[Analysis and evaluation on Paeoniae Radix Alba from different cultivars by UPLC-Q-TOF-MS and HPLC].

In this study, an established ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) method was combined with multivariate statistical analysis to investigate the commonality and difference of main chemical components in the medicinal parts of Paeonia lactiflora from different cultivars; in addition, a high performance liquid chromatography(HPLC) method was established to simultaneously determine the content of eight active components in Paeoniae Radix Alba. Non-targeted analysis was carried out by UPLC-Q-TOF-MS on a Waters ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm, 1.7 µm) column with a gradient elution of 0.1% aqueous formic acid(A)-acetonitrile(B) as the mobile phase at a flow rate of 0.2 mL·min~(-1). The column temperature was 30 ℃, and an electrospray ionization source was used to acquire mass spectrometry data in positive and negative ion modes. According to the accurate molecular weight and fragment ion information provided by multi-stage mass spectrometry and by comparison with reference substances and literature reports, thirty-six identical components were identified in Paeoniae Radix Alba from different cultivars with positive and negative ion modes. In the negative ion mode, two groups of samples were well separated; specifically, seventeen components with significant differences in content were screened and identified, and one component unique in "Bobaishao" was obtained. Quantitative analysis was conducted by high-performance liquid chromatography(HPLC) on an Agilent HC-C_(18)(4.6 mm×250 mm, 5 µm) column with a gradient elution of 0.1% aqueous phosphoric acid(A)-acetonitrile(B) as the mobile phase at a flow rate of 1.0 mL·min~(-1). The column temperature was 30 ℃ and the detection wavelength was at 230 nm. An HPLC method was developed for the simultaneous determination of eight active components(gallic acid, oxypaeoniflorin, catechin, albiflorin, paeoniflorin, galloylpaeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, benzoyl-paeoniflorin) in Paeoniae Radix Albaa from different cultivars. Satisfactory linearity was achieved within the investigated linear ranges and with fine coefficients(r>0.999 0), and the methodological investigation showed that the method had good precision, repeatability and stability. The mean recoveries were 90.61% to 101.7% with RSD of 0.12% to 3.6%(n=6). UPLC-Q-OF-MS provided a rapid and efficient qualitative analytical method for the identification of the chemical components in Paeoniae Radix Alba, and the developed HPLC method was simple, rapid and accurate, which could provide a scientific basis for the evaluation of the germplasm resources and herbal quality of Paeoniae Radix Alba from different cultivars.

[Comparison of transcriptome of Atractylodes lancea rhizome and exploration of genes for sesquiterpenoid biosynthesis].

This study compared the transcriptome of Atractylodes lancea rhizome at different development stages and explored genes encoding the key enzymes of the sesquiterpenoid biosynthesis pathway. Specifically, Illumina NovaSeq 6000 was employed for sequencing the cDNA libraries of A. lancea rhizome samples at the growth stage(SZ), flowering stage(KH), and harvesting stage(CS), respectively. Finally, a total of 388 201 748 clean reads were obtained, and 16 925, 8 616, and 13 702 differentially expressed genes(DEGs) were identified between SZ and KH, KH and CS, and SZ and CS, separately. Among them, 53 genes were involved in the sesquiterpenoid biosynthesis pathways: 9 encoding 6 enzymes of the mevalonic acid(MVA) pathway, 15 encoding 7 enzymes of the 2-C-methyl-D-erythritol-4-phosphate(MEP) pathway, and 29 of sesquiterpenoid and triterpenoid biosynthesis pathway. Weighted gene co-expression network analysis(WGCNA) yielded 12 genes related to sesquiterpenoid biosynthesis for the SZ, 1 gene for the KH, and 1 gene for CS, and several candidate genes for sesquiterpenoid biosynthesis were discovered based on the co-expression network. This study laid a solid foundation for further research on the sesquiterpenoid biosynthesis pathway, analysis of the regulation mechanism, and mechanism for the accumulation of sesquiterpenoids in A. lancea.

[Multiplex PCR method for simultaneous identification of Xanthii Fructus and its adulterants].

The purpose of this study is to establish a molecular method to identify Xanthii Fructus and two adulterants, the fruits of Xanthium mongolicum and X. italicum. Xanthii Fructus is the fruit of X. sibiricum, which is a Chinese herbal medicine used clinically to treat allergic rhinitis. The fruits of X. mongolicum and X. italicum have strong morphological similarities with Xanthii Fructus, while their safety of medication cannot be guaranteed. The genomes of X. sibiricum, X. mongolicum, and X. italicum were sequenced, which generated sequences of 2.21, 2.24, and 2.54 Gb, respectively. Based on the 76 specific contigs screened out by BLASTN and Bowtie 2, the corresponding primers were designed by Primer 5.0. Three pairs of primers with stable amplification efficiency and good reproducibility were screened out to establish a multiplex PCR method based on the PCR amplification results. Further, the annealing temperature, the amount of DNA template, the number of cycles, different DNA polymerases, and different PCR thermal cyclers were optimized. Fragments of 262 bp and 458 bp from X. sibiricum, 260, 454, and 927 bp from X. mongolicum, and 260 bp and 926 bp from X. italicum were amplified under the following conditions: the annealing temperature of 52 ℃, 35 cycles, 30 ng template DNA. Then, the established method was used to detect 18 samples of X. sibiricum, 17 samples of X. mongolicum, and 12 samples of X. italicum. The results showed that all the samples had positive results, which were consistent with the morphological identification results, thus proving the stability and reliability of the established method. Combining genome sequencing technology and multiplex PCR method to identify Xanthii Fructus and its adulterants can not only obtain the difference in genetic background but also facilitate the design of reliable primers. The multiplex PCR have high specificity and repeatability, providing a new method for the molecular identification of Xanthii Fructus.

Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum.

Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs' biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, and the enzyme reaction products were detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) methods. The results indicated that both XsGAO1 and XsGAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum.

[Germacrene-derived sesquiterpene lactones:opportunities and challenges for biosynthesis].

Sesquiterpene lactones are a kind of widely distributed natural organic compounds with anti-tumor, anti-malarial and other significant biological activities. Based on their carbocylic skeletons, sesquiterpene lactones are classified into germacranolide, guaia-nolide, xanthanolide, pseudo-guaianolide, elemonolide and eudesmanolide, etc. In recent years, with the development of various omics and synthetic biology technologies, the biosynthetic pathways of sesquiterpene lactone compounds of different structural types have gradually been resolved. Among them, the researches on germacrene-derived sesquiterpene lactones are relatively more than others. Therefore, this article focused on the germacrene-derived sesquiterpene lactone biosynthesis pathways and their key enzyme genes, which can lay the foundation for in-depth analysis of sesquiterpene lactone biosynthetic pathways, functional gene mining and heterologous synthesis of active ingredients.

[Cloning and quantitative expression analysis of GMPP gene from Dendrobium huoshanense].

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.

[Next generation sequencing and transcriptome analysis of root bark from Paeonia suffruticosa cv. Feng Dan].

Moutan Cortex is an important traditional Chinese medicine, "Fengdan Pi" was known as Dao-di herbs from the root bark of Paeonia suffruticosa cv. Feng Dan for its extracted various active components. However, the genetic basis for their activity is virtually unknown. The transcriptome of the root bark from "Fengdan" was sequenced using the Illumina HiSeq 4000 sequencing platform. The clean reads were then de novo assembled into 72 997 unigenes. Among them, the number of unigenes which could been annotated by dataset Nr and GO was 41 139 and 34 592. The 20 016 unigenes could been annotated by KEGG dataset, which were involved in 5 major categories, 34 middle categories, and 352 metabolism pathways. The number of unigenes which were mapped to the phenylpropanoid biosynthesis pathway, terpenoid backbone biosynthesis pathway, terpenoid biosynthesis pathway, alkaloid biosynthesis pathway, and flavonoid biosynthesis pathway was 214, 104, 152, 55 and 36 respectively, suggesting that they are involves in these pathways of pharmaceutically important. Furthermore, there also showed remarkable differences in groups which enrichment ratio of the different expressed gene compared. In addition, a total of 9 939 SSRs were identified from the sequence of 72 997 unigenes. This study not only provides many valuable basal data which was important gene in the synthesis pathway of secondary metabolites with gene searching, but also has important significance to find molecular marker in germplasm for breeding and improvement.

[Screening target genes for bimolecular marking methods of Magnolia quality].

The study used use bimolecular marking methods to evaluate the lignans of Magnolia officinalis and M. officinalis var. biloba. First, we compare the chemical constituents between M. officinalis and M. officinalis var.biloba. There were significant differences in concentration of magnolignan I between leaves of these two varieties. Then we further select the p-hydroxyphenyl lignin to mining the key enzyme genes of biosynthesis from Magnolia transcriptome, and screened an encoding cinnamyl alcohol dehydrogease gene as the candidate marker of bimolecular marking methods of Magnolia quality by comparing of the expression level and structure variation in homologous gene between M. officinalis and M. officinalis var.biloba. The established method provides the technical support for bimolecular marking methods of Magnolia quality evaluation.

[Herbal textural research on species of Xanthii Fructus].

Xanthii Fructus is a traditional medicine for the treatment of nasal diseases in clinic, mainly come from the burs of Xanthium sibiricum with a worldwide distribution. By sorting and studying literature of Chinese medicine and comparing different figures recorded with the morphological description of several species from Xanthium (Asteraceae) in the Flora of China, combining the biological investigation in resource survey, the article pointed out that the burs or the whole herbs of X. mongolicum, as well as X. sibiricum, has been used by the traditional Chinese medicine in ancient time. It provides a reference for further studies in the future.

[Introduction of traditional medicinal plants in Kyrgyzstan].

Kyrgyzstan is a mountainous country in the northeastern part of Central Asia which shares borders to the southeast with China. Due to their extreme environment and climate, there are a diverse range of species of plants. Many of the plants used in Kyrgyz folk medicine have not been studied using modern scientific techniques. This paper introduced the basic situation of medicinal herbs in Kyrgyzstan by comparing the differences traditional use between China and Kyrgyzstan, and looked for traditional medicinal plant research to provide basis for the development and cooperation of China and Kyrgyzstan.