Eicosanoids, which mediate various physiological and pathophysiological processes, are mainly formed from C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA,20:4n-6) and eicosapentaenoic acid (EPA,20:5n - 3) through cyclooxygenases (COX), lipoxygenases (LOXs) and cytochrome P450 (CYPs) pathways, including prostaglandins (PGs), thromboxanes (TXs) and the lipoxin/leukotriene family of eicosanoids such as hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs). Vast knowledge of eicosanoids stems from works in mammals. Lipid signaling that complicates our understanding of fatty acid signaling is highly complex and fine-tuned in mammal species. Fortunately, the small genetic model Drosophila melanogaster is considered to be an ideal model to investigate the flexible nature of eicosanoids signaling pathways. However, it seems that Drosophila possess a special lipid metabolic system which is different from mammals. Thus, before studying the physiological mechanism of eicosanoids by using Drosophila, it is necessary to clarify its metabolic characteristics to C20 PUFAs based on the detection of eicosanoids in Drosophila. Therefore, this study is aimed to develop a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of eicosanoids in Drosophila.Fifteen metabolites of AA and EPA produced by human in COX, LOX and CYP pathway were chosen, and each pathway contained 1 or 2 metabolites as delegates to ensure the representative of the method. These eicosanoids included PGF2α, PGE2, PGF3α, PGE3, 15 (S)-HETE, LTB4, 15 (S)-HEPE,11 (12)-EET,20-HETE,17 (18)-EpETE,17,18-DiHETE,15 (S)-HpEPE,15 (S)-HpETE, PGH2 and 5 (S)-HpETE, and PGE2-d4, 15 (S)-HETE-d8 and 20-HETE-d6 were used as internal standards. Samples were prepared by solid phase extraction and separated on an EndeavorsilTM C18 column (100 mm × 2.1 mm,1.8 μm). The analytes were detected by using multiple reaction monitoring (MRM) in a negative electrospray ion mode. The HPLC-MS/MS method to analyze 15 selected eicosanoids in Drosophila qualitatively and quantitatively was established by optimizing the sample pretreatment and the detection conditions.It was found that the recoveries were significantly influenced by the pH of sample adjusted by 1 mol/L sodium acetate buffer containing 5% methanol, and the optimized pH was at 6. The response values of analytes separated on a mobile phase of ultrapure water with 0.1% formic acid-acetonitrile were higher than these of ultrapure water with 0.1% formic acid-methanol, ultrapure water-acetonitrile or ultrapure water-methanol. To reduce the matrix interference, the blank Drosophila matrix was used to prepare the standard working solution. Obtained results showed that the calibration curves were of good linearity for the 15 metabolites in the range of 2.5-100 ng/mL with the correlation coefficient (r) of 0.991. The limits of detection and quantitation were about 0.1-2.6 ng/g and 0.3-8.7 ng/g, respectively. Spiked recovery experiments showed that both recoveries (89.3%-111.5%) and relative standard deviations (1.0%-15.0%) met the requirements of analytical methods.In conclusion, our study has established a simple, specific and sensitive method that suitable for the determination of eicosanoids in Drosophila which serves an approach to clarify the metabolic characteristics of Drosophila to C20 PUFAs.

基于单纯形法求解目标的线性规划模型,开发了替代数学建模的调和油配料软件系统.该软件由原料油脂肪酸数据库和运算系统2个模块组成,数据库用于存储各种原料油的SFA、MUFA、PUFA、n-3 PUFA和n-6 PUFA百分率;运算系统可根据所输入的SFA:MUFA:PUFA和n-3 PUFA:n-6 PUFA比例指令给出所选原料油的投料量.然后以经气相色谱分析的山茶油、亚麻籽油、红花籽油和菜籽油为原料,通过该软件运算输出符合浙江、江苏和上海居民营养需求的3种调和油的原料油投料量,在通过Matlab软件建模运算验证后,取原料油进行调和油调配.经分析验证,所配调和油的脂肪酸比例与期望值相符.该软件使用便捷,既适合于食用油企业新产品开发,也适合于为居民家庭提供个性化营养服务.

Drosophila melanogaster has been a widely used as a model system for its powerful genetic tools. However, it remains to be illustrated if Drosophila can be used to examine the biochemical and physiological metabolism of eicosanoids. Thus, the analysis on the metabolism of C20 polyunsaturated fatty acids (PUFA) in Drosophila was implemented with high performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Fatty acid (FA) analysis of the whole body, head, and thorax-abdomen in Drosophila showed C20 PUFA could only be found in Drosophila fed diets supplemented with eicosapentaenoic acid (EPA) and arachidonic acid (ARA), but not in Drosophila fed base diets. The C20 PUFA were found in abundance in the head. Drosophila fed ARA- and EPA-supplemented diets yielded 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid [15(S)-HETE] and 15S-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid [15(S)-HEPE], respectively, while other sampled eicosanoids could not be detected. Similar results were obtained by incubating fly tissue supplemented with ARA or EPA. Furthermore, a genome sequence scan indicated that no gene encoding the key enzymes synthesizing eicosanoids were found in Drosophila. These findings demonstrate that Drosophila may possess a special lipid metabolic system, which is different from mammals.

Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.

Summary Royal jelly(RJ)is the exclusive food for the Western honeybee(Apis mellifera)queen throughout its lifespan and the primary food for the honeybee larvae during the first three days of life,which is secreted by hypopharyngeal and mandibular glands of nurse honey bees.The chemical composition of RJ is very complicate, while the protein components have been found to play critical roles in RJ.Around 50% of dry RJ is made up of proteins which could be divided into two parts,water-soluble proteins and water-insoluble proteins.The water-soluble proteins,accounting for 82% to 90% of the total RJ proteins,are named major royal jelly proteins(MRJPs) which belong to the same family composed of nine members.It has been proved that MRJPs have effects such as anti-aging,immunoregulation and anti-fatigue,et al.In addition,MRJPs,especially MRJP1 are authenticated as the critical constituents to reveal the freshness of RJ and will be hopeful as one new functional component in future. In previous studies,various methods were developed for isolation of MRJPs,which included centrifugation, alkali extraction acid precipitation and column chromatography.However,a series of obstacles,such as low purity, solvent contamination and low productivity limited the application of those methods in honeybee industry. Ultrafiltration as a novel bio-separation technology has many advantages,such as simple operation,easy scale-up, and hence has a huge potentiality for large-scale isolation of bio-macromolecules.Our effort was to obtain the separation technology of MRJPs by using ultrafiltration and provide new knowledge for the industrial application of RJ process in future. Since the molecular masses of MRJPs are in the range of 4.9×104 8.7×104,the poly-sulfone hollow fiber membrane component used in the ultrafiltration equipment was comprised of two types with different molecular mass cut-off membranes.The ultrafiltration membrane with a 1 × 105 molecular mass cut-off was employed to remove macromolecular impurities,whereas the 4.9 × 104 cut-off membrane was employed to retain most of MRJPs.In this study,disposals were taken to separate soluble proteins,MRJPs from RJ by ultrafiltration.The control process included pH,ion intensity and ratio of water to material.In order to obtain the optimal conditions to extract MRJPs,orthogonal test was employed during investigation on experimental factors. The results showed that the optimal condition was as follows,ratio of water to material 5∶1 ,ion intensity 0.5 mol/L and pH 7.0.Under the optimal condition,82.63% of MRJPs was extracted from fresh RJ.It was determined that the lyophilized powder of MRJPs contained 91.00% soluble protein,0.01% moisture,2.00% 10-HDA and 15.22% total sugar.Besides,the by-products contained 10-HDA and total sugar were recycled,and the recycling rate of the two components reached 3.60% and 2.11%,respectively. This method with many advantages,such as no solvent pollution and fully utilization of 10-HDA,has provided a new technology for MRJPs separation by first using ultrafiltration in deep process of RJ,and will be hopeful for large scale application in RJ process industry.

A rapid spectrophotometric method was proposed to determine borax in foods,which was based on that azomethine-H reacted with salicylaldehyde to form methylenimine-H,and the latter reacted with boron to form a yellow complex.The yellow complex could be quantized by spectropotometric method.The results showed that the optimum absorption wavelength of this yellow complex was 413 nm.The optimum reaction conditions were azomethine-H volume of 2.5 mL,salicylaldehyde volume of 4.0 mL,room temperature(25 ℃),time of 15 min,termination solution of 1.2 mL 1.0N HCl.The linear relationship between absorbance and borax content was kept in the range of 0 μg/mL~5.5 μg/mL with relationship index of R2=0.995 1.The detection limit of this proposed method was 0.27μg/mL and the average recovery rate of 106.7%.