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High throughput sequencing technology is also called Next Generation Sequencing (NGS), which can sequence hundreds and thousands sequences in different samples at the same time. In the present study, the culture-independent high throughput sequencing technology was applied to sequence the fungi metagenomic DNA of the fungal internal transcribed spacer 1(ITS 1) in the root of Sinopodophyllum hexandrum. Sequencing data suggested that after the quality control, 22 565 reads were remained. Cluster similarity analysis was done based on 97% sequence similarity, which obtained 517 OTUs for the three samples (LD1, LD2 and LD3). All the fungi which identified from all the reads of OTUs based on 0.8 classification thresholds using the software of RDP classifier were classified as 13 classes, 35 orders, 44 family, 55 genera. Among these genera, the genus of Tetracladium was the dominant genera in all samples(35.49%, 68.55% and 12.96%).The Shannon's diversity indices and the Simpson indices of the endophytic fungi in the samples ranged from 1.75-2.92, 0.11-0.32, respectively.This is the first time for applying high through put sequencing technol-ogyto analyze the community composition and diversity of endophytic fungi in the medicinal plant, and the results showed that there were hyper diver sity and high community composition complexity of endophytic fungi in the root of S. hexandrum. It is also proved that the high through put sequencing technology has great advantage for analyzing ecommunity composition and diversity of endophtye in the plant.
A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(<i>p</i>-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-<i>f</i>]-9,10-phenanthrene (TSPP) in <i>N</i>,<i>N</i>-dimethylformamide (DMF) at 90 °C with anhydrous K<sub>2</sub>CO<sub>3</sub> as catalyst. A mixture of C<sub>1</sub>-C<sub>30</sub> fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C<sub>8</sub> column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines <i>Gentiana straminea</i> and <i>G. dahurica</i> was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were >0.9991. Relative standard deviations (RSDs, <i>n</i> = 6) for the fatty acid derivatives were <3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1-38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.
<br>Display Omitted<br>• Optimal dosages of phosphate and potassium fertilizer on <b>R. tanguticum</b> were firstly explored. • The U-shaped fluctuation curve of total anthraquinone content is firstly proposed. • Optimization of chromatographic columns was firstly proposed when detecting index constituents. • Total anthraquinone content of two-year-old plants had reached <b>Chinese Pharmacopoeia</b> standard.<br>The dried root of <b>Rheum tanguticum</b> plays an important role in formulations and prescriptions in traditional Chinese medicine and Kampo medicine. Due to over-exploitation, <b>R. tanguticum</b> resources have decreased sharply in recent years. The main objective of our investigation (a 3-year field experiment) was to explore the effect of different levels of phosphorus (superphosphate) and potassium (potassium sulfate) fertilizer on the biomass (root fresh weight, root increment, and root dry weight), yield, dry matter content, and anthraquinone content of this plant at different harvesting stages (green stage, growth stage, and wilting stage) under alpine conditions. The root fresh weight and root dry weight increased significantly at the wilting stage following treatment with 90 kg P2O5/ha (100% and 59%, respectively) in 2016 and 75 kg K2O/ha (43% and 41%, respectively) in 2015 compared to the control. The yield of root dry weight obtained from three-year-old <b>R. tanguticum</b> plants was 9200 kg/ha when 90 kg P2O5/ha of phosphorus fertilizer was applied, and 10,400 kg/ha when 75 kg K2O/ha of potassium fertilizer was applied. This yield reached a maximum at the wilting stage. The anthraquinone content of two-year-old <b>R. tanguticum</b> plants had already reached the standard level of the <b>Chinese Pharmacopoeia</b>; however, three-year-old plants had double the anthraquinone content of two-year-old plants. Phosphorus and potassium fertilizers had no obvious influence on the anthraquinone content of <b>R. tanguticum</b> at the same harvesting stage.
Concentrations of 20 free amino aicds (FAAs) in a famous Tibetan medicine Gentiana dahurica was first investigated using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) as the pre-column fluorescence derivatization reagent by reversed-phase high performance liquid chromatography (RP-LC). 20 amino acid derivatives (AAD) were separated on a Hypersil BDS C<sub>18</sub> column with a good baseline resolution within 65 min. Identification of 20 AAD was by online post-column mass spectrometry with an electrospray ionization (ESI) source. The validation of the method was examined by linearity, repeatability, and detection limits. Most linear correlation coefficients for AAD were >0.9990, and detection limits (at signal-to-noise of 3:1) were 6.5-178.2 fmol. There were 18 FAAs found in G. dahurica, of which seven FAAs were necessary to the people's health and related to the treatment of liver and gall disease. Variation of concentrations of the 20 FAAs showed geographical distribution difference among populations. Meanwhile a stable genetic diversity of FAAs composition of G. dahurica was also revealed at the species level. Results of the present study proved that the established method was rapid and reproducible for further separation and determination of FAAs in more medicinal plants.