). The sedimentation rate (13.55 ± 0.30 t ha⁻¹ yr⁻¹) was quantified by the ¹³⁷Cs cores (0–60 cm) at the outlet of this catchment. The total N and total P in sediment were both mostly derived from maize farmland and least from channel banks. The channel banks are significant sediment sources but contribute little to the input of sediment N and P for eutrophication. It implies that chemically-applied farmlands are the main hotspots for catchment erosion control and pollution prevention. The novel application of FRNs and CSSI techniques cost-effectively quantified sediment N and P loads from different sources with a single visit to the catchment, enabling rapid assessment for optimizing soil conservation strategies and land management practices. Keywords: Sediment sources, Land use, N and P loads, Compound-specific stable isotope, Fallout radionuclides.     

大孔树脂精制苦荞总黄酮工艺条件的优化研究

为了研究DM-2型大孔树脂精制苦荞总黄酮的优化工艺条件，以树脂对苦荞总黄酮的吸附率、解吸率为评价指标，采用响应曲面法(response surface methodology，RSM)建立树脂法精制苦荞总黄酮的二次多项回归模型方程，对精制苦荞总黄酮吸附解吸工艺条件进行优化。结果表明吸附最佳工艺条件为吸附速率2 mL/min，pH值2，上样液浓度2 mg/mL。解吸最佳工艺条件：乙醇浓度71.05%，pH值8.76，解吸速率1 mL/min。该方法简单可行，精制效果好，适合于工业化生产。     

Quantitative colorimetric assay for total protein applied to the red wine Pinot noir

A standard method for assaying protein in red wine is currently lacking. The method described here is based on protein precipitation followed by dye binding quantification. Improvements over existing approaches include minimal sample processing prior to protein precipitation with cold trichloroacetic acid/acetone and quantification based on absorbance relative to a commercially available standard representative of proteins likely to be found in wine, the yeast mannoprotein invertase. The precipitation method shortened preparation time relative to currently published methods and the mannoprotein standard yielded values comparable to those obtained by micro-Kjeldahl analysis. The assay was used to measure protein in 48 Pinot noir wines from 6 to 32 years old. The protein content of these wines was found to range from 50 to 102 mg/L with a mean value of 70 mg/L. The availability of a simple and relatively rapid procedure for assaying protein provides a practical tool to quantify a wine component that has been overlooked in routine analyses of red wines.     

大孔吸附树脂一次性分离杜仲叶中杜仲总苷和杜仲黄酮的研究

本文就9种大孔树脂吸附分离杜仲总苷和杜仲黄酮的工艺进行了研究，结果表明：一次性分离杜仲总苷(富含绿原酸、桃叶珊瑚苷等活性物质)和杜仲黄酮的适宜的大孔树脂为XDA-1和X-5。适宜的吸附解吸条件分别为：上柱液pH 6，吸附时间应大于10 h；XDA-1树脂用40%乙醇洗脱杜仲总苷，用80%的乙醇洗脱黄酮；X-5树脂用30%的乙醇洗脱杜仲总苷，用70%的乙醇洗脱黄酮。两种树脂的分离效果分别为：XDA-1分离杜仲总苷(其中绿原酸含量为14.23%，桃叶珊瑚苷含量为7.69%)粗品得率为8.01%，杜仲总黄酮粗品得率为4.76%，含量为15.82%；X-5分离杜仲总苷(其中绿原酸含量为15.39%，桃叶珊瑚苷含量为9.07%)粗品得率为7.35%，杜仲总黄酮粗品得率为5.11%，含量为16.15%。     

Sensitive spectrophotometric assay for 3-hydroxy-substituted flavonoids, based on their binding with molybdenum, antimony, or bismuth

A sensitive spectrophotometric assay has been developed for flavonoids based on their binding with molybdenum, antimony, or bismuth. Acetylation of the hydroxyl group of flavonoids abolished metal binding, thus suggesting a direct role of the hydroxyl groups. From a comparison of several related flavonoids differing in the position of hydroxyl substitutions, the hydroxyl group at position 3 was found to be an important requirement for the formation of a yellow complex. This flavonoid metal complex showed that a specific and significant bathochromic shift in the visible spectrum of the native flavonoid and the corresponding lambda(max) value was used for the colorimetric assays with different metal salts. The molybdenum complex was found to yield higher absorbance compared to antimony and bismuth complexes of various flavonoids. The present method offers a sensitive assay in the 5-25 nM range for these flavonoids and gave comparable results with HPLC quantitative determination.     

Analytical procedure for quantifying five compounds suspected as possible contaminants in recycled paper/paperboard for food packaging

Because contaminants in recycled paper intended for food packaging could be a risk to public health, analytical methods are needed to identify and quantify residues of concern in paper/paperboard. The U. S. Food and Drug Administration is considering development of a guidance document for testing levels of contaminants that might be retained through paper recycling processes. An analytical procedure was developed using paper spiked with suspected contaminants at concentrations of 1-50 ppm in the paper. Benzophenone, dimethyl phthalate, anthracene, methyl stearate, and pentachlorophenol were introduced by soaking the paper in a solution in acetone at 25 degrees C for 24 h; the paper was removed and dried by evaporating the solvent with nitrogen. The model contaminant residues were extracted from the paper using ultrasonication and quantified by GC with flame ionization and electron capture detectors. Recoveries from the spiked paper were 80-109% with a repeatability of +/-4%. The method was also used to analyze commercial recycled paperboard to validate its applicability.     

Simplifying the evaporation method for quantifying soil hydraulic properties

Since about 25 years, we have measured the unsaturated hydraulic conductivity function and water retention curve with the evaporation method of more than 1500 mineral and organic soils samples. From this data base, 104 representative samples of varying texture and dry bulk density were selected and the temporal dynamics of the basic measured values (mass or water loss, respectively, and tension change over time) was analyzed. With the exception of sand, water loss per time interval was constant in all other mineral and organic soils during the measuring time in the tension range between 0 and about 60 kPa. In sands, the nonlinear water loss over time by evaporation can be described by a quadratic function with high accuracy (r² > 0.99). For all other soils, a linear function is sufficient (r² > 0.99). The use of evaporation functions enables extending weighing intervals. This reduces costs for the measuring equipment and increases the effectiveness of the method while maintaining the same quality of unsaturated hydraulic conductivity and water retention functions. It was confirmed that measuring with two tensiometers is sufficient for accurate hydraulic conductivity and water retention function. Reducing evaporation by screening the sample surface helps to decrease hydraulic gradients and keeps tension distributions approximately linear with depth. This is recommended in particular for clayey soils.     

Tools for quantifying N2O emissions from agroecosystems

The importance of constraining the global budget of nitrous oxide (N₂O) has been well established. The current global estimate of the contribution of N₂O to total anthropogenic greenhouse gas emissions from agriculture is about 69%. Considerable progress has been made over the past few years in developing tools for quantifying the emissions from agricultural sources, at the local and field scale (i.e., chamber and tower-based measurements) as well as at the landscape and regional levels (i.e., aircraft-based measurement and modelling). However, aggregating these emissions over space and time remains a challenge because of the high degree of temporal and spatial variability. Emissions of N₂O in temperate climate are largely event driven, e.g., in Eastern Canada, large emissions are observed right after snowmelt. The average emissions during the snowmelt period vary considerably, reflecting the influence of many controlling factors. Cumulative emissions reported here range from 0.05 kg N₂O-N ha⁻¹ in Western Canada to 1.26 kg N₂O-N ha⁻¹ in Eastern Canada, values that reflect differences in climatic zones and fertilizer management practices. This paper describes the tools for refining the global N₂O budget and provides examples of measurements at various scales. Tower-based and aircraft measurement platforms provide good data for quantifying the variability associated with the measurements. Chamber-based methods lack the temporal and spatial resolution required to follow the event driven nature of N₂O fluxes but provide valuable information for evaluating management practices. The model DeNitrification and DeComposition is an example of a technique to estimate N₂O emissions when no data is available.     

Improved method for quantifying the avicide 3-chloro-p-toluidine hydrochloride in bird tissues using a deuterated surrogate/GC/MS method

A method using a deuterated surrogate of the avicide 3-chloro-p-toluidine hydrochloride (CPTH) was developed to quantify the CPTH residues in the gastrointestinal (GI) tract and breast muscle tissues in birds collected in CPTH-baited sunflower and rice fields. This method increased the range of a previous surrogate/gas chromatography/mass spectroscopy method from 0-2 to 0-20 microg/g in tissue samples and greatly simplified the extraction procedure. The modified method also sought to increase recoveries over a range of matrix effects introduced by analyzing tissues from birds collected in the field, where the GI tract contents would be affected by varying diet. The new method was used to determine the CPTH concentration in GI tract samples fortified with CPTH-treated rice bait to simulate the consumption of varying amounts of treated bait by two nontargeted bird species, pigeon (Columbia livia) and house sparrow (Passer domesticus). The new method was then used to examine the CPTH concentrations in the gizzard contents of the targeted bird species, red-winged black bird (Agelaius phoeniceus) and brown-headed cowbird (Molothrus ater), that were collected after feeding at a treated bait site. The method proved sufficiently sensitive to quantify CPTH in the breast muscle tissues and the gizzard contents of red-winged blackbirds and brown-headed cowbirds during an operational baiting program. The levels of CPTH determined for these birds in both tissue samples were determined to be highly correlated. The appearance of CPTH in the breast muscle tissue immediately after feeding was not anticipated. The potential secondary hazard posed by the targeted birds to potential scavengers and predators was also evaluated.     

Unfermented rooibos tea: quantitative characterization of flavonoids by HPLC-UV and determination of the total antioxidant activity

Unfermented rooibos originates from the leaves and the stems of the indigenous South African plant, Aspalathus linearis, and it has been reported to have a higher content of flavonoids compared to that of fermented rooibos. The HPLC/UV method developed in our laboratory for the analysis of the fermented rooibos was applied to the quantitative characterization of the major flavonoids present in the unfermented rooibos. Main compounds determined were aspalathin (49.92 +/- 0.80 mg/g), isoorientin (3.57 +/- 0.18 mg/g), orientin (2.336 +/- 0. 049 mg/g), and rutin (1.69 +/- 0.14 mg/g), followed in order by isovitexin, vitexin, isoquercitrin and hyperoside, quercetin, luteolin and chrysoeryol. The identity of detected flavonoids was confirmed by comparing their retention times and UV spectra with those of corresponding standards. The total antioxidant activity (TAA) of the tea infusions was measured by the ABTS*+ radical cation decolorization assay. The TAA of unfermented rooibos (0.8 Trolox meq/g) resulted 2-fold higher than that of the fermented rooibos. When compared with different water infusions of Camellia sinensis (green and black tea), this TAA value was about 50% lower.     

Methodology for quantifying residues of chlorhexidine in raw dairy milk

A residue method was developed as part of a pharmacokinetics study to determine the elimination of chlorhexidine in raw milk after intramammary infusion into dairy cows affected with bovine mastitis. The developed liquid/liquid and solid-phase extraction procedures effectively reduced sources of milk product interferences in the final extract. By optimizing mobile-phase pH buffer/acetonitrile gradient conditions and employing an end-capped reverse-phase polar embedded-phase chromatographic column, excellent peak resolution was achieved without the additional need of mobile-phase amine modifiers or ion-pairing reagents. The combined cleanup and chromatographic method steps reported herein were sensitive and reliable for determining the pharmacokinetic elimination of chlorhexidine following intramammary infusion. The residue method was found to be rugged with a lower detection limit of 0.1 ppm.     

十字花科黑腐病菌RNA提取与质量鉴定

十字花科黑腐病菌,学名野油菜黄单胞菌野油菜致病变种（Xanthomonas campestris pv.campestris,简称Xcc）,是一类γ-变形菌纲的革兰氏阴性细菌,能在世界范围内侵染十字花科植物,给农业生产造成重大损失。RNA是分子生物学的主要研究对象之一,提取高质量的RNA是研究十字花科黑腐病菌基因表达调控机理的基础。由于Xcc能产生大量胞外多糖及黄单胞色素,且细菌RNA半衰期较短,目前尚缺少一种大量提取Xcc高质量RNA的有效方法。该研究在参考多种RNA提取方法的基础上,建立了一种简单、有效的适合十字花科黑腐病菌RNA的提取方法。得到的RNA样品经过紫外分光光度法和甲醛变性凝胶电泳检测,证实为完整、均一的总RNA,达到了表达谱分析及cDNA文库构建的要求。     

Multivariate calibration for the determination of total azadirachtin-related limonoids and simple terpenoids in neem extracts using vanillin assay

Two-component and multivariate calibration techniques were developed for the simultaneous quantification of total azadirachtin-related limonoids (AZRL) and simple terpenoids (ST) in neem extracts using vanillin assay. A mathematical modeling method was also developed to aid in the analysis of the spectra and to simplify the calculations. The mathematical models were used in a two-component calibration (using azadirachtin and limonene as standards) for samples containing mainly limonoids and terpenoids (such as neem seed kernel extracts). However, for the extracts from other parts of neem, such as neem leaf, a multivariate calibration was necessary to eliminate the possible interference from phenolics and other components in order to obtain the accurate content of AZRL and ST. It was demonstrated that the accuracy of the vanillin assay in predicting the content of azadirachtin in a model mixture containing limonene (25% w/w) can be improved from 50% overestimation to 95% accuracy using the two-component calibration, while predicting the content of limonene with 98% accuracy. Both calibration techniques were applied to estimate the content of AZRL and ST in different parts of the neem plant. The results of this study indicated that the relative content of limonoids was much higher than that of the terpenoids in all parts of the neem plant studied.     

Simplified method for quantifying the hydraulic properties of shrinking soils

In general, soils and their pore size systems are assumed to be rigid during the loss of water on drying. In reality, it is not the case for most soils, especially for soils with high quantities of clay or organic matter. As a result of shrinking, there are changes in the bulk density, the porosity, the pore size distribution, and the hydraulic properties of these soils. Currently, only a few methods enable the shrinkage behavior of soil samples to be determined while simultaneously quantifying the corresponding soil hydraulic properties. Either the methods need proprietary software for data processing, the equipment used is expensive or the calculation of the hydraulic properties is executed by inverse modelling. The aim of this study was to develop an alternative, simplified method for the simultaneous and automatic determination of the soil hydraulic properties, taking shrinkage into account. The HYPROP® evaporative device was combined with a circumference meter. A preliminary investigation found that the diameter of the cylindrical samples used for the HYPROP decreased linearly during evaporation from the bottom to the top. To sum up, recording the perimeter change in the middle position of the sample during drying‐out, together with the corresponding tension and water content, was sufficient to determine the hydraulic functions taking shrinkage into account. Measurements are presented for 6 samples which are different in texture and geological origin. The maximum shrinkage (19.5% by vol. between saturation and 5,000 hPa) was measured in the peat samples. The minimum shrinkage was quantified at 0.68% by vol. for the silty loam samples from Chile. The advantages of the method presented are: (1) the water retention curve and the hydraulic conductivity function can be determined simultaneously in the range between saturation and close to the wilting point, at a high resolution and taking into consideration shrinkage; (2) the method and device are simple and robust to use; (3) little time is required for measurement, between 3 and at most 10 d; (4) the functions are described over the whole tension range, using more than 100 user‐defined data points; (5) the evaluation of the volumetric soil water content measurement in shrinking soils is improved; and (6) common data models can be fitted to the hydraulic data as well as to the shrinkage data.