A New Method of Extracting Polygonatum sibiricum Polysaccharide with Antioxidant Function: Ultrasound-Assisted Extraction-Deep Eutectic Solvents Method.

Polygonatum sibiricum Polysaccharide (PsP) with antioxidant function is the main active component of Polygonatum sibiricum (P.sibiricum). The currently poor extraction yield and extraction methods of PsP cannot meet the application of that in food industrial production. In this research, an ultrasound-assisted extraction-deep eutectic solvents (UAE-DESs) method, which has never been used in the PsP industry, was first used to extract PsP. The extraction conditions were optimized by the response surface method (RSM). Both the extraction yield and antioxidant function were simultaneously considered during the optimization process. The indicators of PsP's level and antioxidant activity in vitro were used to present the extraction yield of the UAE-DESs method, the purity, and the antioxidant effect of PsP. Under the optimal conditions, which included a liquid-solid ratio of 26:1 (mL:g), extraction temperature of 80 °C, ultrasonic time of 51 min, and ultrasonic power of 82 W, the PsP extraction yield could reach (43.61 ± 0.09)%, which was obviously higher than single DESs (33.81%) and UAE (5.83%), respectively, and the PsP appeared favorably antioxidant function. This research proposed an efficient extraction method for PsP, filled the basic research gap, and further improved the development of PsP as a dietary supplement with antioxidant function in the food industry.

[Conditional optimization and methodological evaluation of liquid protein microarray in detecting cystatin C].

OBJECTIVE: To optimize the detection conditions and evaluate of cystatin C(CysC) by liquid protein microarray. METHODS: CysC was detected by double antibody sandwich method using liquid protein microarray. On the basis of determining the optimal concentration combination of captured antibody and detected antibody, the detection conditions were optimized by determining the biological detection limit and lower detection limit, drawing the S-shaped curve and judging the linear range, and establishing the standard curve and regression equation. Methodsologically evaluate the accuracy, precision, reportable range and analytical specificity of the detection method. RESULTS: The optimal concentration combinations of CycC trapping-detection antibodies were 26.6 µg/mL-1∶800. The lower limits of detection and biologic limits of detection of the CysC were 0.037 and 0.237 ng/mL, respectively. Regression equation were as followes: y=-3.315x~2+283.04x+160.89, R~2=0.9921. The relative bias of CysC which was detected on the liquid protein microarry was 5.81%. The dilution recovery and recovery were 70.35%-84.91%(n=3)and 79.94%-122.41%(n=3)respectively. The correlation coefficient of method ology comparison experiment was r=0.616, P<0.05, and there was no significant difference between the two method(t=0.948, P=0.358); The within-run precision range from 3.54% to 4.03%(n=10); The between-run precision range from 12.07% to 15.05%(D=5, n=3); The reportable range was 0.26-3784.04 ng/mL. The analysis of interference test result showed that the both concentrations of hemoglobin(160.00, 71.11 g/L) had interference to the result of CysC detected on the chip. CONCLUSION: This study completed the optimization of conditions and methodological evaluation of liquid protein microarray in detecting CysC.

Construction and Evaluation of a Novel MAP Immunoassay for 9 Nutrition-and-Health-Related Protein Markers Based on Multiplex Liquid Protein Chip Technique.

We attempted to construct and evaluate a novel detection method to realize simultaneous detection based on a multiplex liquid protein chip technique for nine nutrition-and-health-related protein markers to meet the requirement of an accurate, simultaneous and comprehensive analysis of the proteomics of nutrition and health. The lower limits of detection, biological limits of detection and regression equations of serum ferritin (SF), soluble transferrin receptor (sTfR), c-reactive protein (CRP), retinol-binding protein4 (RBP4), apolipoprotein B (ApoB), alpha-fetoprotein (AFP), prealbumin (PA), carcino-embryonic antigen (CEA) and D-Dimmer (D-D) were determined after a series of optimal experiments. Then, the results of the methodological evaluation for this novel method indicated that the accuracies were between 70.12% and 127.07%, the within-run precisions were between 0.85% and 7.31%, the between-run precisions were between 3.53% and 19.07%, the correlation coefficients between this method and other methods were above 0.504 (p < 0.05), and the direct bilirubin (DBIL) of low concentration and the indirect bilirubin (IBIL) of high concentration could not interfere with the detected results of nine indicators. The novel multiplex detection method, which can increase accuracy and improve the ability of comprehensive analysis, can basically meet the requirement of detection and the diagnosis of the proteomics of nutrition and health.

[Serum metabolomics of iron deficiency anemia in infants based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry].

OBJECTIVE: To explore the differential metabolites in the serum of infants with iron deficiency anemia(IDA) and non iron deficiency anemia, and to explore the potential biomarkers. METHODS: Non-targeted metabolomics of 30 infants with iron deficiency anemia aged 6-11 months and 30 infants with non iron deficiency anemia aged 6-11 months were analyzed by ultra-high performance liquid chromatography with time of flight mass spectrometry using Acquity UPLC BEH C_(18) column(100 mm×2.1 mm, 1.8 µm). The differences of metabolites between the two groups were analyzed by principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). Differential metabolites were screened according to OPLS-DA variable importance projection(VIP) >1. The related metabolic pathways involved in the markers were analyzed based on the KEGG database. RESULTS: Differences in serum metabolic profiles between iron deficiency anemia group and non iron deficiency anemia group were observed. The 44 potential biomarkers were mainly lipids. Combined with pathway analysis, the metabolic pathways related to different metabolites included glycerophosphingolipid metabolism and sphingolipid metabolism. CONCLUSION: There are differences in lipid metabolites between infants with non iron deficiency anemia and iron deficiency anemia, suggesting that the occurrence and progress of iron deficiency anemia are related to lipid metabolism.

Intervention Effect of a Soybean-Based Complementary Food Supplement on Anemic Infants in a Poor Rural Region in China: Evidence from Quasi-RCT.

The soybean-based Yingyang Bao complementary food supplement represents a special nutritional improvement method for anemic infants in many intervention projects across China, while its benefits lack rigorous evidence. Using a quasi-randomized controlled trial design, which adhered to randomization and control except for the blinding method, 248 anemic infants were divided randomly into an intervention group (128 cases received the Yingyang Bao intervention based on routine feeding) and a control group (120 cases only received routine feeding). Anthropometric indicators and 16 blood indicators were measured at baseline and 1 year after intervention. The levels of hemoglobin, 1,25-dihydroxy vitamin D, homocysteine, retinol, vitamin D3, and soluble transferrin receptor and the height-age-Z score and weight-age-Z score of the intervention group were significantly improved after the intervention (p < 0.05). The homocysteine level improvement appeared to be moderately negatively correlated with the cobalamin level improvement (p < 0.05). The improvements of five indicators were significant correlated with the intervention duration (p < 0.05), and the corresponding three significant regression equations could predict the intervention effect and the intervention duration to a certain extent. This quasi-randomized controlled trial provided more convincing evidence that Yingyang Bao can effectively improve three kinds of malnutrition compared to previous research which only adopted self before and after comparison.

Optimization and Application of A Bionic System of Dynamic Co-Culture with Hepatocytes and Renal Cells Based on Microfluidic Chip Technique in Evaluating Materials of Health Food.

We aimed to explore the optimization and application of a bionic system of dynamic co-culture with hepatocytes and renal cells based on the microfluidic chip technique in evaluating emodin, which might replace the conventionally cytological evaluation technique of health food. After optimal experiments, the improved bionic system was composed of human hepatocellular carcinoma cells (HepG2), human renal glomerular endothelial cells (HRGECs), rat tail collagen type I, and gelatin with optimized concentrations (1.3 mg/mL + 7.5%). The applicability of the bionic system indicated that the growth stability was appropriate (CV: 7.36%), and the cell viability of that gradually decreased with the increasing of emodin concentration from 0−100 µM, which statistic significances were at 50 and 100 µM (p < 0.05), and the stained results of dead/live cells also showed the same trend. The LDH level appeared rising trend after decline between 0 µM and 100 µM emodi, and the level of that at 100 µM emodin was significantly higher than that at 25 µM and 50 µM emodin, respectively. The BUN level continuously and significantly declined with the increasing of emodin concentration (p < 0.05). Our research realized the application of this optimized bionic system in evaluating emodin, and provided a useful platform and reference for further in vitro alternative research with regard to evaluating the efficacies of health food in the future.

Effect Assessment of Aurantio-Obtusin on Novel Human Renal Glomerular Endothelial Cells Model Using a Microfluidic Chip.

Cassiae semen is widely used as a raw material of health food. Anthraquinone compounds, the main components in cassiae semen, have been reported to show nephrotoxicity. Aurantio-obtusin (AO) is a major anthraquinone compound extracted from cassiae semen. This study investigates the effects of AO on the morphology and physiological function of human renal glomerular endothelial cells (HRGECs) on a microfluidic chip device for the first time. HRGECs were cultured on a microfluidic plate and exposed to a series of AO concentrations. Compared with traditional 96-well culture, HRGECs cultured on the microfluidic chip appeared to better mimic the glomerular microenvironment in vivo. AO induced different degrees of damage to cellular morphology and physiological function. The leakage of lactate dehydrogenase (LDH), as well as the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), and monocyte chemoattractant protein 1 (MCP-1), increased in the AO treated groups. At the same time, cell viability and expression of ZO-1 in the AO treated groups decreased in a dose-dependent manner. The innovative device enables direct visualization and quantification to evaluate the cytotoxic effects of AO on HRGECs, and provides a useful visual in vitro model for studying health effect of health food.

Construction and Application of Hepatocyte Model Based on Microfluidic Chip Technique in Evaluating Emodin.

The current cytological evaluation technique of health food raw materials does not entirely meet the needs of evaluating health food. Our study adopted the microfluidic chip technique for the first time to construct a hepatocyte model of evaluating emodin, which was composed of a human hepatocellular carcinoma cell (HepG2) and microfluidic chip. The mixed glue of a model with rat tail collagen type I (1.3 mg/mL) + gelatin (7.5%) was used to simulate the microenvironment of a cell. The validity of this model was evaluated by cell proliferation activity and cell staining, and the toxicity of emodin was evaluated by a series of metabolic indicators on this model. The results indicated that the repeatability of the constructed hepatocyte model was favorable, with a coefficient of variation (CV) of 2.8%. After emodin continuously was exposed for 48 h, the cell inhibition was obvious at 100 and 200 µM, and the number of dead cells gradually increased with the increasing of emodin concentration, and the difference of BUN was significant between the emodin group and blank group (p < 0.05). The constructed model has a favorable applicability in evaluating emodin. This study provides an important platform and a potential in vitro alternative model for assessing and predicting the health effects of health food.

[Optimization of platform conditions for simultaneous detection of multiple nutritional marker proteins by liquid protein microarray].

OBJECTIVE: To optimize the technical conditions for simultaneous detection of serum ferritin(SF), soluble transferrin receptor(sTfR), C-reactive protein(CRP)and retinol-binding protein four(RBP4)by liquid protein microarray. METHODS: The trapping antibodies of the four proteins were coupled to magnetic beads with different codes, and the samples were added to the 96-well plate. The antibodies were detected by double antibody sandwich method. The serum of 5 patients were diluted with commercial diluent, 1% albumin from bovine serum(BSA) and phosphate buffer saline(PBS) to detect 4 target proteins, and the results were compared. The antibody specific binding ability was tested by antibody specific validation test. The interference between proteins was verified by the paired t test of the signal values of the single reaction system and the mixed reaction system. The lower limit of detection and the limit of biological detection of each protein were found by using multiple dilution method. The standard curve and regression equation were established. RESULTS: 1%BSA and PBS were selected to replace commercial diluent as diluents for the detection of 4 proteins in this experiment. The cross-reaction rate of the four antigens with other capture antibodies and detection antibodies was less than 2%. There was no significant difference in the signal value of each protein in the single reaction system and the mixed reaction system. The limit of detection and the limit of biological detection of SF were 1.155 and 1.625 ng/mL, respectively. The lower limit of detection and the limit of biological detection of sTfR were 2.682 and 5.208 ng/mL, respectively. The detection limit and biological detection limit of CRP were 0.302 and 0.391 ng/mL, respectively. The lower limit of detection and the limit of biological detection for RBP4 were 1.814 and 3.540 ng/mL, respectively. The standard curve and regression equation of the four proteins within the common linear range were as follows: SF y=172.5x-39.65, R~2=0.9968;sTfR y=60.10x+77.38, R~2=0.9972;CRP y=-6.000x~2+210.3x+246.1, R~2=0.9063;RBP4 y=-0.6998x~2+64.31x+134.8, R~2=0.9748. CONCLUSION: The conditions of the detection platform for four proteins such as SF, sTfR, CRP and RBP4 were optimized by using liquid protein chip technology.

[Improved effect of comprehensive nutritional intervention of whole covering for Kazak's pregnant women, lactating women and infants in Altay farming and stockbreeding region].

OBJECTIVE: To study the effect of nutritional intervention for Kazak's pregnant women, lactating women and infants in farming and stockbreeding region of Jeminay County, Altay City. METHODS: 24 h record method was conducted to implement dietary survey, and results were used to analyze dietary structure and nutrient intake level of pregnant women, lactating women and infants. Pregnant women, lactating women and infants over 6 months were intervened with iron fortified soy sauce nutrients supplement and Yingyangbao(YYB) for 2. 4 years. Hemoglobin was detected for pregnant women, lactating women and infants by using HemoCue analyzer. RESULTS: The nutrient intakes of the pregnant women, lactating women and infants were averagely lower than that of the recommended levels. In these infants who received breast feeding, the least acceptability diet quality rate was 42. 1%, and in other infants who didn't receive breast feeding, the rate was 25%. After intervention, anemia prevalence of pregnant women, lactating women and infants were significantly reduced compared with the base line levels at 2 survey time points(2014:pregnant women χ~2=26. 27, lactating women χ~2=18. 06, infants χ~2=44. 46, 2015:pregnant women χ~2=35. 62, lactating women χ~2=25. 05, infants χ~2=39. 61;all P<0. 001). CONCLUSION: The nutrition intervention of whole covering could improve nutrition status of Kazak's pregnant women, lactating women and infants.

[Meta-analysis on relationship between the Chinese maternal MTHFR gene polymorphism(C677T) and neural tube defects in offspring].

OBJECTIVE: To explore the association between maternal MTHFR gene polymorphism( C677T) and neural tube defects in offspring through Meta-analysis in China. METHODS: CNKI, Pub Med, Web of Science, Chinese Wan Fang Data databases, CBM, VIP for published articles were searched from the time of Database establishment to July 5 th 2017. The search strategy was based on combinations of the English and/or Chinese keywords, 'MTHFR'and 'folate pathway'and 'polymorphism'or 'SNP'and'NTDs or Neural Tube Defects'. References of reviews and retrieved studies were also scanned. All the case-control studies about MTHFR gene C677T polymorphism and susceptibility of neural tube defect were collected, which were fulfilled the followinginclusion criteria: case-control study and cohort study design, presentation of data necessary for calculating odds ratios( ORs). Data were extracted from studies and analyzed by Rev Man 5. 3 software. RESULTS: A total of 13 papers were selected, including1500 patients and 1654 controls. Meta-analysis result showed that the combined odds ratio values of neural tube defect for offspring with maternal TT, TT + CT and T allele genotypes were 1. 94, 1. 65 and 1. 39, respectively. CONCLUSION: The present Meta-analysis suggests that MTHFR C677T is significantly associated with maternal risk for NTDs in the Chinese population, supplemental folic acid supplementation based on MTHFR polymorphisms will be an important means to further reduce the birth defects of newborns.

Effects of MTHFR A1298C polymorphism on peripheral blood folate concentration in healthy populations: a meta-analysis of observational studies.

BACKGROUND AND OBJECTIVES: Methylenetetrahydrofolate reductase (MTHFR) irreversibly converts 5,10- methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the main form of folate used in the body. Previous studies suggest that MTHFR polymorphism influences folate metabolism, but conflicting results are reported. We performed a meta-analysis to accurately characterize the association between MTHFR A1298C polymorphism and peripheral blood folate concentration in healthy populations. METHODS AND STUDY DESIGN: Studies focusing on MTHFR A1298C polymorphism and folate concentrations were identified and subjected to a metaanalysis using Review Manager 5.1. Standard mean differences (SMD) with 95% confidence intervals (95% CI) were used to assess the association between these variables. RESULTS: A total of 14 studies with 5616 healthy individuals were included in this meta-analysis. Significant differences in folate concentration were found in the MTHFR homozygote model (SMD=0.12, 95% CI=0.00-0.24, I2=17%, p=0.04) and the dominant model (SMD=0.07, 95% CI=0.01-0.14, I2=22%, p=0.02) in the general population excluding the elderly. While abnormal folate concentrations are more common in elderly, no association between MTHFR A1298C polymorphism and peripheral blood folate concentration was found in the meta-analysis when elderly were included. CONCLUSIONS: This meta-analysis indicates that, in the general population excluding the elderly, the C allele of MTHFR 1298 polymorphism is associated with the risk for an increased folate concentration.

[Optimizing the detection of bovine milk ß-Lactoglobulin with protein chip].

OBJECTIVE: To optimize the conditions of protein chip assay for bovine milk ß-Lactoglobulin( ß-Lg). METHODS: A microarrayer was used for printing anti-ß-Lg as antibody I on each 3-dimensional-slide, another antis ß-Lg antibody was used as detection antibody II and goat antibody coupled to Cy3 was used as antibody III. The standard ß-Lg was detected by double antibody sandwich technique. RESULTS: Mouse monoclonal ß-Lg antibody66# was chosen as the probe and contact printing as the printing method. The range between 42 and 92 spots was chosen as the basic printing condition. The concentration of ß-Lg probes was 0. 5 mg / mL. The ß-Lg detection antibody titre was 1∶2000. One percent no protein blocking solution was choosen as the blocking buffer. The lower detection limit and the biological detection limit of ß-Lg were 17. 54 ng / m L and 55. 31 ng / m L respectively. The linear range was determined according to the S type curve of ß-Lg and the best fitting models and standard curve were established for ß-Lg( R~2=0. 9993). CONCLUSION: The study optimizes conditions of a quantitative analysis system for measurement of ß-Lg with protein chip, thus establishing the protein chip platform for quantitative detection of bovine milk ß-Lactoglobulin.

[Evaluation of iron fortified soy sauce on changes of anemia prevalence in 2004-2013].

OBJECTIVE: To understand the role of iron fortified soy sauce( IFSS) on the decrease of anemia prevalence in Chinese population in 2004- 2013. METHODS: Meta analysis was used to analyze the effect of IFSS on anemia through published randomcontrol studies on the population intervention with IFSS. Integrated with other data from IFSS statistic on IFSS production and consumption, the recovered population from IFSS was identified and anemia decrease determined, therefore, IFSS contribution to anemia change could be estimated. RESULTS: By Meta analysis, the anemia rate of the population consumed IFSS was 27% compared with the anemia rate of the population non-consumed IFSS. In the past 10 years, IFSS had covered more than 186 millions population with effect validity time. A total of 28. 49 millions anemic population recovered from anemia. To recover one anemic with IFSS, the cost was 0. 12 RMB. CONCLUSION: IFSS has showed effect on the decline of anemia prevalence in last decade, 23. 2% of the decrease was estimated owe to IFSS project, but there are unidentified factors which may bring bias to the result.

[Study on detection function of protein microarray for iron deficiency diagnosis].

OBJECTIVE: To study the reality detect function of protein microarray which diagnosis the iron deficiency (ID). METHODS: Evaluated anti-interference ability with interference experiment, analyzed the detection stability with many times repeated experiment, detected the same experiment result repeatedly to analyze the detection indate. RESULTS: There was not interference to serum ferritin and soluble transferrin respector from distraction. The protein microarry can be using in 38 d after print probes. The detection indate should be in 22 d. CONCLUSION: The protein microarry that used as diagnosis ID opposed the reality detection ability.

[Establishment of mammalian cell lines for constitutive expression of influenza virus matrix protein 2].

To establish a mammalian cell line for stable expression of the matrix protein 2 (M2) of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR product was cloned into eukaryotic expression vector pcDNA5/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for 10 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of influenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.

Enhanced immune response induced by a potential influenza A vaccine based on branched M2e polypeptides linked to tuftsin.

Vaccination is the most effective means for preventing influenza-associated morbidity and mortality. Since the influenza virus mutates frequently, the virus strains for new vaccine production should be changed according to predicted epidemic strains. The extracellular domain of matrix protein 2 (M2e) is 24 amino acids long, which is highly conserved and therefore a good target for the development of a universal vaccine which may protect against a much wider range of influenza A virus strains. However its low antigenicity and immunogenicity, which are related to its small size, poses a big challenge for vaccine development. Multiple antigen peptide system (MAP) is based on an inert core molecule of radially branching lysine dendrites onto which a number of peptide antigens are anchored. Tuftsin is an immuno-stimulant molecule peptide. Here we developed a novel peptide vaccine by connecting a tuftsin to a branched, four-copy M2e. Not only did this increase the molecular mass, but also potentiate the immunogenicity. Two branched peptides, (M2e)4-tuftsin and (M2e)4-G4(tuftsin was replaced with four glycines), and a M2e monomer were synthesized using standard solid-phase methods. In vitro and in vivo studies were performed to compare their antigenicity and immunogenicity. Experiments in BALB/c mice demonstrated that the branched M2e could induce stronger humoral and cellular immune responses than the M2e monomer, and (M2e)4-tuftsin induced stronger humoral and cellular immune response than (M2e)4-G4. After lethal challenge with influenza virus PR8 strain, up to 80% of the animals in the (M2e)4-tuftsin vaccinated group still survived, in contrast to 44% in the (M2e)4-G4 group and 30% in the M2e monomer group. The combination of branched polypeptides and tuftsin in vaccine design is presented here for the first time, and the results show that the new construct is a promising candidate for a universal vaccine against the influenza A virus.

[Optimizing the detection of soluble transferrin receptor with protein microarray].

OBJECTIVE: To optimize the conditions of protein microarray assay for soluble transferrin receptor (sTfR). METHODS: A microarrayer was used for printing anti-sTfR as antibody I on each protein microarray; anti-sTfR antibody was used as detection antibody II and goat antibody coupled to Cy3 was used as antibody III. The standard sTfR was detected by double antibody sandwich technique. RESULTS: Mouse monoclon sTfR antibody was chosen as a probe and contact printing as the printing method. A better homogenicity was appeared after pre-printing for 40 spots. The concentration of sTfR probes was 0.25 mg/ml. The concentration of sTfR detection antibody was 0.18 microg/ml. 3% skim milk powder and goat anti rabbit antibody made by GE was chosen as blocking buffer and secondary antibody. The lowest detection limits and the biological detection limits of sTfR were 0.253 ng/ml and 0.78 ng/ml respectively. The best fitting models and standard curve were established for sTfR (r = 0.99699). CONCLUSION: Conditions of a quantitative analysis system for measurement of sTfR with protein microarray were optimized.