小麦花药蛋白质组双向电泳技术体系的优化

以小麦单核期花药为材料,比较了两种不同蛋白质提取方法TCA/丙酮法和酚提取法及不同的蛋白质裂解液组成对双向电泳的影响,并在蛋白质上样量和SDS-PAGE胶浓度等方面也进行了探索与优化。结果表明,采用TCA/丙酮提取法比用酚提取法提取的蛋白质所得的2-DE胶图谱上蛋白质点数增加,图谱背景也比较清晰;样品溶解于含有硫脲和TBP的蛋白质裂解液Ⅱ中,可显著提高蛋白质的溶解性,在2-DE图谱上可分辨出554个蛋白质点,比用蛋白质裂解液Ⅰ提取的蛋白多39个点。以TCA-丙酮法提取小麦花药组织中的蛋白,用蛋白质裂解液Ⅱ[7mol/L尿素、2mol/L硫脲、4%CHAPS、2%TBP、65mmol/L DTT、0.2%载体两性电解质(其中0.1%pH 3~10,0.1%pH4~6)]溶解蛋白,以pH4~7线性17cm的IPG胶条进行双向凝胶电泳,在上样量为800μg,13%SDS-PAGE胶浓度下,蛋白质得到了更好的分离,2-DE图谱上可分辨出631个蛋白点,图谱质量最佳。优化后的双向电泳技术体系,适合于小麦花药全蛋白质的双向电泳分析。     

Optimization of extraction of apple aroma by dynamic headspace and influence of saliva on extraction of volatiles

The dynamic headspace procedure of aroma extraction was optimized on Gala apples (Malus domestica). Two parameters affecting the extractability of compounds were studied: temperature and purge time. The influence of artificial saliva was also included. An increase in purge time and temperature caused an increase in the extraction of volatiles from the apple matrix. The optimum point of extraction was 40 degrees C and 70 min of purge. The study also showed that the addition of saliva influenced the extraction of volatile compounds, but this effect was different from one compound to another. To verify that the headspace extracts presented a global odor representativeness of fresh apple under these conditions of extraction, eight assessors compared the odor of extracts with fresh fruit odor for three different cultivars. With regard to the sensory profiles of extracts, the optimal conditions of extraction were suitable for extraction of volatile compounds, even if cooked apple odor appeared in some extracts. The similarity marks of extracts were low but acceptable.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率（浓度）高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Antioxidant and antiproliferative activities of common fruits

Consumption of fruits and vegetables has been associated with reduced risk of chronic diseases such as cardiovascular disease and cancer. Phytochemicals, especially phenolics, in fruits and vegetables are suggested to be the major bioactive compounds for the health benefits. However, the phenolic contents and their antioxidant activities in fruits and vegetables were underestimated in the literature, because bound phenolics were not included. This study was designed to investigate the profiles of total phenolics, including both soluble free and bound forms in common fruits, by applying solvent extraction, base digestion, and solid-phase extraction methods. Cranberry had the highest total phenolic content, followed by apple, red grape, strawberry, pineapple, banana, peach, lemon, orange, pear, and grapefruit. Total antioxidant activity was measured using the TOSC assay. Cranberry had the highest total antioxidant activity (177.0 +/- 4.3 micromol of vitamin C equiv/g of fruit), followed by apple, red grape, strawberry, peach, lemon, pear, banana, orange, grapefruit, and pineapple. Antiproliferation activities were also studied in vitro using HepG(2) human liver-cancer cells, and cranberry showed the highest inhibitory effect with an EC(50) of 14.5 +/- 0.5 mg/mL, followed by lemon, apple, strawberry, red grape, banana, grapefruit, and peach. A bioactivity index (BI) for dietary cancer prevention is proposed to provide a new alternative biomarker for future epidemiological studies in dietary cancer prevention and health promotion.     

Latent and active polyphenol oxidase (PPO) in red clover (Trifolium pratense) and use of a low PPO mutant to study the role of PPO in proteolysis reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme's own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.     

不同提取方法及上样量对牡丹试管苗茎基部蛋白双向电泳的影响

为建立一套适合于牡丹试管苗茎基部蛋白的双向电泳技术,以便更好地利用蛋白质组技术研究牡丹试管苗不定根的发生机理,本研究比较了三种不同蛋白质提取方法对双向电泳结果的影响,并在蛋白质上样量方面进行了比较。结果表明,乙酸铵/甲醇酚提取法所得2-DE图谱的蛋白点很少,仅检测到45个,且较模糊,有明显的拖尾现象,分辨率很低;乙醇/乙醚丙酮法所得的蛋白点也较少（101个）,较模糊,且横竖纹干扰较大;三氯乙酸/丙酮法所得蛋白点数较多,可检测到434个清晰的蛋白点,且形状规则,重复性好,适合后续分析,操作也较为简便。用三氯乙酸/丙酮法提取蛋白,采用800μg、1000μg和1200μg三个不同的上样量进行双向电泳,在上样量为1200μg时（IPGpH3～10,24cm）,蛋白质在12%SDS-PAGE胶上得到了较好的分离,在2-DE图谱上可分辨出562个蛋白点。因此,三氯乙酸/丙酮法是较适合于牡丹试管苗茎基部蛋白质提取的方法,1200μg是较为合适的上样量。     

Determination of low levels of perchlorate in lettuce and spinach using ion chromatography-electrospray ionization mass spectrometry (IC-ESI-MS)

A sample preparation method was developed to quantify environmentally relevant (low micrograms per liter) concentrations of perchlorate (ClO4(-)) in leafy vegetables using IC-ESI-MS. Lettuce and spinach were macerated, centrifuged, and filtered, and the aqueous extracts were rendered water-clear using a one-step solid-phase extraction method. Total time for extraction and sample preparation was 6 h. Ion suppression was demonstrated and was likely due to unknown organics still present in the extract solution after cleanup. However, this interference was readily eliminated using a Cl(18)O4(-) internal standard at 1 microg/L in all standards and samples. Hydroponically grown perchlorate-free butterhead lettuce was spiked to either 10.3 or 37.7 microg/kg of fresh weight (FW), and recoveries were between 91 and 98% and between 93 and 101%, respectively. Five types of lettuce and spinach from a local grocery store were then analyzed; they contained from 0.6 to 6.4 microg/kg of FW. Spike recoveries using the store-bought samples ranged from 89 to 100%. The method detection limit for perchlorate in plant extracts is 40 ng/L, and the corresponding minimum reporting limit is 200 ng/L or 0.8 microg/kg of FW.     

Detection and quantification of apple adulteration in diluted and sulfited strawberry and raspberry purées using visible and near-infrared spectroscopy

Adulteration of sulfited strawberry and raspberry purées by apple is a commercial problem. Strawberry (n = 31) and raspberry (n = 30) purées were prepared from Irish-grown fruit and adulterated at levels of 10-75% w/w using cooking apples. Visible and near-infrared transflectance spectra were recorded using a 0.1 mm sample thickness. Classification and quantification models were developed using raw and scatter-corrected and/or derivatized spectral data. Classification as pure strawberry or raspberry was attempted using soft independent modeling of class analogy. The best models used spectral data in the wavelength ranges 400-1098 nm (strawberry) and 750-1098 nm (raspberry) and produced total correct classification rates of 75% (strawberry) and 95% (raspberry). Quantification of apple content was performed using partial least-squares regression. Lowest predictive errors obtained were 11.3% (raspberry) and 9.0% (strawberry). These results were obtained using spectral data in the wavelength ranges 400-1880 and 1100-1880 nm, respectively. These results suggest minimum detection levels of apple in soft fruit purées of approximately 25 and 20% w/w for raspberry and strawberry, respectively.     

猪肌肉组织双向电泳分离条件的建立及常见问题分析

为建立和优化猪肌肉组织蛋白质双向电泳技术,从提取方法、加样量、IPG胶条的选择、SDS-PAGE胶浓度、染色方法等多个方面对双向电泳分离效果进行比较研究.结果显示,液氮研磨+超声破碎法提取猪肌肉组织蛋白效果优于液氮研磨法,前者细胞破碎彻底蛋白质溶解性好且核酸污染少,图谱质量较好;18cm pH 4～7的IPG胶条分离效果比pH 3～10非线性IPG胶条好;对于银染,18 cm pH 4～7的IPG胶条150μg上样量比较适宜;同一肌肉样品的三块胶的重复性可达70％以上;同时,对双向电泳过程中的典型问题作了详尽分析.研究结果表明,通过优化的双向电泳条件获得了较高分辨率和较好重复性的猪肌肉组织双向电泳图谱,可用于后续蛋白质组学分析.     

Effect of ascorbic acid and dehydration on concentrations of total phenolics, antioxidant capacity, anthocyanins, and color in fruits

The purpose of this investigation was to report on the total phenolics, anthocyanins, and oxygen radical absorbance capacity (ORAC) of strawberry, peach, and apple, the influence of dehydration and ascorbic acid treatments on the levels of these compounds, and the effect of these treatments on fruit color. Results showed that fresh strawberry had the highest levels for total phenolics [5317.9 mg of chlorogenic acid equivalents (CAE)/kg], whereas lower levels were found in fresh apple and peach (3392.1 and 1973.1 mg of CAE/kg, respectively), and for anthocyanins (138.8 mg/kg), whereas lower levels were found in fresh apple and peaches (11.0 and 18.9 mg/kg, respectively; fresh strawberry had an ORAC value of 62.9 mM/kg Trolox equivalents. The fresh apple and peach were found to have ORAC values of 14.7 and 11.4 mM/kg of Trolox equivalents, respectively. The color values indicated that the addition of 0.1% ascorbic acid increased the lightness (L) and decreased the redness (a) and yellowness (b) color values of fresh strawberry, peach, and apple, sliced samples, and the puree made from them. Also, results showed that dehydration is a good method to keep the concentrations of total phenolics and anthocyanins and ORAC values at high levels.     

Variation in ascorbic acid and oxalate levels in the fruit of Actinidia chinensis tissues and genotypes

Ascorbic acid and total oxalate were measured in fruit from six genotypes of Actinidia chinensis. Ascorbic acid was separated from oxalate in fruit extracts by HPLC and quantified from absorbance at 245 nm, whereas oxalate was measured enzymatically in the HPLC eluate. Levels of whole fruit mean ascorbic acid in the different genotypes ranged from 98 to 163 mg/100 g of fresh weight (FW), whereas mean oxalate varied between 18 and 45 mg/100 g of FW. Ascorbic acid was highest in the inner and outer pericarp, whereas oxalate was concentrated in the skin, inner pericarp, and seed. Essentially no ascorbic acid was found in the seed. Each tissue clustered separately when the tissue ascorbic acid and oxalate data were normalized to the whole fruit level of ascorbic acid and oxalate in that genotype and plotted against each other, suggesting that oxalate is not a sink for excess ascorbic acid but that oxalate formation is regulated.     

Comparative study of health properties and nutritional value of durian, mangosteen, and snake fruit: experiments in vitro and in vivo

In vitro and in vivo studies of the health and nutritional properties of durian (Durio zibethinus Murr.) were compared with snake fruit (Salacca edulis Reinw.) and mangosteen (Garcinia mangostana). Dietary fibers, minerals, and trace metals were comparable. Total polyphenols (mg of GAE/100 g of FW) and flavonoids (85.1+/-6.1) were significantly higher (p<0.05) than in snake fruit (217.1+/-13.2 (mg of CE/100 g of FW)), durian (309.7+/-19.3 and 61.2+/-4.9), and mangosteen (190.3+/-12.1 and 54.1+/-3.8). Antioxidant activity (microM TE/100 g of FW) of durian measured by DPPH and ABTS assays (228.2+/-13.4 and 2016.3+/-81.1) was significantly higher (p<0.05) than in snake fruit (110.4+/-7.9 and 1507.5+/-70.1) and mangosteen (79.1+/-5.9 and 1268.6+/-62.3). HPLC/DAD analysis of durian (microg/100 g of FW) showed that quercetin (1214.23+/-116.7) was present at levels three times that of caffeic acid, and twice as high as p-coumaric and cinnamic acids. The correlation coefficients between the bioactive compounds of fruits and their antioxidant activities were high (R2=0.99). Male Wistar rats (25) were divided into five dietary groups: the control group was fed the basal diet (BD); in addition to BD, the cholesterol (Chol) group was supplemented with 1% of Chol; the diets of the Chol/Durian, Chol/Snake, and Chol/Mangosteen groups were supplemanted with 5% of these fruits, respectively. It was found that diets supplemented with durian, and to a lesser degree with snake fruit and mangosteen, significantly hindered the rise in plasma lipids and the decrease in antioxidant activity. The nutritional values were comparably high. In conclusion, it could be suggested that inclusion of studied tropical fruits, especially durian, in known disease-preventing diets could be beneficial.     

Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2)

Consumption of fruits and vegetables, which are rich in polyphenols, has been associated with a reduced risk of chronic diseases such as cancer. Dietary polyphenols have antioxidant and antiproliferative properties that might explain their beneficial effect on cancer prevention. The aim of this study was to investigate the effects of different pure polyphenols [quercetin, chlorogenic acid, and (-)-epicatechin] and natural fruit extracts (strawberry and plum) on viability or apoptosis of human hepatoma HepG2 cells. The treatment of cells for 18 h with quercetin and fruit extracts reduced cell viability in a dose-dependent manner; however, chlorogenic acid and (-)-epicatechin had no prominent effects on the cell death rate. Similarly, quercetin and strawberry and plum extracts, rather than chlorogenic acid and (-)-epicatechin, induced apoptosis in HepG2 cells. Moreover, quercetin and fruit extracts arrested the G1 phase in the cell cycle progression prior to apoptosis. Quercetin and strawberry and plum extracts may induce apoptosis and contribute to a reduced cell viability in HepG2 cells.     

Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.     

Neutral cleanup procedure for 2,3,7,8-tetrachlorodibenzo-p-dioxin residues in bovine fat and milk.

A neutral cleanup method for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in milk and animal tissue was developed involving solvent extraction and liquid adsorption chromatography on magnesia-Celite 545, alumina, and Florisil. Cleaned up extracts were subjected to dual-ion analysis in a direct probe high resolution mass spectrometer, interfaced to a multi-channel analyzer for signal averaging. Calibration experiments were carried out with bovine milk and beef fat samples containing added TCDD. The 37CI isotopic isomer of TCDD was added as an internal standard. The response was linear for concentrations in the ppt range, with recoveries about 80%. Milk from a cow fed TCDD was cleaned up by the neutral procedure or, alternatively, a base-acid extraction procedure. The TCDD recoveries for both procedures were essentially the same. Recoveries of TCDD from liver samples of a rat given 14C-TCDD intraperitoneally, subjected to neutral cleanup and radioactive counting, were about 70%.     

Simplified extraction of ergovaline and peramine for analysis of tissue distribution in endophyte-infected grass tillers

An improved extraction and cleanup procedure for quantitative analysis of ergovaline in Neotyphodium-infected grass tissues by high-performance liquid chromatography was developed, utilizing aqueous 2-propanol-lactic acid as extraction solvent. Losses of sample material and time requirements were significantly reduced, handling procedures simplified, and ergovaline and internal standard ergotamine recovered with similar efficiency from extracts. Analyses can be carried out on very small amounts (2-5 mg of dry weight) of samples and another endophyte-alkaloid, peramine, determined in the same extracts. Calibration curves with 2-propanol-lactic acid were linear over the range 0.004-0.938 microM ergovaline (= 2-500 ng/mL) in extracts, corresponding to 0.04-10 microg/g in samples. The distribution of ergovaline in the plant was extremely heterogeneous, indicating low in-planta mobility and strong regulation of accumulation by the internal plant environment. In contrast, peramine was much more uniformly distributed. These results clearly demonstrate very large differences in the tissue specificities of ergovaline and peramine.     

Effects of Extraction Conditions on the Content of Anthocyanins and Bioelements in Berry Fruit Extracts

Abstract

Two methods for extracting anthocyanins and bioelements [calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe)] from black chokeberry, black currant, strawberry, and bilberry fruits were compared in this study. Correlations between the content of anthocyanins and bioelements in fruit extracts were determined using method A [extraction with hydrochloric acid (HCl)] and method B [extraction with water (H₂O)]. Relationships between the content of anthocyanins and particular nutrients, as well as between Mg and Ca and between Zn and Fe, in fruit extracts obtained by method A and by method B were also established.

The mean content of anthocyanins and bioelements (Ca, Mg, Zn, Fe) was the highest in black chokeberry fruits, regardless of the extraction method used. Bilberry fruits had the lowest nutrient content, despite a high anthocyanin content. The mean content of anthocyanins and bioelements was higher in fruit extracts obtained by method A than method B. The coefficients used to recalculate method A into method B were anthocyanins, 5.1; Ca, 1.1; Mg, 1.2; and Zn, 2.2. The statistical analysis showed that the effect of a given extraction method on the content of anthocyanins and bioelements (except for Ca) in fruit extracts depended on fruit species. The correlations between the content of anthocyanins, Ca, Mg and Zn in fruit extracts obtained by method A were found to be positive and highly significant, in comparison with method B. There was no correlation between the content of anthocyanins and particular nutrients in fruit extracts obtained by method A and by method B. The Mg content of fruit extracts was highly significantly higher than the Ca content, irrespective of the extraction method applied.     

Quantitation of polyphenols in different apple varieties

Forty-one apple samples, representing eight of the most widely cultivated varieties in western Europe, were collected in Trentino, Italy. Samples were extracted from fresh fruit with a mixture of acetone/water to achieve a good extraction of polyphenols, including proanthocyanidin oligomers which were analyzed by normal-phase HPLC. Up to 20 compounds including catechin, epicatechin, B2 procyanidin, hydroxycinnamates, flavonols, anthocyanins, and dihydrochalcones were analyzed by reversed-phase HPLC and LC-MS. Total polyphenol content was independently measured with an optimized Folin-Ciocalteu assay. The mean content of total polyphenols lay between 66.2 and 211.9 mg/100 g of FW depending on the variety. With chromatographic analysis, it was possible to explain the whole amount of total polyphenols measured by the FC assay. Flavanols (catechin and proanthocyanidins) are the major class of apple polyphenols (71-90%), followed by hydroxycinnamates (4-18%), flavonols (1-11%), dihydrochalcones (2-6%), and in red apples anthocyanins (1-3%).     

Use of SDS to Extract Sorghum and Maize Proteins for Free Zone Capillary Electrophoresis (FZCE) Analysis

Two different extraction methods for extracting sorghum (Sorghum bicolor L. Moench.) storage proteins for free zone capillary electrophoresis (FZCE) analysis were compared. A traditional solvent based on 60% t‐butanol was compared with a pH 10 borate buffer containing the anionic detergent SDS followed by precipitation of nonkafirins using 60% t‐butanol. FZCE analysis of both types of extracts showed identical patterns, despite the fact that the SDS should have given all proteins equal charge‐to‐mass ratios. This methodology was also successfully applied to maize proteins. The use of t‐butanol to precipitate nonkafirins, combined with electrophoresis at low pH, is thought to have removed the SDS from the storage proteins. The SDS extraction procedure produced more stable extracts for FZCE analysis. These extracts could also be used directly for SDS capillary electrophoresis (SDS‐CE) separations. Kafirins from 15 genotypes were extracted with this procedure and analyzed by FZCE and SDS‐CE. Resolution of the kafirins by FZCE was much higher than the SDS‐CE, demonstrating that the kafirin proteins possessed a high level of charge density variability within a relatively small molecular size distribution. Two distinct groups of α‐kafirins could be seen in the FZCE electropherograms.     

棉花胚珠蛋白三氯乙酸丙酮法与酚抽提法的比较分析

以亚洲棉（Gossypium arboreum）DPL971及DPL972初生壁合成期的胚珠为材料，通过蛋白质产率、纯度测定以及凝胶电泳图谱对三氯乙酸丙酮沉淀法和酚抽提法进行了比较分析，结果表明：1.两种方法均能获得大量的可溶性蛋白，但是酚抽提法获得的蛋白产率高、质量好、种类多；2.两种方法提取的蛋白种类有所不同，但酚抽提法在酸性区域以及对高分子量和低分子量蛋白的提取有更好的表现；3.酚抽提法可以更加有效地清除干扰物质，获得高质量的凝胶图谱，适合棉胚珠组织蛋白的提取。实验为棉纤维发育期蛋白质组学的研究提供了参考，同时对包含多种干扰物质的其它非模式植物蛋白提取也具有一定的借鉴意义。