Determination of Veterinary Drug Residues in Animal-derived Foods by Liquid Chromatography-Mass Spectrometry

[Objectives]This study was conducted to establish a rapid and effective method for simultaneous extraction of 54 kinds of veterinary drug residues in animal-derived food, including sulfonamides, quinolones, tetracyclines, malachite greens, penicillins, nitroimidazoles, tranquilizers and macrolides, by HPLC-MS. [Methods] The samples were extracted with 80% acetonitrile water(containing 0.1% formic acid), combined with QuEChERS extraction technology and C18 and PSA purification, analyzed by high performance liquid chromatography-mass spectrometry, and quantified by external standard method. The target substances were analyzed on ZORBAX Eclipse C18 chromatographic column using 0.2% formic acid water and 0.2% methanol as mobile phases. The gradient elution mode was used for chromatographic separation and multiple reaction detection. [Results] In the linear range of 0.5-50.0 ng/ml, the linear relationship of the 54 kinds of veterinary drug residues was good, with correlation coefficients(r~2) greater than 0.995, and the detection limits ranged from 0.30 to 1.00 μg/kg. The results showed that the recovery ranged from 75.4% to 118.2% when different matrixes were added for recovery. [Conclusions] This method is simple, efficient, accurate, stable, and highly operable. It is applicable to simultaneous batch screening of veterinary drug residues in animal-derived food, and has high practical application value.

Structural Characterization and Biological Activity of Oligopeptides from Isochrysis zhanjiangensis:Based on Quantum Mechanics,Molecular Docking and in Vitro Experiments

DAPTMGY(DTY)is an oligopeptide derived from marine microalgae with proven potential to combat oxidative stress in previous research.The composition,ordering,and active sites of amino acids play a key role in activity studies and are also the research trends in recent years.As an oligopeptide with a molecular weight of less than 1000 Da,DTY is of great significance to explore the active site and structure-activity relationship.This study used quantum mechanics to optimize DTY’s structure and predict the active site through molecular orbits,energy,and charge.In addition,an LPS-treated HUVEC cell was established as an oxidative-stress model.DTY could reduce mitochondrial oxidative stress and inhibit ROS production by enhancing the antioxidant enzymes SOD,GPX,and HO-1.Moreover,it was confirmed to inhibit inflammation and apoptosis through the NF-κB and MAPK signaling pathways.Lastly,the correlation of the oligopeptide DTY’s active site and antioxidative-stress activity was verified by molecular docking,showing that hydrogen bonding is the main force,which was also the main factor for antioxidant activity.

Bat-derived oligopeptide LE6 inhibits the contact-kinin pathway and harbors anti-thromboinflammation and stroke potential

Thrombosis and inflammation are primary contributors to the onset and progression of ischemic stroke.The contact-kinin pathway,initiated by plasma kallikrein(PK)and activated factor XII(FXIIa),functions bidirectionally with the coagulation and inflammation cascades,providing a novel target for therapeutic drug development in ischemic stroke.In this study,we identified a bat-derived oligopeptide from Myotis myotis(Borkhausen,1797),designated LE6(Leu-Ser-Glu-Glu-Pro-Glu,702 Da),with considerable potential in stroke therapy due to its effects on the contact kinin pathway.Notably,LE6 demonstrated significant inhibitory effects on PK and FXIIa,with inhibition constants of 43.97μmol/L and 6.37μmol/L,respectively.In vitro analyses revealed that LE6 prolonged plasma recalcification time and activated partial thromboplastin time.In murine models,LE6 effectively inhibited carrageenan-induced mouse tail thrombosis,FeCl3-induced carotid artery thrombosis,and photochemically induced intracerebral thrombosis.Furthermore,LE6 significantly decreased inflammation and stroke injury in transient middle cerebral artery occlusion models.Notably,the low toxicity,hemolytic activity,and bleeding risk of LE6,along with its synthetic simplicity,underscore its clinical applicability.In conclusion,as an inhibitor of FXIIa and PK,LE6 offers potential therapeutic benefits in stroke treatment by mitigating inflammation and preventing thrombus formation.

Structure-activity Relationships of Vasorelaxing Effect of Polyarginine Oligopeptides

Based on the EDRF(endothelium derived relaxing factor)-like effects for polyarginine Arg-Arg-oH was selected as the lead compound and its derivatives Arg-Arg- OCH_3.Arg Arg-Arg-OH,HO-ArgCOCH_2CH_2COArg-OH,HO-ArgCOCH_2COArg-OH,were synthesized.These substances showed on bioassay various degrees of vasorelaxant activities. With protection for the C-terminal of Arg-Arg-OH with a methyl ester.the vasorelaxing ac- tivitv was decreased.In contrast.when the N-terminal of Arg-OH was protected with mal- onic acid or butane diacid.the biological activites were lower than those of Arg-Arg-OH due to the lowered metabolic rate.With protection of N-terminal of Arg-Arg-OH with L-Arg residue.Arg-Arg-Arg-OH was obtained,which showed a vasorelaxing activity better than that of Arg-Arg-OH.The bioactivities observed on the Wister's rats for the former com- pound become the experimental basis for prodrug design of EDRF.

Isoflavone Aglycones and Oligopeptides in Lactic Acid-Fermented Soy Milk Differentially Regulate Lipid Metabolism-Related Gene Expression

We previously investigated the physiological effect of an ethanol extract of fermented soymilk on rats and clarified that this extract modulated their hepatic lipid metabolism. Although the soy isoflavones and oligopeptides are representative functional components of the ethanol extract, it remained unclear whether these substances share a role in lipid metabolism modulation. Therefore, we attempted to clarify the effects of isoflavones and oligopeptides in lactic acid-fermented soymilk on lipid metabolism-related gene expression in rats and HepG2 cells. The fermented soymilk extract had a higher isoflavone aglycone content than the soymilk extract. Sevenweek-old male Sprague-Dawley rats were fed an AIN-93G diet, a diet plus 70% soymilk ethanol extract, or a diet plus 70% fermented soymilk ethanol extract for 5 weeks. Although both the soymilk and fermented soymilk ethanol extracts did not significantly affect plasma and hepatic lipid profiles, the expression levels of the genes encoding CYP7a1 and SREBP-2 were significantly upregulated in the livers of rats fed with the fermented soymilk extract. Whereas isoflavone aglycones upregulated CYP7a1-encoding gene expression in HepG2, oligopeptides in soymilk and fermented soymilk downregulated this expression. Oligopeptides in fermented soymilk downregulated the expression stronger than that observed with soymilk. On the other hand, no significant change in FAS expression was observed in the livers of rats fed the fermented soymilk extract. Although isoflavone aglycones did not affect FAS expression in HepG2 cells, oligopeptides in fermented soymilk downregulated FAS expression. The downregulation of FAS with oligopeptides from fermented soymilk was stronger than that from soymilk. In the present animal experiment, the effect on reduction of fat synthesis was not found because of insufficient amount of peptides derived from digestion of soy protein. These results suggest that isoflavone aglycones increase CYP7a1 gene expression, whereas oligopeptides decrease FAS expression. Isoflavone glycosides and proteins in soymilk were converted to isoflavone aglycones and oligopeptides by lactic acid fermentation, respectively, and these functional components independently improved the lipid metabolism. In the present study, it was found that isoflavone aglycones and oligopeptides in fermented soymilk differentially regulate hepatic lipid metabolism-related gene expression. Therefore, the consumption of fermented soymilk containing isoflavone aglycones and soy oligopeptides might prevent dyslipidemia more effectively than that of any other soy food. Fermented soymilk is a superior functional food modulating lipid metabolism.

A PRELIMINARY STUDY OF THE WATER—SOLUBLE OLIGOPEPTIDES AND AMINO ACIDS OF GINSENG

γ-Amino butyric acid(GABA)and several γ-glutamyl oligopeptides were isolated and identified from water-extract of ginseng.

Extrahepatic bile duct reconstruction in pigs with heterogenous animal-derived artificial bile ducts:A preliminary experience

BACKGROUND Extrahepatic biliary duct injury(BDI)remains a complicated issue for surgeons.Although several approaches have been explored to address this problem,the high incidence of complications affects postoperative recovery.As a nonimmunogenic scaffold,an animal-derived artificial bile duct(ada-BD)could replace the defect,providing good physiological conditions for the regeneration of autologous bile duct structures without changing the original anatomical and physiologic conditions.AIM To evaluate the long-term feasibility of a novel heterogenous ada-BD for treating extrahepatic BDI in pigs.METHODS Eight pigs were randomly divided into two groups in the study.The animal injury model was developed with an approximately 2 cm segmental defect of various parts of the common bile duct(CBD)for all pigs.A 2 cm long novel heterogenous animal-derived bile duct was used to repair this segmental defect(group A,ada-BD-to-duodenum anastomosis to repair the distal CBD defect;group B,ada-BD-to-CBD anastomosis to repair the intermedial CBD defect).The endpoint for observation was 6 mo(group A)and 12 mo(group B)after the operation.Liver function was regularly tested.Animals were euthanized at the above endpoints.Histological analysis was carried out to assess the efficacy of the repair.RESULTS The median operative time was 2.45 h(2-3 h),with a median anastomosis time of 60.5 min(55-73 min).All experimental animals survived until the endpoints for observation.The liver function was almost regular.Histologic analysis indicated a marked biliary epithelial layer covering the neo-bile duct and regeneration of the submucosal connective tissue and smooth muscle without significant signs of immune rejection.In comparison,the submucosal connective tissue was more regular and thicker in group B than in group A,and there was superior integrity of the regeneration of the biliary epithelial layer.Despite the advantages of the regeneration of the bile duct smooth muscle observed in group A,the effect on the patency of the ada-BD grafts in group B was not confirmed by macroscopic assessment and cholangiography.CONCLUSION This approach appears to be feasible for repairing a CBD defect with an ada-BD.A large sample study is needed to confirm the durability and safety of these preliminary results.

OLIGOPEPTIDES FROM THE ROOTS OF ASTER TATARICUS L. f.

Two new oligopeptides, Asterin A and B, have been isolated from the roots of Aster tataricus L. f., and their structures were elucidated on the basis of spectroscop- ic, chemical and enzymatic methods.

Determination of Sodium Pentachlorophenoxide Residues in Animal-derived Foods by Ultra-performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS)

[Objectives]This study was conducted to establish an ultra-high performance liquid chromatography-tandem mass spectrometry for the rapid extraction of sodium pentachlorophenoxide from animal-derived food.[Methods]The samples were extracted with an acetonitrile water solution(8∶2),0.1 mol/L hydrochloric acid and a purification extraction bag with shaking.Centrifugation was performed to obtain supernatants,which were added to purification tubes containing PSA and C_(18) for purification,and then filtered with membranes for determination.Each test solution was separated by a ZORBAX Eclipse plus C_(18) column with acetonitrile and 5 mmol/L ammonium acetate as mobile phases,and determined with electrospray ionization and multiple reaction monitoring.[Results]The method had good linearity in the concentration range of 1.0-50 ng/ml,and the correlation coefficient was 0.9997.The limit of detection was 0.25μg/kg and the limit of quantification was 0.75μg/kg.The recovery was between 87.4%and 112.5%,and the RSD%was between 0.5%and 10.0%.[Conclusions]The method has simple operation and high sensitivity,and is suitable for trace detection of sodium pentachlorophenoxide in large quantities of animal-derived food.

Determination of Sym-triazine Residues in Animal-derived Food by Liquid Chromatography-High Resolution Mass Spectrometry

[Objective]The research aimed to establish a liquid chromatography-high-resolution mass spectrometry for detecting residues of diclazuril,toltrazuril and its metabolites in animal-derived food.[Method]The samples were pre-treated by QuEChERS technology and detected by Exactive liquid chromatography-high resolution mass spectrometry.The liquid chromatography-high-resolution mass spectrometry for detecting the residues of diclazuril,toltrazuril,toltrazuril sulfone and toltrazuril sulfoxide in animal-derived food was established.[Result]Diclazuril,toltrazuril,toltrazuril sulfone and toltrazuril sulfoxide showed a good linear correlation in the range of 2.0-50.0μg/L（r^2〉0.99）.The average recoveries of these four compounds in bovine power at the spiked levels of 2,10 and 20μg/kg were 74.5%-90.1%,and the relative standard deviations（RSD）were 15.4%-17.5%.[Conclusion]This method was stable and reliable,and could be used to detect diclazuril,toltrazuril,toltrazuril sulfone and toltrazuril sulfoxide in animal-derived food.

Impact of China’s sanitary and phytosanitary measures on animal-derived food imports

This paper takes the imports of animal-derived food as the example,and uses the 6-digit-code HS data of the CEPII BACI database ranging from 2002 to 2018 in an extended gravity model to study the impact of the sanitary and phytosanitary(SPS)measures implemented in China on the import trade and trade margins of animal-derived food.The results show that(1)China’s SPS measures have not significantly restricted the import trade of animal-derived food but rather enhanced import trade by alleviating negative externalities and information asymmetry;(2)SPS measures have positive effects on the extensive,price,and quantity margins for developed countries;while(3)SPS measures have negative effects on the quantity margin for developing countries.Thus,China’s SPS measures have not yet constituted a trade restriction.

HPLC of Amino Acids and Oligopeptides by Pre-Column Fluorescence Derivatization with 9-Acridine Formyl Chloride

A highly sensitive HPLC method for the detection of amino acids and oligopeptides with 9-acridine formyl chloride by pre-column fluorescence derivatization has been developed. Glycine, glycylglycine, histidine, triglycine and glutathione were separated on a reversed-phase C-18 column with methanol-water-triethylamine eluent, derivatization and chromatographic conditions were optimized. The five derivatives were eluted in 28 min with a good reproducibility. Linear range of the calibration graph was 0.08-260 nmol/ml(-1). The relative standard deviations(n=6) are < 5%. Detection limits (signal-to-noise ratio=3) for the five derivatives are 20-40 fmol.

大豆肽和豌豆肽对胰岛素抵抗HepG2细胞糖代谢及其相关机制的研究

通过细胞实验探讨大豆肽与豌豆肽的复合物对II型糖尿病胰岛素抵抗作用,并初步探明其作用机制。采用1×10^(-6)mol/L的人胰岛素处理HepG2细胞24 h建立胰岛素抵抗模型;CCK-8法确定大豆肽及豌豆肽对HepG2细胞的安全剂量;检测不同复配比例的大豆肽和豌豆肽实验组的葡萄糖消耗、葡萄糖吸收及细胞中的糖原含量反应糖代谢情况,通过Western Blot法检测各蛋白的表达。结果表明,与对照组相比,HepG2细胞置于含1×10^(-6)mol/L人胰岛素培养液孵育24 h,葡萄糖消耗量、葡萄糖吸收量、糖原含量都降低,表明建模成功。大豆肽及豌豆肽干预均能改善HepG2细胞的胰岛素抵抗状况,提高细胞对葡萄糖的吸收和消耗能力、糖原合成能力,并能够提高葡萄糖转运蛋白GLUT2、GLUT4的表达量及胰岛素受体底物-1(insulin receptor substrate-1,IRS-1)和蛋白激酶B(Akt)蛋白的磷酸化水平,其中质量浓度为200μg/mL、比例为2∶1的大豆肽和豌豆肽效果最好。大豆肽与豌豆肽以2∶1的质量比进行复配可以有效改善HepG2细胞的胰岛素抵抗状况,可能与肽提高细胞中GLUT2和GLUT4的表达量,以及提高IRS-1和Akt蛋白的磷酸化水平有关。

利用乳酸乳球菌发酵大豆蛋白产低聚肽的研究

筛选可以发酵分解大豆蛋白的食源性微生物,对分解产生的多肽进行分子量分析,通过分离纯化获得低聚肽并研究其抗氧化活性。结果表明:从自制泡菜中分离到一株食源性乳酸菌PZ1,经形态和16S rDNA鉴定为乳酸乳球菌;全基因组分析PZ1菌株具有多种肽酶和蛋白酶基因,具备分解蛋白的潜在能力;利用PZ1发酵大豆分离蛋白,采用凝胶渗透色谱分析发酵产生的多肽,分子量1000 Da以下占比达85%;通过超滤纯化获得300~1000 Da的低聚肽;研究大豆低聚肽的抗氧化活性,发现低聚肽对DPPH自由基、羟基自由基(·OH)和超氧阴离子自由基(O_(2)^(−)·)均有较好的清除作用,浓度为2 mg/mL时,清除率分别为79.31%、78.27%和84.62%。因此乳酸乳球菌PZ1能够高效降解大豆蛋白,可作为益生菌应用于大豆功能产品的开发。

熬煮条件对鸡汤中低聚肽含量和感官品质的影响

为探究熬煮条件对鸡汤品质的影响,文章使用微量双缩脲法测定鸡汤中低聚肽含量,并结合感官评分等指标探究低温煮、超声波低温煮和常压蒸煮对鸡汤品质的影响。结果表明,超声波低温煮的低聚肽含量远远高于低温煮和常压蒸煮,并且3种加工方式的低聚肽含量都呈现逐渐上升的趋势。超声波低温煮在90℃熬制3 h时低聚肽含量达到最高,为0.90 mg/mL;低温煮在90℃熬制9 h时低聚肽含量达到最高,为0.751 mg/mL;常压蒸煮在熬制3 h时低聚肽含量达到最高,为0.747 mg/mL。由感官评价结果可知,超声波低温煮在80℃熬制2 h时感官评分达到最高,为92.8分;低温煮在90℃熬制6 h时感官评分达到最高,为92.4分;常压蒸煮在熬制2 h时感官评分达到最高,为90.6分。

大豆低聚肽对小麦淀粉理化性质的影响

为改善小麦粉的品质,提升小麦食品的蛋白质含量,将具有营养价值和生物活性的大豆低聚肽添加到小麦淀粉中,对小麦淀粉糊化特性、流变学特性、热力学特性和结构特性进行研究。结果表明,大豆低聚肽的添加(0%~40%)使复合物的峰值黏度、低谷黏度和终值黏度降低,糊化温度显著升高(P<0.05)。复合物表现为弱凝胶性质,具有假塑性剪切稀化行为。与对照组相比,大豆低聚肽添加量为40%时,复合物的峰值黏度最小,糊化温度最大,分别为396 cp和77.5℃,焓变下降了79.6%,Zeta-电位的绝对值下降了89.6%,粒径增加至924.1 nm。大豆低聚肽增加了复合物氨基酸含量和热稳定性,降低了复合物的凝胶特性,为功能性谷物食品的开发和加工提供了参考。

小肽对动物生长性能、肠道健康和品质影响的研究进展

小肽是蛋白质分解产物之一,包括二肽和三肽,其在动物肠道中不仅具有较高的吸收利用效率,还具有多种生物活性,能够对动物的抗氧化、免疫、肌纤维和肠道发育等方面发挥积极调节作用,在动物饲料添加剂领域具有广阔的应用前景。本文综述了小肽对动物生长性能和肠道健康影响的研究进展,介绍了小肽对动物生理功能和肌肉品质调控的作用机理,并对小肽研究前景进行展望,旨在为后续小肽药物、改良小肽饲料配方的研究提供参考。

鱼胶原低聚肽对急诊复杂手外伤手术患者预后的影响

目的探讨早期肠内营养添加鱼胶原低聚肽对急诊复杂手外伤手术患者预后的影响。方法回顾性分析急诊收治的122例接受手术治疗的复杂开放性手外伤患者资料,所有患者术后均早期应用肠内营养制剂,根据配方不同分为2组。对照组60例,肠内营养配方为口服均衡营养素联合乳清蛋白;试验组62例,肠内营养配方为对照组配方加用鱼胶原低聚肽。比较2组患者临床基线资料,治疗前后血红蛋白(Hb)、淋巴细胞计数(Lym)、中性粒细胞与淋巴细胞比值(NLR)、白蛋白(ALB)、前白蛋白(PA)、丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、尿素氮(BUN)、肌酐(Cr)、甘油三酯(TG)、血清总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)等水平变化;比较2组患者住院时间和术后感染性并发症发生率。结果与治疗前比较,对照组和试验组营养治疗后Hb、ALB、肝肾功能及脂质代谢指标无显著变化,PA、Lym显著提高,NLR显著降低;治疗后与对照组相比,试验组NLR下降更明显(P<0.01)。试验组感染性并发症发生率低于对照组,住院时间缩短(P<0.05)。结论急诊复杂手外伤术后早期肠内营养添加鱼胶原低聚肽可促进PA合成,降低炎症反应及伤口感染发生率,缩短住院时间。

小麦肽及其与大豆肽、豌豆肽复配肽的体外组合对TNF-α刺激C2C12骨骼肌细胞蛋白表达的影响

通过细胞实验探讨小麦肽、大豆肽、豌豆肽复合物对TNF-α刺激C2C12骨骼肌细胞蛋白表达的影响,并初步探明作用通路。采用终质量浓度为25 ng/mL的TNF-α处理C2C12细胞4 d建立TNF-α刺激模型,CCK-8法确定小麦肽、大豆肽、豌豆肽对C1C12细胞的安全剂量;检测不同复配比例的小麦肽、大豆肽、豌豆肽实验组的肌球蛋白相关指标(MyoD、MyoG、MyHC)、泛素-蛋白酶体系统肌肉萎缩相关指标(TRIM63和MAFbx)的蛋白表达、Akt、mTOR和其磷酸化蛋白表达情况。结果表明,与对照组相比,TNF-α刺激的C2C12细胞中MyoD、MyoG和MyHC的蛋白表达量均有下降,其中MyHC显著降低;MAFbx和TRIM63表达量显著升高;Akt和mTOR的磷酸化水平显著降低,而经过肽样品处理后,各指标均有缓解,且以小麦肽与大豆肽+豌豆肽质量比1∶3效果最好。小麦肽与大豆肽+豌豆肽以1∶3的质量比进行复配在几种配比中可以最有效地改善由TNF-α造成的C2C12细胞肌蛋白含量降低,可通过调节E3泛素连接酶的表达和提高Akt/mTOR的蛋白磷酸化水平的通路,从而降低细胞中蛋白降解和促进蛋白合成。

酸性条件下小麦低聚肽增强紫薯花色苷抗氧化性的研究

在pH4.0酸性条件下,添加25~100g/L的小麦低聚肽(WOPs)虽降低了紫薯花色苷(PSPAs)的热稳定性,但两者相互作用增强了其抗氧化能力,并随着其添加量的增加而增大。当添加量为100g/L时,PSPAs溶液在体外模拟消化后的DPPH、ABTS自由基清除能力和总抗氧化能力(FRAP)分别提高了1.5倍、34倍和34%,且分别在37℃和45℃贮藏7d后,其抗氧化能力没有出现显著性降低,相比于单独的PSPAs具有更强的生物活性以及更高的稳定性。紫外光谱和荧光光谱表明在实验浓度下,PSPAs可以引起WOPs特定波长下吸收峰的红移和荧光猝灭,证明WOPs和PSPAs之间存在相互作用。综上可知,WOPs具有应用于酸性PSPAs饮料开发的潜力。