Widely targeted metabolomics analysis of Sanghuangporus vaninii mycelia and fruiting bodies at different harvest stages.

Sanghuangprous vaninii is a medicinal macrofungus cultivated extensively in China. Both the mycelia and fruiting bodies of S. vaninii have remarkable therapeutic properties, but it remains unclear whether the mycelia may serve as a substitute for the fruiting bodies. Furthermore, S. vaninii is a perennial fungus with therapeutic components that vary significantly depending on the growing year of the fruiting bodies. Hence, it is critical to select an appropriate harvest stage for S. vaninii fruiting bodies for a specific purpose. With the aid of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), metabolomics based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS) was used to preliminarily determine 81 key active metabolites and 157 active pharmaceutical metabolites in S. vaninii responsible for resistance to the six major diseases. To evaluate the substitutability of the mycelia and fruiting bodies of S. vaninii and to select an appropriate harvest stage for the fruiting bodies of S. vaninii, we analyzed the metabolite differences, especially active metabolite differences, among the mycelia and fruiting bodies during three different harvest stages (1-year-old, 2-year-old, and 3-year-old). Moreover, we also determined the most prominent and crucial metabolites in each sample of S. vaninii. These results suggested that the mycelia show promise as a substitute for the fruiting bodies of S. vaninii and that extending the growth year does not necessarily lead to higher accumulation levels of active metabolites in the S. vaninii fruiting bodies. This study provided a theoretical basis for developing and using S. vaninii.

[Simultaneous determination of 11 active components in Lonicera japonica flowers and leaves at different development stages by HPLC-DAD].

This study aims to develop an HPLC-DAD method for simultaneous determination of 11 components(6 phenolic acids and 5 iridoids) in Lonicera japonica flowers(LjF) and leaves(LjL), and compare the content differences of LjF at different development stages, LjL at different maturity levels, and between LjF and LjL. One-way ANOVA, principal component analysis(PCA), and orthogonal partial least-squares discriminant analysis(OPLS-DA) were employed to compare the content of the 11 components. The content of total phenolic acids, total iridoid glycosides, and total 11 components in LjF showed an overall downward trend with the development of flowers. The content of total phenolic acids, total iridoid glycosides, and total 11 components in young leaves were higher than those in mature leaves. The results of PCA showed that the samples at different flowering stages had distinguishable differences in component content. The VIP value of OPLS-DA showed that isochlorogenic acid A, chlorogenic acid, and secologanic acid were the main differential components of LjF at different development stages or LjL with different maturity levels. LjF and LjL have certain similarities in chemical composition while significant differences in component content. The content of total phenolic acids in young leaves was significantly higher than that in LjF at various development stages. The content of total iridoid glycosides in young leaves was similar to that in LjF before white flower bud stage. The total content of 11 components in young leaves was significantly higher than that in LjF at green flower bud stage, before and during completely white flower bud stage. LjL have great potential for development. Follow-up research on the pharmacodynamic equivalence of LjF and LjL(especially young leaves) should be carried out to speed up the development and application of LjL.

Suppression of Helicobacter pylori infection by daily cranberry intake: A double-blind, randomized, placebo-controlled trial.

BACKGROUND AND AIM: Dietary strategies that contribute to reducing incidence of Helicobacter pylori infection without negative side effects are highly desirable owing to worldwide bacterial prevalence and carcinogenesis potential. The aim of this study was to determine dosage effect of daily cranberry consumption on H. pylori suppression over time in infected adults to assess the potential of this complementary management strategy in a region with high gastric cancer risk and high prevalence of H. pylori infection. METHODS: This double-blind, randomized, placebo-controlled trial on 522 H. pylori-positive adults evaluated dose-response effects of proanthocyanidin-standardized cranberry juice, cranberry powder, or their placebos on suppression of H. pylori at 2 and 8 weeks by 13 C-urea breath testing and eradication at 45 days post-intervention. RESULTS: H. pylori-negative rates in placebo, low-proanthocyanidin, medium-proanthocyanidin, and high-proanthocyanidin cranberry juice groups at week 2 were 13.24%, 7.58%, 1.49%, and 13.85% and at week 8 were 7.35%, 7.58%, 4.48%, and 20.00%, respectively. Consumption of high-proanthocyanidin juice twice daily (44 mg proanthocyanidin/240-mL serving) for 8 weeks resulted in decreased H. pylori infection rate by 20% as compared with other dosages and placebo (P < 0.05). Percentage of H. pylori-negative participants increased from 2 to 8 weeks in subjects who consumed 44 mg proanthocyanidin/day juice once or twice daily, showing a statistically significant positive trend over time. Encapsulated cranberry powder doses were not significantly effective at either time point. Overall trial compliance was 94.25%. Cranberry juice and powder were well-tolerated. CONCLUSIONS: Twice-daily consumption of proanthocyanidin-standardized cranberry juice may help potentiate suppression of H. pylori infection. TRIAL REGISTRATION: ChiCTR1800017522, per WHO ICTRP.

Dissecting Efficacy and Metabolic Characteristic Mechanism of Taxifolin on Renal Fibrosis by Multivariate Approach and Ultra-Performance Liquid Chromatography Coupled With Mass Spectrometry-Based Metabolomics Strategy.

Taxifolin (TFN) is an important natural compound with antifibrotic activity; however, its pharmacological mechanism is not clear. In this study, our aim is to gain insight into the effects of TFN and its potential mechanisms in unilateral ureteral obstruction (UUO) animal model using metabolomics approach to identify the metabolic biomarkers and perturbed pathways. Serum metabolomics analysis by UPLC-Q-TOF/MS was carried out to discover the changes in the metabolic profile. It showed that TFN has a significant protective effect on UUO-induced renal fibrosis and a total of 32 potential biomarkers were identified and related to RF progression. Of note, 27 biomarkers were regulated by TFN treatment, which participate in eight metabolic pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, and phenylalanine metabolism. It also showed that metabolomics was a promising strategy to better dissect metabolic characteristics and pharmacological mechanisms of natural compounds by multivariate approach and ultra-performance liquid chromatography coupled with mass spectrometry.

Dinuclear cobalt and nickel complexes of a mercaptoacetic acid substituted 1,2,4-triazole ligand: syntheses, structures and urease inhibitory studies.

Because of its versatile coordination modes and strong coordination ability, the mercaptoacetic acid substituted 1,2,4-triazole 2-{[5-(pyridin-2-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetic acid (H2L) was synthesized and characterized. Treatment of H2L with cobalt and nickel acetate afforded the dinuclear complexes {µ-3-[(carboxylatomethyl)sulfanyl]-5-(pyridin-2-yl)-4H-1,2,4-triazol-4-ido-κ2N1,N5:N2,O}bis[aqua(methanol-κO)cobalt(II)] methanol disolvate, [Co2(C9H6N4O2S)2(CH3OH)2(H2O)2]·2CH3OH (1), and {µ-3-[(carboxylatomethyl)sulfanyl]-5-(pyridin-2-yl)-4H-1,2,4-triazol-4-ido-κ2N1,N5:N2,O}bis[diaquanickel(II)] methanol disolvate dihydrate, [Ni2(C9H6N4O2S)2(H2O)4]·2CH3OH·2H2O (2), respectively. Complex 1 crystallized in the monoclinic space group P21/c, while 2 crystallized in the tetragonal space group I41/a. Single-crystal X-ray diffraction studies revealed that H2L is doubly deprotonated and acts as a tetradentate bridging ligand in complexes 1 and 2. For both of the obtained complexes, extensive hydrogen-bond interactions contribute to the formation of their three-dimensional supermolecular structures. Hirshfeld surface analysis was used to illustrate the intermolecular interactions. Additionally, the urease inhibitory activities of 1, 2 and H2L were investigated against jack bean urease, where the two complexes revealed strong urease inhibition activities.

Identification of the Perturbed Metabolic Pathways Associating With Renal Fibrosis and Evaluating Metabolome Changes of Pretreatment With Astragalus polysaccharide Through Liquid Chromatography Quadrupole Time-Of-Flight Mass Spectrometry.

Renal fibrosis is glomerulosclerosis and renal tubulointerstitial fibrosis caused by the increase of interstitial cells and intercellular substances and the accumulation of extracellular matrix, and is a common pathological manifestation of renal disease progressing to end-stage renal failure. It has proved that Astragalus polysaccharide (AP) has curative effect on renal disease; however, its therapeutic mechanism on renal fibrosis is still unclear. Metabolomics approach provides an opportunity to identify novel molecular biomarkers. The purpose of this study is to study the changes of serum metabolic profile of rats with unilateral tubal ligation and replication of renal fibrosis model and the therapeutic effect of AP on it. The blood samples of rats in the control group, renal fibrosis model group, and AP treatment group collected on the 21st day were analyzed by metabolomics method based on UPLC-Q-TOF-MS. Principal component analysis (PCA) showed that clustering was obvious and significantly separated, and paired partial least squares discriminant analysis (OPLS-DA) was used for further analysis. Combined with the network databases such as HMDB and KEGG and a large number of literatures, 32 potential biomarkers related to renal fibrosis were preliminarily screened out and further verified by MS/MS secondary debris information. After pretreatment with AP, 20 biomarkers were significantly regulated, and correlated with phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, etc. It also revealed the metabolic changes of renal fibrosis and intervention effect of AP. These data uncover a link between metabolism and the molecular mechanism with potential implications in the understanding of the intervention effect of AP. Conclusively, UPLC-Q-TOF-MS-based metabolomics can be valuable and promising strategy to understand the disease mechanism and natural drug pretreatment.

[Microbial Community Diversity Analysis During Composting of Lincomycin Mycelia Dreg with Manure].

Aerobic composting experiments were conducted using lincomycin mycelia wastes (dreg) and manure (T), using sewage sludge with manure as a control (CK). High performance liquid phase methods and high throughput sequencing were used to determine the concentration of lincomycin residue and to characterize the microbial community. The results showed that lincomycin was reduced significantly, with the concentration decreasing from 1800 mg·kg-1 to 483 mg·kg-1, accounting for 73% degradation. In addition, the bacterial community abundance and diversity indices were all lower than that of sludge-manure at the mesophilic and thermophilic phases, because of the high concentration of lincomycin residue in lincomycin mycelia dreg. By contrast, the fungal community abundance and diversity indices showed the reverse, due to the high content of organic matter and nitrogen in lincomycin mycelia dreg. Therefore, the microbial communities were greatly different between T and CK treatment with the domain genera of Paucisalibacillus, Cerasibacillus, Bacillus, Virgibacillus, Ureibacillus, Paenibacillus, and Sinibacillus in T compost and Truepera, Actinomadura, Pseudosphingobacterium, Pseudomonas, Luteimonas and Ureibacillus in CK compost. However, as the composting continued to a mature phase, most of the lincomycin was reduced, and the differences between the two microbial communities gradually decreased. This showed that composting could make lincomycin mycelia dreg harmless and could be used to turn it into a resource.

[Effects of the ultra-filtration extract mixture from Hedysarum Polybotrys on human liver cells HepG2 radiosensitivity].

OBJECTIVE: To investigate the effects of the ultra-filtration extract mixture from Hedysarum Polybotrys (UEMHP) on the radiosensitivity of HepG2 cells, and to explore its possible mechanisms. METHODS: The proliferation inhibition effects of UEMHP on HepG2 cells was detected by CCK-8 assay. The colony formation assay was used for the survival fraction (SF) analysis. The distribution of the cell cycle and the apoptosis rate were detected using flow cytometry (FCM). The survivin mRNA expression level was detected using reverse transcription-PCR assay. RESULTS: The inhibition of UEMHP on HepG2 cells was time-and dose-dependent at the concentration ranging between 5 -50 mg/L (P < 0.05). The parameters of the two curve for SF (P < 0.05) showed statistical difference between the irradiation group and the UEMHP irradiation group. UEMHP could inhibit the clone formation of HepG2 cells and enhance the radiosensitivity of HepG2 cells. The results of FCM showed that UEMHP could induce G2/M phase arrest. The apoptosis rate in the UEMHP irradiation group (21.42% +/- 3.74%) was higher than that in the control group (5.35% +/- 0.41%), the only UEMHP group (10.36% +/- 1.75%), or the irradiation group (10.58% +/- 2.01%) (P < 0.01). RT-PCR showed that the survivin mRNA expression level was lower in the UEMHP irradiation group (0.31 +/- 0.02) than in the control group (0.82 +/- 0.06) and the irradiation group (0.58 +/- 0.04) respectively, showing statistical difference (P < 0.01). CONCLUSION: UEMHP can enhance the radiosensitivity of HepG2 cells, and its possible mechanisms might be correlated to down-regulating the survivin mRNA expression and promoting the apoptosis.

[Study on growth of height among students during their adolescence in Zhongshan, Guangdong].

OBJECTIVE: To discuss characteristics of height growth such as Peak Height Velocity (PHV) and Age at Peak Height Velocity (PHA) during adolescence, and to compare the results with other research findings. METHODS: Primary and middle school students' annual physical examination data of Zhongshan in 2005 - 2010 was used. The height velocity by age, PHV, PHA, height velocity by PHA were calculated. RESULTS: The average peak height velocity boys was (10.03 ± 1.67) cm/yr. and that of the girls was (8.39 ± 1.05) cm/yr. Both findings were close to the results from previous similar findings. The average age at which peak height velocity reached 12.28 ± 1.30 years for boys and 10.78 ± 1.04 years for girls, both lower than the previous findings. The correlation coefficients, between height level and PHA were -0.357 (P < 0.001) for boys and -0.338 (P < 0.001) for girls. CONCLUSION: The height levels were positively related to the height velocity before PHA. The Zhongshan students' PHA was lower than the Beijing, Shanghai and Shenyang students, also lower than American and Britain students', but their PHVs were similar.

[Effect of fluoride on the expression of MMP-20/TIMP-2 in ameloblast of rat incisor and the antagonistic effect of melatonin against fluorosis].

PURPOSE: To study the effect of concentration of fluoride on the expression of matrix metalloproteinase-20(MMP-20) and tissue inhibitors of metalloproteinase-2 (TIMP-2) in the ameloblast of rat incisor,and explore the formation mechanism of dental fluorosis. By comparing the different expression of MMP-20,TIMP-2 between fluoride group and the melatonin group,to decide whether melatonin has antagonitic effect on dental fluorosis. METHODS: Forty Wistar rats were randomly divided into 6 groups. The groups were as follows: control group,low-dose group, high-dose group,normal saline group and melatonin group. The animals were sacrificed 10 weeks after treatment. HE and immunohistochemical staining were used to observe the changes of ameloblasts and the expression of MMP-20 and TIMP-2 in rat incisors. MetaMorph microscope images analysis system was used to analyze the images, and SPSS12.0 software package was used for data analysis. RESULTS: The surface of rat incisors fed with fluoride had chalky color change and cross stritations could be seen on the enamel surface.In the fluoride group,the ameloblasts were disarranged, cells arranged in multi-layer,even showing vacuolar change.The changes in the high-dose group was severer than the low-dose group. MMP-20, TIMP-2 were expressed both in the secretory ameloblasts, and in the odontoblasts.The expression of MMP-20 in rat's ameloblasts in the experimental group was significantly lower than that in the control group (P < 0.01); and no significant difference was found between the low-dose and high-dose groups(P > 0.05). The difference of expression of TIMP-2 was not significant among all the groups. The difference of expression of MMP-20 and TIMP-2 was not significant between the melatonin and the fluoride groups. CONCLUSIONS: The excessive fluoride can inhibit the secretion of MMP-20 and disturb the balance between MMP-20 and TIMP-2,which lead to the delay of amelogenin removal and enamel demineralization. Melatonin has no antagonistic effect on the dental fluorosis. Supported by National Natural Science Foundation of China (30600509) and Natural Science Foundation of Liaoning Province (20102278).

[Expression and analysis of biological activity of wild-type and mutant interleukin-13 in E.coli].

AIM: To express recombinant wild-type human interleukin-13 (rhIL-13) and mutant interleukin-13 (rhIL-13') in E.coli BL21 (DE3) and get active proteins through purification and renaturation. METHODS: IL-13 and IL-13' gene fragments were amplified by PCR and site-directed mutagenesis PCR, respectively and then were inserted into expression vector pET30a(+). Recombinant plasmids were transformed to E.coli BL21 (DE3) and were expressed under IPTG induction. Expressed products were purified through Ni-NTA chromatographic column. The purified proteins were renatured by GSH-GSSG (reduced glutathione, oxidized glutathione) system and dialysis and their bioactivity was detected by MTT colorimetry. RESULTS: The expressed recombinant proteins existed in the form of inclusion body with relative molecular mass about 17 000. The recombinant proteins with higher purity were obtained after purification. The renatured inclusion bodies were biologically active. CONCLUSION: rhIL-13/rhIL-13' with biological activity have been obtained successfully, which lays the foundation for further study on their function.

[Chemiluminescent detection of E. coli O157:H7 in food based on magnetic enzyme-linked immuno method].

AIM: To establish a new chemiluminescent assay with high sensitivity for detecting E. coli O157:H7 in food. METHODS: The assay was established based on the alkaline phosphatase (ALP) labeled anti-E. coli O157:H7 antibody, which reacted with an excellent chemiluminescent reagent, 3(2' spiroadamantane)4 methoxy 4(3''phosphoryloxy) phenyl 1,2 dioxetane (AMPPD) buffer solution and emitted the photons. RESULTS: The sensitivity of chemiluminescence assay was 850, and the linear range was 1000-50,000. The intra- and interassay CV values (CVs) were respectively below 15% and 20%. The correlation coefficient was 0.9807 by comparing with the conventional counting method. CONCLUSION: The chemiluminescent immunoassay is more accurate than other methods in the detection of concentration of E. coli O157:H7 in samples.

[The effect of high dose fluoride on the rat offspring osteoblasts which ingested by female rats].

OBJECTIVE: To investigate the effect of high dose fluoride which ingested by female rats on morphologic change in rat offspring's bone and osteoblast, discuss the relation between the mechanism of fluorosis and cell cycle, cell apoptosis. METHODS: In stock diets condition, Wistar female rats drank distilled water containing 0,50,100,150 mg/L NaF for 2 months, then they are mated with normal rats. The calvarium and osteoblast of offsprings were used to investigate the effects of fluoride on ultrastructure by LM and TEM. FCM was used to analysis cell cycle and apoptosis. RESULTS: The Electron microscope revealed the number of microvilli of osteoblasts were overall decreased in rat offsprings with fluorosis. There was mitochondrial swelling and dilation of the rough endoplasmic reticulum (RER). The matrix of calvarium was hyperplasia and collagen was accumulated and turbulenced. The nuclear manifested the apoptosis character. NaF at 150 mg/ L increased the osteoblast number of S phase with relative decrease of cell number of G2/M phase, but did not change that in G0/G1 phase. The apoptosis percentage increased in this group. CONCLUSION: Excessive fluoride can directly through the placental barrier, influence cell structure and cell cycle distribution of fluorosis rat offspring and render the cell cycle stagnant in S phase, induce apoptosis.

[Effect of fluoride on activities of enzyme and ultrastructure in primary cultured rat hepatocytes].

OBJECTIVE: To study the cell viability, activities of enzyme and ultrastructure changes induced by sodium fluoride in primary cultured rat hepatocytes. METHODS: Hepatocytes were isolated using half-in situ collagenase digestion method. Cellular viability was determined by MTT method. The activities of ALT and AST were determined by spectrophotography method. The ultrastructure changes of hepatocyte were observed under transmission electron microscope. RESULTS: After cultured with various concentrations of fluoride for 24 hours, a dose-dependent decrease of cell viability was detected in the hepatocytes. The activities of AST and ALT in the 2 mmol/L and 4 mmol/L groups were significantly higher than the control group (P < 0.05). Transmission electron microscope study showed that in fluoride treated hepatocytes the changes included swollen mitochondria and disordered, disrupted endoplasm reticulum. CONCLUSION: Excessive fluoride induced significant toxicity in primary cultured hepatocytes which manifested the injuries of membrane and organell plasma membrane.

[A novel microbeads-based chemiluminescence immunoassay for detecting human thyrotropin].

AIM: To establish a low cost and sensitive microbeads-based chemiluminescence immunoassay (CLEIA) for detecting human thyrotropin. METHODS: Thyrotropin in sera was captured by an alkaline phosphase-labeled mAb against TSH beta subunit and a FITC-labeled mAb against TSH alpha subunit. Captured thyrotropin was then isolated with immuno-magnetic beads conjugated with anti-FITC antibody and then quantified by chemiluminescence using adamanatane amine as luminescent substrate. RESULTS: The sensitivity of this assay is 0.004 mIU/L. The intra-assay CV and the inter-assay CV of the assay were 7.45% and 10.45%, respectively. The recovery rate was 91.4% -102.4%. The examination result of the assay correlates well with that of ACS-180 system. CONCLUSION: The CLELA for detecting TSH is low-cost, sensitive, specific and stable, and therefore may have a promising prospect in clinical application.