A comprehensive study of the differences in protein expression and chemical constituents in tea leaves (Camellia sinensis var. sinensis) with different maturity using a combined proteomics and metabolomics method.

The maturity of tea leaves has a great influence on the flavor quality and commercial price of tea. In this work, a combined proteomics and metabolomics analysis was applied to investigate the differences in protein expression and metabolites among tea leaves with different maturity. Integrated analysis showed that there were significant differences in 112 nonvolatile components related to the pathways of photosynthesis, glycolysis, tricarboxylic acid cycle, and the biosynthesis of amino acids, phenylpropanoids and flavonoids. The bud had higher expression levels of most enzymes related to the biosynthesis of amino acids, phenylpropanoids, and flavonoids, leading to higher levels of amino acids, most flavanols, and procyanidins compared with the leaves. The 1st leaf showed a higher expression level of flavonol synthase, which produces higher levels of flavonol-3-glycosides. This study offers deep insight into the maturity of tea at both the protein and metabolite levels and provides a guideline for tea manufacturing.

Metabolomics combined with proteomics provides a novel interpretation of the compound differences among Chinese tea cultivars (Camellia sinensis var. sinensis) with different manufacturing suitabilities.

Different tea cultivars differ in their manufacturing suitability. In this study, metabolomics and proteomics were applied to investigate the metabolite and protein differences in fresh leaves from 23 Chinese tea cultivars suitable for manufacturing green, white, oolong, and black teas. The combined analysis revealed 115 differential metabolites and significant differences in the biosynthesis pathways for amino acids, phenylpropanoids, flavonoids, and terpenoids, and in the peroxidases abundances among these four groups. Green tea cultivars had higher abundances of amino acids and amino acids biosynthesis-related enzymes but lower abundances of flavanols and flavonoids biosynthesis-related enzymes. Black tea cultivars presented higher abundances of flavanols, flavanol-O-glycosides, flavonoids biosynthesis-related enzymes, and peroxidases. Oolong tea cultivars showed higher abundances of enzymes involved in terpenoids biosynthesis. Our study provides a novel interpretation of the manufacturing suitability of tea cultivars from the perspective of both metabolites and proteins and will be helpful for cultivar breeding.

Metabolomics Analysis Reveals Four Novel N-Ethyl-2-pyrrolidinone-Substituted Theaflavins as Storage-Related Marker Compounds in Black Tea.

Tea market is currently oversupplied, and unsold tea often needs to be properly stored for a period of time. However, the chemical changes occurring in black tea during storage are limitedly understood. In this study, a comprehensive nontargeted and targeted metabolomics approach was used to investigate the dynamic changes in compounds in time-series (0-19 months)-stored black teas. The contents of flavanols, theaflavins (TFs), theasinensins, procyanidins, most phenolic acids, amino acids, quercetin-O-glycosides, and myricetin-O-glycosides decreased during storage, while the contents of N-ethyl-2-pyrrolidinone-substituted flavanols, flavone-C-glycosides, and most kaempferol-O-glycosides increased. More importantly, four novel compounds strongly positively correlated with storage duration (r = 0.922-0.969) were structurally assigned as N-ethyl-2-pyrrolidinone-substituted TFs and validated with synthetic reactions of TFs and theanine standards. The content of N-ethyl-2-pyrrolidinone-substituted TFs was 51.54 µg/g in black tea stored for 19 months. To the best of our knowledge, N-ethyl-2-pyrrolidinone-substituted TFs were discovered in tea for the first time.

Antidepressant effects of total iridoids of Valeriana jatamansi via the intestinal flora-blood-brain barrier pathway.

CONTEXT: Valeriana jatamansi Jones [syn. V. wallichii DC, (Valerianaceae)] (VJJ) is used to treat depression. OBJECTIVE: To explore the effects of total iridoids of VJJ extract (TIV) on chronic unpredictable mild stress (CUMS) in mice. MATERIALS AND METHODS: VJJ roots and rhizomes were extracted with 70% ethanol. CUMS rats were treated daily with fluoxetine (2.6 mg/kg, i.g.) or TIV (5.7, 11.4, and 22.8 mg/kg, i.g.) for 14 days. Male Kun Ming mice on normal chow and 0.5% CMC-Na solution were used as a control. Behavioural tests included the tail suspension (TST) and sucrose preference tests (SPT). Evans blue staining was used to evaluate blood-brain barrier (BBB) permeability. Western blotting was used to measure zonula occludens-1 (ZO-1) and occludin expression. 16S rRNA sequencing was used to analyse intestinal flora abundance. Tax4Fun was used to predict KEGG metabolic pathways. RESULTS: TIV treatment reduced TST time (117.35 ± 8.23 or 108.95 ± 6.76 vs. 144.45 ± 10.30 s), increased SPT (55.83 ± 7.24 or 53.12 ± 13.85 vs. 38.98 ± 5.43%), increased the abundance of phylum Firmicutes (86.99 ± 0.03 vs. 60.88 ± 0.19%) and genus Lactobacillus (75.20 ± 0.19 vs. 62.10 ± 0.13%), reduced the abundance of phylum Bacteroidetes (6.69 ± 0.06 or 11.50 ± 0.09 vs. 25.07 ± 0.20%). TIV increased carbohydrate metabolism (14.50 ± 3.00 × 10-3 or 14.60 ± 2.00 × 10-3 or 14.90 ± 2.00 × 10-3 vs.13.80 ± 4.00 × 10-3%), replication and repair functions (5.60 ± 1.00 × 10-3 or 5.60 ± 1.00 × 10-3 vs. 5.10 ± 4.00 × 10-3%), reduced the frequency of infectious disease (1.60 ± 2.00 × 10-4 or 1.90 ± 5.00 × 10-4 or 1.80 ± 3.00 × 10-4 vs. 2.20 ± 7.00 × 10-3%), BBB permeability (0.77 ± 0.30 vs. 1.81 ± 0.33 µg/g), and up-regulated the expression of ZO-1 (1.42-fold, 1.60-fold, 1.71-fold) and occludin (1.79-fold, 2.20-fold). CONCLUSIONS: TIV may modulate the intestinal flora, thereby inducing the expression of ZO-1 and occludin, protecting the BBB and exerting an antidepressant effect.

Kaempferol attenuates streptozotocin-induced diabetic nephropathy by downregulating TRAF6 expression: The role of TRAF6 in diabetic nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Kaempferia rhizome is a famous traditional herbal medical in tropical and subtropical areas. Kaempferol (KPF) is one of the main bioactive compounds in Kaempferia rhizome, with anti-oxidant/anti-inflammatory effects demonstrated in various disease models, including cancers, obesity and diabetes. AIM OF THE STUDY: Inflammation plays an important role in the pathogenesis of diabetic nephropathy (DN). TRAF6 functions as a signal transducer in toll-like receptor 4 and NF-κB pro-inflammatory signaling pathway. We aimed at investigate whether KPF is able to mitigate inflammatory responses by regulating TRAF6 in DN. MATERIAL AND METHODS: C57BL/6 mice were injected with streptozotocin to induce type 1 DN. NRK-52E, a tubular epithelial cell line, was used for in vitro analysis. TRAF6 was knockdown using siRNA in vitro and AAV2/2-shRNA in vivo. The anti-DN and inflammatory effects of KPF or knockdown of TRAF6 were evaluated by investigating renal filtration index, pathological changes of kidney tissue. Proinflammatory cytokine levels were detected using ELISA. NF-κB pathway and protein levels of related pathways were detected through Western blot. RESULTS: KPF significantly reduced renal inflammation, fibrosis, and kidney dysfunction in diabetic mice. These effects were associated with a downregulation of TRAF6 in diabetic mouse kidneys, indicating the potential role of TRAF6. Knockdown of TRAF6 in mice through AAV2-shTRAF6 confirmed the importance of TRAF6 in DN. In vitro, treatment of KPF in NRK-52E cells attenuated high glucose (HG)-induced inflammatory and fibrogenic responses, associated with downregulated TRAF6 expression. The conclusion was further confirmed in NRK-52E cells by knocking down the expression and by overexpression of TRAF6. CONCLUSION: Our findings provide direct evidence that TRAF6 mediates diabetes-induced inflammation leading to renal dysfunction. We also show that KPF is a potential therapeutic agent to reduce inflammatory responses in DN. Also, TRAF6 may represent an interesting target to combat DN.

Transcriptomic analysis of the orchestrated molecular mechanisms underlying fruiting body initiation in Chinese cordyceps.

Chinese cordyceps, the fruiting body of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is among the most valuable traditional Chinese medicine fungi. Transcriptomic analysis of O. sinensis has revealed several aspects of its life cycle and ecological importance. However, the molecular mechanisms involved in fruiting body initiation remain unclear. The developmental transcriptomes were analyzed from three tissues at the fruiting body initiation stage, namely, the mycelium, sclerotium and primordium. Principal component analysis showed that in the three tissues, the gene expression patterns differed from each other. The functional analysis of differentially expressed genes showed that DNA synthesis and cell division were active in the primordium. In addition, the function of the mycelium was to absorb certain substances from the environment and the sclerotium was the metabolism center of O. sinensis. Genes participating in the mitogen-activated protein kinase (MAPK) signal pathway were involved in fruiting body initiation. Two environmental sensing genes, including a pheromone receptor gene (OSIN6252) and an amino acid sensing gene (OSIN6398), were highly expressed in the primordium, suggesting their important roles in initiation. These results provided insights into the orchestrated functions and gene profiles of different O. sinensis tissues at the key stage. These findings will aid in revealing the underlying mechanisms of fruiting body initiation, which will further benefit artificial cultivation.

A New High-Quality Draft Genome Assembly of the Chinese Cordyceps Ophiocordyceps sinensis.

Ophiocordyceps sinensis (Berk.) is an entomopathogenic fungus endemic to the Qinghai-Tibet Plateau. It parasitizes and mummifies the underground ghost moth larvae, then produces a fruiting body. The fungus-insect complex, called Chinese cordyceps or "DongChongXiaCao," is not only a valuable traditional Chinese medicine, but also a major source of income for numerous Himalayan residents. Here, taking advantage of rapid advances in single-molecule sequencing, we assembled a highly contiguous genome assembly of O. sinensis. The assembly of 23 contigs was ∼110.8 Mb with a N50 length of 18.2 Mb. We used RNA-seq and homologous protein sequences to identify 8,916 protein-coding genes in the IOZ07 assembly. Moreover, 63 secondary metabolite gene clusters were identified in the improved assembly. The improved assembly and genome features described in this study will further inform the evolutionary study and resource utilization of Chinese cordyceps.

Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis.

Ophiocordyceps sinensis is an entomopathogenic fungus that infects ghost moth larva, forming the most valuable and rare traditional Chinese medicine, Chinese cordyceps. Our knowledge of the basic morphology and developmental biology of Chinese cordyceps is limited. In this study, morphological and ultrastructural observations of O. sinensis development in the hemocoel of Thitarodes xiaojinensis were obtained by multiple light and electron microscopy techniques, and the host immune reaction activities were determined. Our results indicated that fungal cells in the host hemocoel underwent morphotype transformations from blastospores to prehyphae to hyphae in sequence. The fusiform yeast-like blastospores were the initial cell type present in the host hemocoel and remained for 5 months or more; the encapsulation reaction and phenoloxidase activity of T. xiaojinensis hemolymph were inhibited during this period. When larvae entered the last instar, the blastospores switched to prehyphae and expanded throughout the host tissues, and then hyphae germinated from the prehyphae and mycelia formed, which finally led to host death. Considering the distinct differences between blastospores and hyphae, we identified prehyphae, which play important roles in fungal expansion, hyphae germination, and fusion formation among filaments. Notably, the elongation of prehyphae was strongly presumed to occur through fission but without separation of the two sister cells, in contrast to blastospore budding. During the morphotype transformation, the amount and composition of lipid droplets changed greatly, suggesting their important roles in these events. Overall, we provide a morphological and ultrastructural characterization of O. sinensis vegetative development within the hemocoel of T. xiaojinensis, identify and name the prehypha fungal cell type in entomopathogenic fungi for the first time, and conclude that O. sinensis infection causes sustained immunosuppression in T. xiaojinensis.

[Metabolic profiling analysis of amino metabolites in plant extract based on pre-column derivatization-ultra high performance liquid chromatography-mass spectrometry].

Amino metabolites are important compounds that play a key role in plant growth and development. A metabolic profiling analysis method of amino metabolites in plant extract was developed based on pre-column derivatization-ultra high performance liquid chromatography- mass spectrometry. Using the tobacco leaf as an example, a total of 87 amino metabolites, including amino acids, amines, peptides, alkaloids etc. were detected. The repeatability of the method was good with RSDs of 85 amino metabolites between 1. 5% and 18. 8%. Forty-three amino metabolites validated by standard samples showed good linearity with the correlation coefficients of 0.993-0.999, covered linear range of four orders of magnitude. The limits of detection were 0.03-6.58 ng/mL. The intra-day and inter-day precisions were 0.7%-15.6% and 0.8%-22.9%, respectively. The recoveries were 74.4%-122.7%. The influence of topping on metabolic profiling of amino metabolites in fresh tobacco was investigated using the developed method. The results showed that the amino metabolites in the upper tobacco leaves were most affected than those in the middle and lower leaves. Metabolism of amino metabolites in the upper leaves after topping was mainly towards the alkaloid synthesis. The method integrated the advantages of triple quadrupole mass spectrometry and high resolution quadrupole-time of flight mass spectrometry. It can be used for metabolic profiling analysis of amino metabolites in plant extract with high sensitivity and selectivity.

Metabolomics study of cured tobacco using liquid chromatography with mass spectrometry: method development and its application in investigating the chemical differences of tobacco from three growing regions.

Cured tobacco is an important plant material. Component studies are a big challenge for its significantly diverse chemical properties and vastly different concentrations. In this work, liquid chromatography with quadrupole time-of-flight mass spectrometry was used to perform a metabolomics study of cured tobacco owing to its efficient separation and detection of semipolar metabolites. A solvent of methanol/water (8:2, v/v) and 30 min of ultrasound time were found to be optimal to perform extraction. 95, 92, and 93% of metabolite features had within 20% of coefficient of variation for repeatability, intraday and interday precision analysis, respectively, indicating a good stability of the method developed. 113 metabolites were identified in cured tobacco based on accurate mass, retention time, and MS/MS fragments. The developed method was applied to a metabolomics study of cured tobacco from three growing regions. Forty three metabolites were found to be contributed to the classification. It is shown that the developed method can be applied to metabolomics analysis of plant materials.