[Simultaneous determination of seven artemisinin-related compounds in Artemisia annua by UPLC-QQQ-MS/MS].

A variety of compounds in Artemisia annua were simultaneously determined to evaluate the quality of A. annua from multiple perspectives. A method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS) was established for the simultaneous determination of seven compounds: amorpha-4,11-diene, artemisinic aldehyde, dihydroartemisinic acid, artemisinic acid, artemisinin B, artemisitene, and artemisinin, in A. annua. The content of the seven compounds in different tissues(roots, stems, leaves, and lateral branches) of A. annua were compared. The roots, stems, leaves, and lateral branches of four-month-old A. annua were collected and the content of seven artemisinin-related compounds in different tissues was determined. A multi-reaction monitoring(MRM) acquisition mode of UPLC-QQQ-MS/MS was used, with a positive ion mode of atmospheric pressure chemical ion source(APCI). Chromatographic separation was achieved on an Eclipse Plus RRHD C_(18) column(2.1 mm×50 mm, 1.8 µm). The gradient elution was performed with the mobile phase consisted of formic acid(0.1%)-ammonium formate(5 mmol·L~(-1))(A) and the methanol(B) gradient program of 0-8 min, 55%-100% B, 8-11 min, 100% B, and equilibrium for 3 min, the flow rate of 0.6 mL·min~(-1), the column temperature of 40 ℃, the injection volume of 5 µL, and the detection time of 8 min. Through methodological investigation, a method based on UPLC-QQQ-MS/MS was established for the simultaneous quantitative determination of seven representative compounds involved in the biosynthesis of artemisinin. The content of artemisinin in A. annua was higher than that of artemisinin B, and the content of artemisinin and dihydroartemisinic acid were high in all the tissues of A. annua. The content of the seven compounds varied considerably in different tissues, with the highest levels in the leaves and neither artemisinene nor artemisinic aldehyde was detected in the roots. In this study, a quantitative method based on UPLC-QQQ-MS/MS for the simultaneous determination of seven representative compounds involved in the biosynthesis of artemisinin was established, which was accurate, sensitive, and highly efficient, and can be used for determining the content of artemisinin-related compounds in A. annua, breeding new varieties, and controlling the quality of Chinese medicinal materials.

AIE/AEE tripodal PEGyl-melphalan supramolecules and detection of trinitrophenol in water.

Herein, the design of a novel aggregation-induced emission (AIE) supramolecular fluorescence sensor (TA-PEGn) based on a tridentate melphalan derivative and three different molecular weight PEGs is presented. The three TA-PEGn sensors could self-assemble into a supramolecular system in water and show sensitive and selective responses toward trinitrophenol.

[Genome-wide identification of bZIP family genes and screening of candidate AarbZIPs involved in terpenoid biosynthesis in Artemisia argyi].

Artemisia argyi is an important medicinal and economic plant in China, with the effects of warming channels, dispersing cold, and relieving pain, inflammation, and allergy. The essential oil of this plant is rich in volatile terpenoids and widely used in moxi-bustion and healthcare products, with huge market potential. The bZIP transcription factors compose a large family in plants and are involved in the regulation of plant growth and development, stress response, and biosynthesis of secondary metabolites such as terpenoids. However, little is known about the bZIPs and their roles in A. argyi. In this study, the bZIP transcription factors in the genome of A. argyi were systematically identified, and their physicochemical properties, phylogenetic relationship, conserved motifs, and promoter-binding elements were analyzed. Candidate AarbZIP genes involved in terpenoid biosynthesis were screened out. The results showed that a total of 156 AarbZIP transcription factors were identified at the genomic level, with the lengths of 99-618 aa, the molecular weights of 11.7-67.8 kDa, and the theoretical isoelectric points of 4.56-10.16. According to the classification of bZIPs in Arabidopsis thaliana, the 156 AarbZIPs were classified into 12 subfamilies, and the members in the same subfamily had similar conserved motifs. The cis-acting elements of promoters showed that AarbZIP genes were possibly involved in light and hormonal pathways. Five AarbZIP genes that may be involved in the regulation of terpenoid biosynthesis were screened out by homologous alignment and phylogenetic analysis. The qRT-PCR results showed that the expression levels of the five AarbZIP genes varied significantly in different tissues of A. argyi. Specifically, AarbZIP29 and AarbZIP55 were highly expressed in the leaves and AarbZIP81, AarbZIP130, and AarbZIP150 in the flower buds. This study lays a foundation for the functional study of bZIP genes and their regulatory roles in the terpenoid biosynthesis in A. argyi.

[Chloroplast genome structure characteristics and phylogenetic analysis of Artemisia indica].

Artemisia indica is an important medicinal plant in the Asteraceae family, but its molecular genetic information has been rarely reported. In this study, the chloroplast genome of A. indica was sequenced, assembled, and annotated by the high-throughput sequencing technology, and its sequence characteristics, repeat sequences, codon usage bias, and phylogeny were analyzed. The results showed that the length of the chloroplast genome for A. indica was 151 161 bp, which was a typical circular four-segment structure, including two inverted repeat regions(IRs), a large single-copy(LSC) region, and a small single-copy(SSC) region, with a GC content of 37.47%. A total of 132 genes were annotated, and 114 were obtained after de-duplication, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Fifty long repeat sequences and 191 SSRs were detected in the chloroplast genome of A. indica, and SSRs were mainly single nucleotides. Codon usage bias analysis showed that leucine was the most frequently used amino acid(10.77%) in the chloroplast genome, and there were 30 codons with relative synonymous codon usage(RSCU)>1 and all ended with A/U. The phylogenetic tree constructed based on the chloroplast genomes of the 19 species from the Asteraceae family showed that A. indica and A. argyi were closest in the genetic relationship, and Artemisia species clustered into separate evolutionary branches. The results of this study are expected to provide a theoretical basis for the genetic diversity and resource conservation of Artemisia medicinal plants.

[Screening of reference genes for quantitative real-time PCR in Artemisia argyi].

Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.

Chemical profiling of Fritillariae thunbergii Miq prepared by different processing methods reveals two new quality markers: Zhebeininoside and imperialine-3-ß-D-glucoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae thunbergii Miq (FTM)exhibit versatile biological activities including the significant antitussive and expectorant activities. As a herbal medicine, the therapeutic effects of FTM may be expressed by multi-components which have complicated integration effects on multi-targets. With the time going, the different processing methods of FTM has been changed a lot. Thus，the study described the effect of processing methods to FTM and its quality. MATERIAL AND METHOD: Studies were undertaken by using UHPLC-LTQ Orbitrap MS and pharmacodynamic models. All reagents were involved of analytical grade. While a HPLC-ELSD's method has been developed and validated, a certified Quality System is conformed to ICH requirements. The experimental animals followed the animal welfare guidelines. AIM OF THE STUDY: We aimed to found the differences after the different processing methods of FTM, and to demonstrate the changes could be selected as quality control indicators, and established a method for simultaneous determination of these for quality control. RESULTS: we have previously found two new steroidal alkaloids: zhebeininoside and imperialine-3-ß-D-glucoside from the different processing methods of FTM, which is the difference between the different processing methods of FTM, mainly on the steroidal alkaloids. The activity analysis of zhebeininoside, imperialine-3-ß-D-glucoside, verticine and verticinone showed that the mouse model of cough expectorant has antitussive effect. The positive drug selected was dextromethorphan syrup. The positive group showed biological activity, but the blank group showed nothing. The model group showed illness which means that the model was effective. There are two ways of the mechanism of action of the expectorant action which can make sputum thin, reduce its viscosity, and be easy to cough up, or can accelerate the movement of mucous cilia in the respiratory tract and promote the discharge of sputum. In our study, the content of phenol red was significantly reduced in the administration group. CONCLUSIONS: To sum up, our results suggest that zhebeininoside and other three components cloud be selected as quality control indicators, and a method for simultaneous determination of zhebeininoside and other three components was established for quality control.

[Molecular genetics research of medicinal plants].

With the development of various biotechnology,the research on molecular genetics of medicinal plants has gradually deepened. In this paper,the research system of molecular genetics of medicinal plants was proposed for the first time,which was elaborated from the aspects of genetic resources,genome,gene function and research methods. The application fields of medicinal plant mainly contain species identification,molecular breeding and biosynthesis. The research directions of molecular genetics of medicinal plants in genetic resources,model platform,synthetic biology and molecular breeding were put forward,which include 1 000 genome projects of medicinal plants,model species and mutant libraries,gene original libraries of heterologous synthetic systems,construction gene original library and specific chassis cells in heterologous synthesis system of active ingredient,breeding of new varieties of medicinal plants with high active ingredient and high resistance based on molecular markers andtransgenes.

[Identification of 23 unknown Li minority medicinal plants based on DNA barcoding].

DNA barcode molecular biological technique is used to identify the species of 23 unknown Li minority medicinal plants.DNA was extracted from 23 unknown medicines using the Plant Genomic DNA Extraction kit. The ITS2 and psbA-trnH regions were amplified and sequenced bi-directionally. The Codon Code Aligner V 7. 0. 1 was used to proofread and assemble the contigs and generated consensus sequences. All the sequences were submitted to Traditional Chinese Medicine DNA Barcode Database and NCBI Gen Bank to get information of the species identifications. If the maximum similarity of the identification result is ≥ 97%,exact species can be known. If it is between 97% and 90%,samples' genus can be confirmed; If it is <90%,then we can only confirm its family. Finally there are 17 samples can be identified to species level,5 can be identified to genus level and 1 can be identified to family level. This shows that DNA barcoding used in medicinal plants molecular identification,can identify unknown species rapidly and accurately.

[Application of DNA metabarcoding in species identification of Chinese herbal medicines].

DNA metabarcoding,one rapid and robust method using specific standard DNA fragments,has been widely used for rapid species identification of a bulk sample through high-throughput sequencing technologies.While it has been widely used in the studies of metagenomics,animal and plant biodiversity,it has gradually come to be used as a profitable method in species identification of mixed Chinese herbal medicines.In this paper,we mainly summarize the current studies of the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines.Moreover,high-throughput sequencing technologies adopted in those studies,such as Sanger,the next-generation,and third-generation sequencing technologies,are discussed.It is conducted to provide a theoretical guidance for the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines and in more other biodiversity studies.

[Statues and research strategy of molecular breeding in Artemisia annua].

Malaria is one of the three most deadly diseases in the world. Artemisinin is the first line and effective drug for treating malaria, and only can be extracted from Artemisia annua. Therefore, it is of great significance to cultivate new varieties of A. annua with high artemisinin content. Based on the germplasm bank and the whole genome, transcriptome and genetic map, the authors can explore high-quality genes, stress-resistant genes and genetic markers which have been used for rapid breeding of superior varieties of A. annua. So these methods of molecular breeding will become the main breeding direction of A. annua in the future. The breeding times of new varieties of A. annua can be shortened with molecular breeding technology. Based on the genetic background and the current situation of molecular breeding of A. annua, the strategy and technical route of molecular breeding were discussed and worked out in this paper, which provided a guidance and scientific reference for molecular breeding of A. annua in the future.

[Effects of wide-range planting on the yield and nitrogen use efficiency of winter wheat cultivar Tainong 18.] / å®½å¹ æ’­ç§å¯¹å†¬å°éº¦'泰农18'äº§é‡å’Œæ°®ç´ åˆ©ç”¨çŽ‡çš„å½±å“.

The effects of wide-range planting (WR) versus drilling-planting (DP) on grain yield, nitrogen use efficiency (NUE), and nitrogen uptake efficiency (UPE) were investigated using winter wheat cultivar Tainong 18 at experimental fields in Tai'an and Yanzhou during the growing seasons of 2015 and 2016. The results showed that planting pattern, experimental field location, and their interaction significantly affected the grain yield, NUE, and related indices of cultivar Tainong 18. Compared to DP, the WR pattern significantly increased grain yield by 22.5% and 15.4% at Tai'an and Yanzhou, respectively, by raising the number of spikes per unit area at maturity (originating from the greater numbers of tillers per plant and per unit area) and the number of spikes per plant. Compared to DP, the WR pattern significantly increased UPE by 27.7% and 17.5% at Tai'an and Yanzhou, respectively. NUE with the WR pattern at Tai'an and Yanzhou was also increased, respectively, by 22.5% and 15.4% by enhancing nitrogen accumulation and increasing the UPE. A stonger positive effect on yield was observed at Tai'an than at Yanzhou. Thus, the popularization and application of a WR pattern would synergistically improve grain yields and NUE in winter wheat.

Complete Chloroplast Genomes from Sanguisorba: Identity and Variation Among Four Species.

The genus Sanguisorba, which contains about 30 species around the world and seven species in China, is the source of the medicinal plant Sanguisorba officinalis, which is commonly used as a hemostatic agent as well as to treat burns and scalds. Here we report the complete chloroplast (cp) genome sequences of four Sanguisorba species (S. officinalis, S. filiformis, S. stipulata, and S. tenuifolia var. alba). These four Sanguisorba cp genomes exhibit typical quadripartite and circular structures, and are 154,282 to 155,479 bp in length, consisting of large single-copy regions (LSC; 84,405⁻85,557 bp), small single-copy regions (SSC; 18,550⁻18,768 bp), and a pair of inverted repeats (IRs; 25,576⁻25,615 bp). The average GC content was ~37.24%. The four Sanguisorba cp genomes harbored 112 different genes arranged in the same order; these identical sections include 78 protein-coding genes, 30 tRNA genes, and four rRNA genes, if duplicated genes in IR regions are counted only once. A total of 39⁻53 long repeats and 79⁻91 simple sequence repeats (SSRs) were identified in the four Sanguisorba cp genomes, which provides opportunities for future studies of the population genetics of Sanguisorba medicinal plants. A phylogenetic analysis using the maximum parsimony (MP) method strongly supports a close relationship between S. officinalis and S. tenuifolia var. alba, followed by S. stipulata, and finally S. filiformis. The availability of these cp genomes provides valuable genetic information for future studies of Sanguisorba identification and provides insights into the evolution of the genus Sanguisorba.

Recent Progress in (Dynamic) Kinetic Resolution Catalyzed by N-Heterocyclic Carbenes.

Catalytic kinetic resolution is often the "method of choice" for the preparation of optically enriched functionalized compounds and, over the past decade, kinetic resolutions and dynamic kinetic resolutions catalyzed by N-heterocyclic carbenes (NHCs) have been employed for the synthesis of a variety of optically enriched functionalized molecules. Herein we provide an overview of reported NHC-catalyzed kinetic resolutions in terms of the structure of the substrate, the reaction type, and the activation mode. This Focus Review is arranged according to the structure of the racemic compounds and includes the kinetic resolution of alcohols, amines, imines, and aldehydes, as well as dynamic kinetic resolution. In each section, the activation mode and reaction mechanism are discussed. We conclude with a summary of the current state-of-play in NHC-catalyzed kinetic resolution and provide an outlook for the development of this field in the future.

[Influence of light on gene expression of key synthesis enzyme genes FtANR and FtLAR about proanthocyanidin in seeds of homologous plant of food and medicine Fagopyrum tataricum].

Tartary buckwheat Fagopyrum tataricum is an important medicinal and functional herb due to its rich content of flavonoids in the seeds. F.tataricum exhibited good functions for free radicals scavenging， anti-oxidation， anti-aging activities. Although much genetic knowledge of the synthesis， regulation， accumulation of rutin， the genetic basis of proanthocyanidins(PAs) in tartary buckwheat and their related gene expression changes under different lights(blue， red， far red， ultraviolet light) remain largely unexplored. In this study， we cloned one anthocyanidin reductase gene(ANR) and two leucocyanidin reductase gene(LAR) named FtANR，FtLAR1，FtLAR3 involved in formation of(+)-catechin and(-)-epicatechin precusor proanthocyanidin by digging out F. tataricum seed transcriptome data. The expression data showed that the opposite influence of red light on these gene transcript level compared to others lights. The expression levels of FtANR and FtLAR1 decreased and FtLAR3 appeared increment after exposed in the red light， while the expression levels of those genes appeared opposite result after exposed in the blue and far red light.

[Identification of herbal tea ingredient Plumeria rubra and its adulterants using DNA barcoding].

ITS2 sequence was used as a barcode to identify herbal tea ingredient Plumeria rubra and its adulterants. Genomic DNAs from forty eight samples were extracted, the ITS2 sequences were amplified and sequenced bi-direstionlly, and then assembled and obtained using CodonCode Aligner. The sequences were aligned using ClustalW, the genetic distances were computed by kimura 2-parameter (K2P) model and the Neighbor-joining (NJ) phylogenetic trees were constructed using MEGA5.0. Results showed that the length of ITS2 sequence of P. rubra were 244 bp. The intra-specific genetic distances (0-0. 016 6) were much smaller than inter-specific ones between P. rubra and its adulterants(0.320 8-0.650 4). The NJ tree indicated that P. rubra and its adulterants could be distinguished clearly. Therefore, Using ITS2 barcode can accurately andeffectively distinguish herbal tea ingredient P. rubra from its adulterants, which providesa new molecular method to identify P. rubra and ensure its safety in use.

Femtosecond laser-assisted deep anterior lamellar keratoplasty for keratoconus and keratectasia.

AIM: To describe the initial outcomes and safety of femtosecond laser-assisted deep anterior lamellar keratoplasty (DALK) for keratoconus and post-LASIK keratectasia. METHODS: In this non-comparative case series, 10 eyes of 9 patients underwent DALK procedures with a femtosecond laser (Carl Zeiss Meditec AG, Jena, Germany). Of the 9 patients, 7 had keratoconus and 2 had post-LASIK keratectasia. A 500 kHz VisuMax femtosecond laser was used to perform corneal cuts on both donor and recipient corneas. The outcome measures were the uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), corneal thickness, astigmatism, endothelial density count (EDC), and corneal power. RESULTS: All eyes were successfully treated. Early postoperative evaluation showed a clear graft in all cases. Intraoperative complications included one case of a small Descemet's membrane perforation. Postoperatively, there was one case of stromal rejection, one of loosened sutures, and one of wound dehiscence. A normal corneal pattern topography and transparency were restored, UCVA and BCVA improved significantly, and astigmatism improved slightly. There was no statistically significant decrease in EDC. CONCLUSION: Our early results indicate that femtosecond laser-assisted deep anterior lamellar keratoplasty could improve UCVA and BCVA in patients with anterior corneal pathology. This approach shows promise as a safe and effective surgical choice in the treatment of keratoconus and post-LASIK keratectasia.

[Estimation on the HIV-1 incidence among high risk groups using the pooling RNA technique].

OBJECTIVE: To timely identify the HIV-1 infection in window-period and to estimate the HIV-1 incidence among people who came for voluntary counseling and testing (VCT) service as well as men who have sex with men (MSM), respectively. METHODS: HIV antibody negative samples that were determined by screening tests between January and October 2012, were collected and tested with pooling HIV-1 RNA testing technique (2-staged pooling by 50:1, 10:1). Positive cases were followed-up for HIV antibody testing while HIV incidence was calculated under Ron Brookmeyer' s method, among VCT and MSM populations. RESULTS: Among 1400 HIV antibody negative samples of VCT, two showed HIV-1 RNA positive during the antibody window period with the HIV-1 incidence as 1.87% per year (95% CI: 1.23%-2.65% ). Among 500 HIV antibody negative samples from MSM population, two showed HIV-1 RNA positive in the antibody window period, with HIV-1 incidence as 5.31% per year (95% CI: 3.52%-7.45% ). CONCLUSION: Pooling HIV-1 RNA testing seemed a powerful tool for HIV antibody testing in the window-period. Measures should be taken to strengthen the HIV diagnostic programs among MSM and other high risk groups,during the HIV antibody window-period. More frequent detection approach as pooling HIV-1 RNA testing might be a good choice.

[Evaluation of PIMA analyzer detecting CD4 cell count of venous and capillary blood in HIV-infected individuals].

OBJECTIVE: This study is aimed at evaluating the utility of the portable CD4 analyzers (PIMA). METHODS: The paired finger prick blood (25 µl) and 5 ml venous blood samples were collected from 196 HIV infected patients, who came to Yunnan CDC voluntary counseling and testing (VCT) clinic for CD4 test services, from May to August, 2012. The absolute CD4 cell counts were measured by PIMA (using venous and finger-prick blood) and by Calibur (using venous blood) as the reference. The PIMA and Calibur CD4 results were compared using the Wilcoxon matched-pairs test, and the Spearman's rank correlation coefficients were estimated. The Bland-Altman plots were used to assess the consistency of the two methods. RESULTS: The median absolute CD4 counts of 196 venous blood samples obtained by PIMA and by Calibur were 268 (range:169-403) cells/µl and 302 (range:181-474) cells/µl respectively, which showed significant difference (Z = -7.31, P < 0.01). The median absolute CD4 counts measured by PIMA and by Calibur (using 188 finger-prick and venous blood samples respectively) were 271 (range: 165-450) cells/µl and 304 (range:188-476) cells/µl, which also showed significant difference (Z = -7.60, P < 0.01). The CD4 counts obtained by PIMA CD4 analyzer (using venous and finger-prick blood) showed strong positive correlation with the CD4 counts obtained by the reference method (using venous blood), and the r values were 0.94 and 0.92 respectively (P < 0.01) . The mean biases (limit of agreement) were -38.7 (-210.9-133.5)cells/µl and -45.4 (-221.8-131.0) cells/µl, respectively.Using 350 CD4 counts as the threshold for ART treatment initiation, the sensitivity and specificity of PIMA were 99.1% and 79.3% for venous blood samples, and 97.2%and 78.5% for finger-prick blood samples, respectively. CONCLUSION: The CD4 counts obtained by PIMA are lower than that obtained by Calibur, while the sensitivity is high.

A ferrocenyl-DHIPOH/Cu(OAc)2 complex for diastereo- and enantioselective catalysis of the 1,4-addition of glycine derivatives to alkylidene malonates.

A planar chiral ferrocene derivative Fc-DHIPOH served as an excellent N,O- chiral ligand for asymmetric copper catalyzed 1,4-addition of glycine derivatives to alkylidene malonates. The corresponding 1,4-adducts were obtained in high yields with excellent enantioselectivities up to 95% ee.