Recovering high value-added anthocyanins from blueberry pomace with ultrasound-assisted extraction.

Food waste is a potential source to replace fresh materials for obtaining functional ingredients. Blueberry pomace contains considerable amounts of anthocyanins. In this study, we investigated ultrasonic-assisted extraction (UAE) of anthocyanins from blueberry pomace. We used a Box-Behnken design (BBD) to screen and optimize the important factors influencing yield. The optimum extraction conditions were a temperature of 40 °C, an ultrasonic power of 400 W and an extraction time of 40 min. The optimum yield was 108.23 mg/100 g DW. In addition, we used a cation column to separate anthocyanins, and optimized the chromatographic conditions of HPLC to analyze and identify the main anthocyanins. Thirteen anthocyanins were found in blueberry pomace, of which Malvidin-3-Galactoside (22.65 %) was the highest. These findings provide a theoretical basis and optimized process parameters for the recovery of high value-added anthocyanins from blueberry pomace with ultrasound-assisted extraction, thus facilitating the comprehensive utilization of blueberry pomace.

Color Stability Enhancement and Antioxidation Improvement of Sanhua Plum Wine under Circulating Ultrasound.

Anthocyanins contribute to the attractive color of fruit wine, and their excessive degradation is deleterious to quality, especially for wine with an inherently low anthocyanin content, such as Sanhua plum wine. Ultrasonic treatment is well recognized for wine color maintenance. In the present study, fresh Sanhua plum wine was ultrasonic-treated and aged in barrels for three months. Our results demonstrate that ultrasonic treatment at 28 and 40 kHz improves color performance, as expressed by an increase in a*, b*, and C* values and color intensity, which is highly related to copigmentation. This successful conservation was attributed to the inactivation of polyphenol oxidase and the corresponding reduction in anthocyanin degradation. Finally, the increased antioxidative ability was verified due to the hydrogen donating ability of the surviving anthocyanins. This study indicates the reliability of ultrasonic treatment for providing superior colorfastness during Sanhua plum wine aging, which is also of great potential in processing different fruit wines.

Using untargeted metabolomics to profile the changes in roselle (Hibiscus sabdariffa L.) anthocyanins during wine fermentation.

In this study, an UHPLC-QE-MS approach in combination with multivariate statistical analyses was used to investigate the metabolic profiles, especially the anthocyanin profiles, during the fermentation of roselle wine. Overall, a large number of different metabolites (e.g., phenols, lipids, carbohydrates, amino acids and peptides, and others) were identified in the fermentation processes. Eight anthocyanin metabolites were identified in roselle wine for the first time, of which six were identified in the main fermentation process and two in the post-fermentation process. In addition, we identified several metabolic pathways during the fermentation process, and the metabolic pathways of anthocyanins in roselle wine are mainly related to flavonoid biosynthesis and to anthocyanin biosynthesis. These findings are expected to be useful for further studies on wine chemistry and yeast metabolism.

Estimation of SPAD value in waterlogged winter wheat based on characteristic indices of hyperspectral and digital image.

To explore the optimal monitoring method for soil and plant analyzer development (SPAD) of winter wheat under waterlogging stress based on hyperspectral and digital image techno-logy, the correlations between SPAD of the waterlogged winter wheat and fifteen indices of hyperspectral characteristic and fourteen indices of digital image feature were analyzed under a micro-plot which could be irrigated and drainage separately. Then, the BP neural network models for SPAD estimation were constructed based on the optimal monitoring feature indices. Compared with the normal winter wheat, SPAD and the value of hyperspectral reflectance did not change under short-term waterlogging (less than 7 d), whereas the SPAD was significantly decreased after more than 12 d waterlogging treatment with the value being close to zero at the late stage of growth. The estimation accuracy based on the digital image characteristics of green minus red, excess red index, norma-lized redness index and excess green index showed similar results compared to that using the BP network model based on the characteristics of the corresponding hyperspectral band. The highest R2 between the measured value and the predicted value was 0.86, while the root mean square error (RMSE) was 3.98. Compared with the BP network models built with the digital image feathers, the accuracy of the models based on the four hyperspectral characteristic indices (carotenoid reflex index, yellow edge amplitude, normalized difference vegetation index and structure insensitive pigment index) for SPAD was significantly improved, with the highest R2 of 0.97 and the lowest RMSE of 1.95. Our results suggest that both hyperspectral and digital image technology could be used to estimate SPAD value of waterlogged winter wheat and that the BP network model based on hyperspectral characteristic indices performed better in the estimation accuracy.

Nitrogen and phosphorus losses from paddy fields and the yield of rice with different water and nitrogen management practices.

The assessment and control of losses of nitrogen (N) and phosphorus (P) from paddy fields is critical to improve the quality of water and atmosphere on earth. A field experiment was conducted to investigate the effect of three N managements (local common N fertilization practice, urea mixed with controlled-release N fertilizer, and optimized and reduced N fertilizer, designated CN, U + CRF and ON, respectively) on N and P losses through runoff and leaching from a paddy field, and yield of rice under shallow-irrigation and deep-sluice (SIDS) and continuous flooding irrigation (FI) in the Jianhan Plain of China in 2016. The results showed that, compared with FI, SIDS significantly reduced the frequency of irrigation and amount of irrigation water, resulting in an increase of 16.2% in rainfall use efficiency, and therefore, a reduction in the amount of surface runoff and water that had leached. This was responsible for the decreased total N (TN) and total P (TP) losses through runoff leaching under SIDS. The U + CRF and ON treatments resulted in a significant reduction in losses of TN through runoff and leaching and the loss of TP through leaching compared to CN. SIDS resulted in comparable or greater soil TN and TP contents in the 0-40 cm soil depths after rice harvest; N and P accumulation at the jointing, filling and maturity stages; and yield of grain compared to FI. Moreover, the U + CRF and ON improved or maintained accumulation of N and P and yield of rice compared to CN. Compared with FI coupled with CN, SIDS coupled with the U + CRF or ON treatments significantly reduced losses of N and P from paddy fields and enhanced or maintained the accumulation of N and P and yield of rice grains. In conclusion, SIDS coupled with the new N management could be an effective approach to reduce losses of N and P from paddy fields and would be a positive improvement for high yield of middle-season rice grains in the Jianhan Plain of central China and other regions with similar environments.

[Effect of alpha-zearalanol on the generation of reactive oxygen species (ROS) and ROS-activated signal transduction in tumor necrosis factor alpha-stimulated human endothelial cells].

OBJECTIVE: To investigate the effects of alpha-zearalanol (alpha-ZAL) on the generation of reactive oxygen species (ROS) and ROS-activated signal transduction in the tumor necrosis factor (TNF-alpha)-stimulated human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were cultured and divided into 4 groups: (1) normal control group, (2) TNF-alpha stimulated group, undergoing TNF-alpha stimulation for 24 h, (3) alpha-ZAL retreatment group, undergoing re-treatment with alpha-ZAL of the concentrations of 1 x 10(-8), 1 x 10(-7), or 1 x 10(-6) mol/L for 1 h, then stimulation of TNF-alpha for 24 h, and (4) plasmid transfection group, transfected with p47(phox) siRNA for 24 h to block the NADPH oxidase protein subunit p47(phox) in the HUVECs, or transfected with blank plasmid as control. The intracellular ROS production was detected by using 2, 7-dichlorofluorescin diacetate as probe. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the mRNA and protein expression of p47(phox). The activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor-kappaB (NF-kappaB), stimulatory protein (SP)-1, and activator protein (AP)-1 were assessed with Western blotting. RESULTS: The ROS level in the HUVECs of the TNF-alpha group was higher than that of the control group by 155.4%, and alpha-ZAL reduced the ROS level dose-dependently. TNF-alpha treatment up-regulated the p47(phox) mRNA expression by 212.8%, and obviously increased the p47(phox) protein expression; and alpha-ZAL pretreatment attenuated the TNF-alpha-induced p47(phox) mRNA expression by 63.0%, and also markedly inhibited the p47(phox) protein expression. No obvious ROS was found in the HUVECs stimulated by TNF-alpha after the transfection of p47(phox) siRNA. The ERK activation and nuclear translocation of transcription factors SP-1 and NF-kappaB induced by TNF-alpha were abolished or markedly inhibited by alpha-ZAL pretreatment. CONCLUSION: alpha-ZAL has a potent inhibitory effect on the ROS production and ROS-activated signaling pathway in the TNF-alpha stimulated endothelial cells, mainly through the inhibition of NADPH oxidase.

Improved Jayaweera-Mikkelsen model to quantify ammonia volatilization from rice paddy fields in China.

Current estimates of China's ammonia (NH3) volatilization from paddy rice differ by more than twofold, mainly due to inappropriate application of chamber-based measurements and improper assumptions within process-based models. Here, we improved the Jayaweera-Mikkelsen (JM) model through multiplying the concentration of aqueous NH3 in ponded water by an activity coefficient that was determined based on high-frequency flux observations at Jingzhou station in Central China. We found that the improved JM model could reproduce the dynamics of observed NH3 flux (R2 = 0.83, n = 228, P < 0.001), while the original JM model without the consideration of activity of aqueous NH3 overstated NH3 flux by 54% during the periods of fertilization and pesticide application. The validity of the improved JM model was supported by a mass-balance-based indirect estimate at Jingzhou station and the independent flux observations from the other five stations across China. The NH3 volatilization losses that were further simulated by the improved JM model forced by actual wind speed were in general a half less than previous chamber-based estimates at six stations. Difference in wind speed between the inside and outside of the chamber and insufficient sampling frequency were identified as the primary and secondary causes for the overestimation in chamber-based estimations, respectively. Together, our findings suggest that an in-depth understanding of NH3 transfer process and its robust representation in models are critical for developing regional emission inventories and practical mitigation strategies of NH3.

Inhibitory effects of alpha-zearalenol on angiotensin II-induced integrin beta3 mRNA via suppression of nuclear factor-kappaB.

OBJECTIVE: To investigate the effect of alpha-zearalenol on angiotensin II-induced beta3 integrin mRNA expression in human umbilical vein endothelial cells (HUVECs). METHODS: The mRNA level in integrin beta3 was determined by reverse transcription-polymerase chain reaction. Endothelial NF-kappaB activity was determined by the luciferase activity assay of plasmid NF-kappaB-LUC. RESULTS: The angiotensin II-induced beta3 integrin mRNA expression was inhibited by alpha-zearalenol and 17beta-estradiol (10 nmol/L -1 micromol/L), but not influenced by ICI 182, 780, a pure competitive antagonist for estrogen receptor or a nitric oxide inhibitor Nomega-Nitro-L-arginine methyl ester hydrochloride. Alpha-zearalenol and 17beta-estradiol suppressed the angiotensin II-induced activation of NF-kappaB in endothelial cells. CONCLUSION: Alpha-zearalenol inhibits angiotensin II-induced integrin beta3 mRNA expression by suppressing NF-kappaB activation in endothelial cells.

[mRNA expression of muscarinic receptors in spinal cord and brainstem in morphine dependent rats].

AIM: To observe mRNA expression of muscarinic acetylcholine receptors in spinal cord and brainstem in morphine dependent or withdrawal rats. METHODS: The mRNA expression level of m1, m2, m3, m4 and m5 were determined by RT-PCR, the beta-actin mRNA expression was used as internal control. RESULTS: The mRNA level of m1, m2, m3, m4 and m5 in spinal cord and m1 and m2 in brainstem were increased significantly during morphine dependence, and the levels of m1, m2, m3 and m4 in spinal cord and m1 in brainstem were decreased 1 h after the injection of naloxone (4 mg.kg-1, i.p.) in morphine dependent rats. Either scopolamine (0.5 mg.kg-1) or pirenzepine (10 mg.kg-1) was shown to significantly decrease the morphine withdrawal symptoms in rats. The levels of m1, m2, m3 and m5 in spinal cord were increased by pretreatment with pirenzepine and the levels of m2, m3 and m4 in spinal cord were increased by pretreatment with scopolamine. CONCLUSION: The adaptive expression of muscarinic receptors at spinal and supraspinal levels play important role in mediating morphine dependence and withdrawal in rats.

Angiotensin II stimulates endothelial integrin beta3 expression via nuclear factor-kappaB activation.

The present study examined the effect and part mechanism of angiotensin II-stimulated integrin beta3 gene expression in human umbilical vein endothelial cells. Protein level and mRNA level of integrin beta3 expression were determined using enzyme-linked immunoadsorbent assay and reverse transcription-polymerase chain reaction. Four plasmids of 5'-different deletion of integrin beta3 gene promoter were constructed to transiently transfected into cells to uncover the region in response to angiotensin II. Blockade of nuclear factor-kappaB (NF-kappaB) signaling pathway effect on integrin beta3 expression was analyzed by cotransfection with mutant plasmids for NF-kappaB-inducing kinase, inhibitory proteins alpha and beta of NF-kappaB kinase, respectively, together with the integrin beta3 plasmid including the sequence -1486 approximately - 900. The study found that 10(-8) mol/L,10(-7) mol/L, 10(-6) mol/L, and 10(-5) mol/L angiotensin II increased integrin beta(3) protein level by 45%, 52%, 62%, and 73% respectively. Angiotensin II at 10(-6) mol/L increased integrin beta3 mRNA level by 67%. The luciferase activity of the integrin beta3 plasmid PGL3 - 1486 approximately - 900 increased by 84.72% in response to angiotensin II. N-acetylcysteine blocked angiotensin II-induced NF-kappaB activity and integrin beta3 expression. Blockade of NF-kappaB signaling pathway abolished the stimulation of angiotensin II. These results suggest that angiotensin II stimulates integrin beta3 expression partly by NF-kappaB activation.

TSC-36/FRP inhibits vascular smooth muscle cell proliferation and migration.

OBJECTIVE: In-stent restenosis is a vascular proliferation/migration disorder characterized by hyperplasia of vascular smooth muscle cells (VSMCs). Because mounting evidence suggests that the therapeutic potential of anti-proliferation and anti-migration therapy, we investigated possible inhibitory effects of the matricellular protein TGF-beta-stimulated clone 36 (TSC-36) on vascular smooth muscle cell proliferation and migration in vitro and in vivo. METHODS: Human umbilical artery smooth muscle cells (SMCs) were treated with inducting agents daidzein or estradiol. TSC-36 expression was detected by nested competitive PCR and in situ hybridization. TSC-36 was expressed in Origami (DE3) cells. The recombinant protein was used to immunize rabbits to produce polyclonal antibodies. VSMCs were treated with various concentrations of recombinant TSC-36 (rTSC-36) protein and daidzein. The MTT assay was used to analyze for cell proliferation. A transwell system was used to detect cell migration. Flow cytometry was used to detect cell phase. A rat carotid artery balloon injury model was duplicated. The rats were treated with daidzein or solvent control. Animals were sacrificed 5 weeks later, and injured arteries were taken for pathology and histology. RESULTS: TSC-36 mRNA and protein expression was induced in SMCs. Cell proliferation and migration were inhibited by rTSC-36. rTSC-36 caused accumulation of SMCs in G2 phase. The inducting agent daidzein decreased neo-intima proliferation. TSC-36 mRNA and protein expression was induced and expressed in the neo-intima. CONCLUSION: TSC-36 can be induced in VSMCs and inhibits VSMCs proliferation in vitro and in vivo.

Endothelial nitric oxide synthase gene polymorphisms associated with susceptibility to high altitude pulmonary edema in Chinese railway construction workers at Qinghai-Tibet over 4 500 meters above sea level / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To examine whether the polymorphisms of endothelial nitric oxide synthase (eNOS) gene are associated with the susceptibility to high altitude pulmonary edema (HAPE) in Chinese railway construction workers at Qinghai-Tibet where the altitude is over 4 500 m above sea level.</p><p><b>METHODS</b>A case-control study was conducted including 149 HAPE patients in the construction workers and 160 healthy controls randomly recruited from their co-workers, matching the patients in ethnicity, age, sex, lifestyle, and working conditions. Three polymorphisms of eNOS gene, T-786C in promoter, 894G/T in exon 7, and 27bp variable number tandem repeat (VNTR) in intron 4, were genotyped using polymerase chain reaction (PCR) and confirmed with DNA sequencing.</p><p><b>RESULTS</b>The frequencies of 894T allele and heterozygous G/T of the 894G/T variant were significantly higher in HAPE patients group than in the control group (P=0.0028 and P=0.0047, respectively). However, the frequencies of the T-786C in promoter and the 27bp VNTR in intron 4 were not significantly different between the two groups. Haplotypic analysis revealed that the frequencies of two haplotypes (H3,T-T-b, b indicates 5 repeats of 27 bp VNTR; H6, C-G-a, a indicates 4 repeats of 27 bp VNTR) were significantly higher in HAPE patients (both Pü0.0001). On the contrary, the frequencies of H1 (T-G-b) and H2 (T-G-a) were lower in HAPE patients than in healthy controls (both Pü0.001).</p><p><b>CONCLUSIONS</b>Two haplotypes (T-T-b and C-G-a) may be strongly associated with susceptibility to HAPE. Compared with the individual alleles of eNOS gene, the interaction of multiple genetic markers within a haplotype may be a major determinant for the susceptibility to HAPE.</p>

Roles of NF-kappaB and SP-1 in oxidative stress-mediated induction of platelet-derived growth factor-B by TNFalpha in human endothelial cells.

Platelet-derived growth factor-B (PDGF-B) is upregulated by proinflamatory stimuli in the early stages of atherosclerosis. However, its mechanisms are not fully elucidated. In the present study, by using the antioxidant N-acetylcysteine (NAC), we investigated in human umbilical vein endothelial cells (HUVECs) the roles of oxidative stress in PDGF-B expression induced by tumor necrosis factor alpha (TNFalpha) and its underlying mechanisms. Exposure of HUVECs to TNFalpha (200 U/ml) for 24 hours caused significant increases of both the PDGF-B expression and its promoter/enhancer activity, which were abolished by NAC (20 mmol/L). Accordingly, a prolonged oxidative stress was induced by TNFalpha and that was prevented by pretreatment with NAC. Electrophoresis mobility shift assay (EMSA) and Western blot analysis showed that both the nuclear factor-kappaB (NF-kappaB) and the specificity protein-1 (SP-1) were activated by TNFalpha. However, NAC only partially inhibited the TNFalpha-induced activation of NF-kappaB, but abolished the activation of SP-1. Mutation of the NF-kappaB binding site resulted in a moderate reduction in the TNFalpha-induced activity of PDGF-B promoter/enhancer, whereas mutation of SP-1 binding site resulted in an absence of induction by TNFalpha. These results suggest that oxidative stress mediates the TNFalpha-induced expression of PDGF-B in HUVECs through redox-sensitive transcription factors, predominantly the SP-1 and possibly, to some extent of NF-kappaB.