Analysis of furan and its major furan derivatives in coffee products on the Chinese market using HS-GC-MS and the estimated exposure of the Chinese population.

The current study analysed concentrations of furan and its derivatives in coffee products commercially available in China based on an improved headspace gas chromatography-mass spectrometry (HS-GC-MS) method and estimated health risks. A total of 101 samples of coffee products on the Chinese market was analysed. Furan (98%, ND-6569 µg/kg) and 2-methylfuran (100%, 2-29639 µg/kg) were the compounds with the highest concentrations and detection rates in coffee products. The mean dietary exposure of Chinese consumers to furan and the sum of furan, 2-methylfuran and 3-methylfuran in coffee products was 0.09 and 0.46 µg/kg bw/day, respectively. For the neoplastic effects of furan, the margin of exposure (MOE) was 14,556 for the mean dietary exposure of consumers. For the non-neoplastic effects of furan, MOEs were 711 and 139 for furan and the sum of furan, 2-methylfuran and 3-methylfuran, respectively. Overall, a health concern is indicated for coffee consumers with MOEs below 10000.

[Clinical observation of Tiaoshen acpuncture for dry eye syndrome with yin deficiency of liver and kidney].

OBJECTIVE: To compare the curative effect of Tiaoshen acupuncture combined with artificial tears and artificial tears alone for dry eye syndrome with yin deficiency of liver and kidney. METHODS: A total of 70 patients of dry eye syndrome with yin deficiency of liver and kidney were randomized into an acupuncture combined with medication group (35 cases, 2 cases dropped off) and a medication group (35 cases, 3 cases dropped off). In the medication group, 0.1% sodium hyaluronate eye drop was applied, 3 times a day, 1 drop each time. On the basis of the treatment as the medication group, Tiaoshen acupuncture was applied at Shenting (GV 24), Shenmen (HT 7), Benshen (GB 13), Taixi (KI 3), Taichong (LR 3), Guangming (GB 37), Fengchi (GB 20), Tongziliao (GB 1), Yuyao (EX-HN 4) in the acupuncture combined with medication group, Tongziliao (GB 1) and Yuyao (EX-HN 4) were connect with electroacupuncture, the needles were retained for 30 min, once a day, 6 times a week. Both groups were treated for 2 weeks. Before and after treatment, the clinical symptom score, SchirmerⅠtest (SⅠT), break-up time (BUT), corneal fluorescein staining (FL) score and Hamilton anxiety scale (HAMA) score were observed in the two groups, and the clinical efficacy was compared in the two groups. RESULTS: Compared before treatment, the clinical symptom scores, corneal FL and HAMA scores were decreased (P<0.05), the levels of SⅠT and BUT were increased after treatment in the two groups (P<0.05). After the treatment, the clinical symptom score, FL and HAMA scores in the acupuncture combined with medication group were lower than the medication group (P<0.05), the levels of SⅠT and BUT in the acupuncture combined with medication group were higher than the medication group (P<0.05). The total effective rate in the acupuncture combined with medication group was 90.9% (30/33), which was higher than 71.9% (23/32) in the medication group (P<0.05). CONCLUSION: Tiaoshen acupuncture combined with artificial tears could promote the secretion of tears for patients of dry eye syndrome with yin deficiency of liver and kidney, repair corneal defect and prolong tear film break-up time, and alleviate anxiety state, its curative effect is better than simple artificial tears.

[Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum].

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

High-density SNP linkage map construction and QTL mapping for flavonoid-related traits in a tea plant (Camellia sinensis) using 2b-RAD sequencing.

BACKGROUND: Flavonoids are important components that confer upon tea plants a unique flavour and health functions. However, the traditional breeding method for selecting a cultivar with a high or unique flavonoid content is time consuming and labour intensive. High-density genetic map construction associated with quantitative trait locus (QTL) mapping provides an effective way to facilitate trait improvement in plant breeding. In this study, an F1 population (LJ43×BHZ) was genotyped using 2b-restriction site-associated DNA (2b-RAD) sequencing to obtain massive single nucleotide polymorphism (SNP) markers to construct a high-density genetic map for a tea plant. Furthermore, QTLs related to flavonoids were identified using our new genetic map. RESULTS: A total of 13,446 polymorphic SNP markers were developed using 2b-RAD sequencing, and 4,463 of these markers were available for constructing the genetic linkage map. A 1,678.52-cM high-density map at an average interval of 0.40 cM with 4,217 markers, including 427 frameset simple sequence repeats (SSRs) and 3,800 novel SNPs, mapped into 15 linkage groups was successfully constructed. After QTL analysis, a total of 27 QTLs related to flavonoids or caffeine content (CAF) were mapped to 8 different linkage groups, LG01, LG03, LG06, LG08, LG10, LG11, LG12, and LG13, with an LOD from 3.14 to 39.54, constituting 7.5% to 42.8% of the phenotypic variation. CONCLUSIONS: To our knowledge, the highest density genetic map ever reported was constructed since the largest mapping population of tea plants was adopted in present study. Moreover, novel QTLs related to flavonoids and CAF were identified based on the new high-density genetic map. In addition, two markers were located in candidate genes that may be involved in flavonoid metabolism. The present study provides valuable information for gene discovery, marker-assisted selection breeding and map-based cloning for functional genes that are related to flavonoid content in tea plants.

Antitumor Effect of By-1 from Spent Broth from Submerged Cultures of Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Higher Basidiomycetes), on A549 Adenocarcinoma Cells.

By-1 was obtained from spent broth from submerged cultures of Taiwanofungus camphoratus. This report evaluates the effects of By-1 on plate clone formation, wound healing, cell cycle, activated caspase-3 expression, and ROS release in A549 lung cancer cells. The result of plate clone formation assay revealed that By-1 could dramatically inhibit the viability of A549 cells in vitro. The inhibitory effect of By-1 on cell migration was tested using a wound healing assay. Proliferation rates of A549 cells were significantly inhibited following exposure to By-1 (12.5, 50, and 80 µg/mL). Flow cytometry revealed that the extracts increased, in a concentration-dependent manner, the number of cells in the G0/G1 phases of the cell cycle. The results of the caspase-3 experiment suggested that By-1 could induce A549 cells apoptosis, and this apoptosis was related to the release of reactive oxygen species by the A549 cells. All these results indicate that By-1 has potential in anti-lung cancer drug development.

Effect of lutein supplementation on visual function in nonproliferative diabetic retinopathy.

BACKGROUND AND OBJECTIVES: The purpose of this study was to determine whether supplementation with lutein improved visual function in patients with nonproliferative diabetic retinopathy (NPDR). METHODS AND STUDY DESIGN: In this randomized, double-blind, placebo-controlled trial, 31 patients with NPDR were assigned randomly to 10 mg/d of lutein or identical placebo for 36 weeks. Visual performance indices, including visual acuity (VA), contrast sensitivity (CS) and glare sensitivity (GS) at four different spatial frequencies, were measured at baseline, week 18 and 36. RESULTS: At 36 weeks, a slight improvement in VA was found in the lutein group. A significant association was observed between the changes in VA and the corresponding baseline values in treatment group (r=-0.53; p=0.04). At 36 weeks, the lutein treatment group increased CS at four spatial frequencies, and the improvement achieved statistical significance at 3 cycles/degree (p=0.02). The changes in CS at 3 cycles/degree for the lutein group was marginally significantly greater than those for the placebo group (p=0.09). There was also a slight increase in GS in the lutein group up to week 36, however, no significant changes were found over time in any cycles/degree. CONCLUSIONS: In patients with NPDR, supplementation with lutein resulted in potential improvements in CS at low spatial frequency. Further studies are required to determine the possibility that such intervention could be used as an adjunct therapy to prevent vision loss in diabetic patients.

[Intervention of Qingshen Granule on NF-KB Signal Pathway of Chronic Renal Failure Patients with Damp-heat Syndrome].

Objective To observe the effect of Qingshen Granule (QG) on expressions of nucle- ar factors-κB p65 (NF-κB p65) and phosphonated inhibitor of nuclear factor-κB (p-lκBα) in peripheral blood NF-κB signal transduction pathway of chronic renal failure (CRF) patients with damp-heat syn- drome (DHS) , and to study possible mechanism. Methods Totally 68 CRF patients with DHS were as- signed to the control group and the treatment group by random digit table, 34 in each group. Actually 63 patients completed, 32 in the treatment group and 31 in the control group. A normal group (20 cases) was set up. All patients received basic treatment of Western medicine (WM) and retention enema of Chi- nese medicine (CM). Patients in the treatment group additionally took QG, 1 package each time, 3 times per day. The therapeutic course for all was 8 weeks. The clinical efficacy, level of serum creatinine (SCr), estimated glomerular filtration rate (eGFR), the levels of NF-κB p65 and p-IκBα in peripheral blood were observed and measured before and after treatment. They were also compared with those of the normal group. Results The clinical efficacy and the total effective rate of CM syndrome were 84. 38% (27/32)and 81. 25% (26/32), superior to those of the control group [54. 84%(17/31), 51. 61% (1631) ; P <0. 01 ]. Compared with before treatment, the level of SCr was obviously lower, and eGFR was obviously higher in the treatment group after treatment (P <0. 01). They were better than those of the control group after treatment (P <0. 05). Compared with the normal group, the levels of NF-κB p65 and p-IκBα were significantly higher in the treatment group and the control group before treatment (P < 0. 01). Compared with before treatment, the levels of NF-κB p65 and p-IκBα were obviously lowered in the treatment group after treatment (P <0. 01). They were also better than those of the control group after treatment (P <0. 05). Conclusions QG could improve clinical symptoms of CRF patients with DHS, de- crease SCr level, and increase eGFR level. It could protect renal function. Its mechanism might possibly be related with reducing peripheral blood levels of NF-κB p65 and p-IκBα.

Transcriptome analysis reveals self-incompatibility in the tea plant (Camellia sinensis) might be under gametophytic control.

BACKGROUND: Self-incompatibility (SI) is under genetic control and prevents inbreeding depression in angiosperms. SI mechanisms are quite complicated and still poorly understood in many plants. Tea (Camellia sinensis L.) belonging to the family of Theaceae, exhibits high levels of SI and high heterozygosity. Uncovering the molecular basis of SI of the tea plant may enhance breeding and simplify genomics research for the whole family. RESULTS: The growth of pollen tubes following selfing and crossing was observed using fluorescence microscopy. Self-pollen tubes grew slower than cross treatments from 24 h to 72 h after pollination. RNA-seq was employed to explore the molecular mechanisms of SI and to identify SI-related genes in C. sinensis. Self and cross-pollinated styles were collected at 24 h, 48 h and 72 h after pollination. Six RNA-seq libraries (SP24, SP48, SP72, CP24 CP48 and CP72; SP = self-pollinated, CP = cross-pollinated) were constructed and separately sequenced. In total, 299.327 million raw reads were generated. Following assembly, 63,762 unigenes were identified, and 27,264 (42.76 %) unigenes were annotated in five public databases: NR, KOG, KEGG, Swiss-Port and GO. To identify SI-related genes, the fragments per kb per million mapped reads (FPKM) values of each unigene were evaluated. Comparisons of CP24 vs. SP24, CP48 vs. SP48 and CP72 vs. SP72 revealed differential expression of 3,182, 3,575 and 3,709 genes, respectively. Consequently, several ubiquitin-mediated proteolysis, Ca(2+) signaling, apoptosis and defense-associated genes were obtained. The temporal expression pattern of genes following CP and SP was analyzed; 6 peroxidase, 1 polyphenol oxidase and 7 salicylic acid biosynthetic process-related genes were identified. The RNA-seq data were validated by qRT-PCR of 15 unigenes. Finally, a unigene (CL25983Contig1) with strong homology to the S-RNase was analyzed. It was mainly expressed in styles, with dramatically higher expression in self-pollinated versus cross-pollinated tissues at 24 h post-pollination. CONCLUSIONS: The present study reports the transcriptome of styles after cross- and self-pollination in tea and offers novel insights into the molecular mechanism behind SI in C. sinensis. We believe that this RNA-seq dataset will be useful for improvement in C. sinensis as well as other plants in the Theaceae family.

[Identification of dominant microbes from traditional Chinese medicine Pinelliae Rhizoma Fermentata].

To investigate the microbial species, amount changes as well as the isolation and identification of domain strains at different fermentation time points of Pinelliae Rhizoma Fermentata, and provide basis for exploring the mechanism of Pinelliae Rhizoma Fermentata processing. Five samples were chosen at the time points (0, 18, 36, 54, 72 h) of Pinelliae Rhizoma Fermentata processing. Bacteria, mold and yeast from the samples were cultured; their colonies were counted, and the dominant strains were isolated and purified. The dominant bacteria and dominant fungi were identified by 16S rDNA and 26S rDNA sequencing respectively. The results showed that the bacteria count was low with slow and smooth changes in the fermentation process;while mold and yeast grew dramatically after 54 h culturing and reached 1×107 CFU•mL⁻¹ at the end of fermentation. Through the NCBI homology alignment and phylogenetic tree construction, the dominant bacteria were identified as Streptomyces sp., Bacillus pumilus, B. subtilis, B. aryabhattai and other Bacillus sp.; the dominant yeast was identified as Meyerozyma guilliermondii; the dominant mold were identified as Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger in the processing of Pinelliae Rhizoma Fermentata. The results indicated that multiple microbe species, especially yeast and mold, played a role in the fermentation processing of Pinelliae Rhizoma Fermentata. M. guilliermondii, P. variotii, P. variotii and A. niger and Bacillus sp. can be the crucial factors in the processing of Pinelliae Rhizoma Fermentata.

[Intervention of Qingshen Granule for Immune Inflammation of Chronic Renal Failure Patients with Damp-heat Syndrome].

Objective To observe the effect of Qingshen Granule (QG) on the immune function of chronic renal failure (CRF) patients with damp-heat syndrome (DHS). Methods A total of 162 CRF patients were assigned to the treated group and the control group by random digit table, 81 in each group. All patients were treated with conventional Western medical therapy. Those in the treated group addition- ally took QG, one package each time (10 g) , thrice per day. The therapeutic course for all was 12 weeks. The clinical efficacy was observed in the two groups. The contents of peripheral blood CD4 ⁺ T cells, CD8 ⁺ T cells, Thl7 cells, and nuclear factor-κB p65 (NF-κBp65) activity were detected using flow cy- tometry. Expressions of serum IL-17, tumor necrosis factor receptor-associated factor 6 (TRAF6), ma- trix metalloproteinase-9 (MMP-9) , matrix metalloproteinase inhibitor-I (TIMP-1 ) , collagen N (Col-V) were detected using ELISA. Results Finally 156 patients completed the trial (77 cases in the treated group and 79 cases in the control group). The total clinical curative effective rate was significantly higher in the treated group (80. 52%, 62/77) than in the control group (68. 35%, 54/79) with statistical differ- ences between the two groups (x² = 54. 849, P <0. 05). Compared with before treatment in the same group, the levels of peripheral blood CD4 ⁺ /CD8 ⁺ , Thl7 cell content, NF-κB p65 activity, serum levels of IL-17, TRAF6, and TIMP-1 , TIMP-1/MMP-9 ratio, Col-IV level all decreased in the treated group after treat- ment (P <0. 05) ; serum MMP-9 level decreased .(P <0. 05) and TIMP-1 /MMP-9 ratio increased (P <0. 05) in the control group. Compared with the control group, CD4⁺/CD8 ⁺ T cell ratio, Th17 cell content, NF-κB p65 activity decreased more obviously in the treated group after treatment (P <0. 05). Serum levels of IL- 17, TRAF6, TIMP-1, TIMP-1/MMP-9 ratio, and Col-IV all decreased (P <0.05) and MMP-9 level increased (P <0. 05) in the treated group (P <0. 05). Conclusion QG could adjust immune dysfunction and disar- ranged immunity mediated inflammatory response, and attenuate renal fibrosis in CKD patients with DHS.

Transcriptome analysis of indole-3-butyric acid-induced adventitious root formation in nodal cuttings of Camellia sinensis (L.).

Tea (Camellia sinensis L.) is a popular world beverage, and propagation of tea plants chiefly depends on the formation of adventitious roots in cuttings. To better understand potential mechanisms involved in adventitious root formation, we performed transcriptome analysis of single nodal cuttings of C. sinensis treated with or without indole-3-butyric acid (IBA) using the Illumina sequencing method. Totally 42.5 million RNA-Seq reads were obtained and these were assembled into 59,931 unigenes, with an average length of 732 bp and an N50 of 1292 bp. In addition, 1091 differentially expressed unigenes were identified in the tea cuttings treated with IBA compared to controls, including 656 up- and 435 down-regulated genes. Further real time RT-PCR analysis confirmed RNA-Seq data. Functional annotation analysis showed that many genes were involved in plant hormone signal transduction, secondary metabolism, cell wall organization and glutathione metabolism, indicating potential contributions to adventitious rooting. Our study presents a global view of transcriptome profiles of tea cuttings in response to IBA treatment and provides new insights into the fundamental mechanisms associated with auxin-induced adventitious rooting. Our data will be a valuable resource for genomic research about adventitious root formation in tea cuttings, which can be used to improve rooting for difficult-to-root varieties.

Floral transcriptome sequencing for SSR marker development and linkage map construction in the tea plant (Camellia sinensis).

Despite the worldwide consumption and high economic importance of tea, the plant (Camellia sinensis) is not well studied in molecular biology. Under the few circumstances in which the plant is studied, C. sinensis flowers, which are important for reproduction and cross-breeding, receive less emphasis than investigation of its leaves or roots. Using high-throughput Illumina RNA sequencing, we analyzed a C. sinensis floral transcriptome, and 26.9 million clean reads were assembled into 75,531 unigenes averaging 402 bp. Among them, 50,792 (67.2%) unigenes were annotated with a BLAST search against the NCBI Non-Redundant (NR) database and 10,290 (16.67%) were detected that contained one or more simple sequence repeats (SSRs). From these SSR-containing sequences, 2,439 candidate SSR markers were developed and 720 were experimentally tested, validating 431 (59.9%) novel polymorphic SSR markers for C. sinensis. Then, a consensus SSR-based linkage map was constructed that covered 1,156.9 cM with 237 SSR markers distributed in 15 linkage groups. Both transcriptome information and the genetic map of C. sinensis presented here offer a valuable foundation for molecular biology investigations such as functional gene isolation, quantitative trait loci mapping, and marker-assisted selection breeding in this important species.

[The determination of phthalic acid esters in edible vegetable oils by gas chromatography-mass spectrometry].

OBJECTIVE: We aimed to establish a quantified method for the 17 phthalate acid esters (PAE) in edible vegetable oil by gas chromatography-mass spectrometry (GC-MS) with the pretreatment of acetonitrile extraction and silica/N-(n-propyl)ethylenediamine (silica/PSA) mixed solid phase extraction column and evaluated the PAE of 25 edible oil samples from supermarkets in Hangzhou city. METHODS: The internal standard solution (D4-DEHP) was added in edible vegetable oil sample. The analytes were extracted by acetonitrile with 1 min vortex, and centrifuged at 3050×g for 5 min. The supernatant was then cleaned with silica/PSA column, and eluted with acetonitrile. The elution was dried with N2 flow at 50°C and diluted to 1.0 ml with hexane. Then, 17 PAE were tested by GC-MS and quantified with internal standards. The repeatability and sensitivity of the assay were evaluated. PAE were then determined in 25 plastic buckets of edible vegetable oil from supermarkets in Hangzhou city. RESULTS: By the quantification of internal standard of D4-DEHP, a good linearity range of related 17 PAE was observed. The correlation coefficient was 0.994-1.000 and the standard lowest quantified level was 0.05-0.15 µg/ml. The spiking recoveries of 17 PAE were 78.3%-108.9% with the RSD of 4.3%-12.1% (n=6). The method detection limits were 0.1-0.2 mg/kg. In 25 plastic buckets of edible vegetable oil from Hangzhou, DMP, DEP, DIBP, DBP and DEHP were detected at the range of <0.1-1.8 mg/kg and the detection rates were 12% (3/25), 24% (6/25), 100% (25/25), 96% (24/25) and 100% (25/25), respectively. Other 12 PAE was not detected. For DBP with the level of <0.1 to 1.3 mg/kg, the results of 16% (4/25) samples exceeded the regular migrating limit of 0.3 mg/kg. For DEHP of <0.2-1.8 mg/kg, the data of 12% (3/25) samples were beyond the regular migrating limit of 1.5 mg/kg. CONCLUSION: The pretreatment by silica/PSA mixed solid phase extraction column can satisfy the PAE determination requirements in edible vegetable oils. The DMP, DIBP, DEP, DBP and DEHP were detected from the survey of 25 edible oil samples in Hangzhou city.

[Identification of Xihu longjing tea by PLS model using near-infrared spectroscopy].

Protection of geographical indication for Xihu longjing tea was necessary and an accurate method of identification of Xihu longjing tea ought to be developed to prevent Xihu longjing tea from being faked and protect the trade mark of Xihu longjing tea. Identification of Xihu longjing tea with near-infrared spectrum was researched and a new method for identification of Xihu longjing tea by a quantitative model was developed. A new variance was first defined for Xihu longjing tea and other flat-shaped tea. And then the near-infrared spectrum without preprocess of tea was analyzed and partial least squares (PLS)was most suitably used to build the model (The number of PLS factors in this model is 9) for quantitative prediction. Compared with the critical numeral value, the result predicted by the PLS model was used to identify true Xihu longjing tea. The model was used to predict for 70 known samples which were used to build the quantitative model and 24 unknown samples. The recognition rate of 100% was achieved. So it is proved that the quantitative model by PLS was reliable and accurate to identify real Xihu longjing tea.