[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.

[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.

[Construction strategies and prospect of the Global Medicinal Plant Stem Cell Bank].

Medicinal plant stem cells are separated from the meristem and vascular cambium of medicinal plants, which can produce active components for preventing and treating diseases and improving body physical functions under certain conditions. Medicinal plant stem cells come from a broad category of medicinal plants, including ethnic medicinal plants, folk medicinal plants, original plants of health products, vegetables, fruits, and other potential medicinal plants. At present, the techniques for the isolation, identification, preservation and culture of medicinal plant stem cells have become increasingly mature, and the mechanism of stem cell differentiation, growth and regulation of secondary metabolites has been studied in depth. Medicinal plant stem cells have a broad application prospect in medicine, health food, food and medical beauty products. As a strategic resource, the construction of the "Global Medicinal Plant Stem Cell Bank" was first proposed to preserve various kinds of medicinal plant resources in the world, and it will go global relying on the internationalization strategy of traditional Chinese medicine. The bank should follow safety, environmental protection, advanced and practical design principles. The main construction contents include the original plant bank, stem cell bank, component resource bank, gene bank, database and resource sharing system, with genetic and data resources incorporated into the scope of protection and utilization. The bank will establish a new strategy for medicinal plant resources protection and regeneration, and provide a new resource for natural products drug discovery and a technology sharing platform for various medicinal plant stem cells. As a resource treasury, a source of innovative technologies and a center of cooperation, it will become the core driving force of the global medicinal plant stem cell industry.

[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.

Microbial transformation of pseudoprotodioscin by Gibberella fujikuroi.

Three new (6, 9, and 12) and nine known steroidal saponins were obtained from the fermentation broth of pseudoprotodioscin (PPD) incubated with a fungus Gibberella fujikuroi CGMCC 3.4663. Structures of the metabolites were elucidated by 1-D (1H, 13C), 2-D (HMBC, HSQC, NOESY) NMR, and HR-MS analyses. The biotransformation pathway of pseudoprotodioscin by Gibberella fujikuroi CGMCC 3.4663 was proposed. Compounds 1-11 were tested in vitro for their cytotoxic activities against two human cancer cell lines (HepG2 and Hela). Compounds 1, 6, 9, and 10 exhibited cytotoxic activity against HepG2 cells. Compound 10 exhibited cytotoxicity to Hela cells.

[Explore microcosmic connection between autophagy mechanism and follicular development based on "kidney governing reproduction" theory].

In the theory of traditional Chinese medicine(TCM) that "kidney storing essence and governing reproduction", reproductive essence is an important part of the kidney essence and acts as the original material of offspring embryos. Sperm, oocyte and zygote should be all included in the range of reproductive essence. Ovum is the essence of reproduction from inborn. The follicles maturation depends on the quality of oocyte and the vigor of kidney essence. Meanwhile, discharge of mature ovum relies on the stimulation and promotion by kidney Qi. Autophagy almost exists in different cells stages and all various of mammalian cells. Many studies have found that autophagy not only participates in the formation of follicles, but also in every phase of the follicles development, and is involved in the occurrence and development of ovarian diseases. Recently, more and more scholars believe that autophagy is a new field to explore the microcosmic relationship between autophagy and TCM. Kidney-nourishing TCM could promote follicular growth and improve variety clinical symptoms by inhibiting the apoptosis of ovarian granulosa cells and reducing follicular atresia. Meanwhile, apoptosis of ovarian granulosa cells is closely related to autophagy of ovarian granulosa cells. In order to provide some theoretical foundation for kidney-nourishing therapy's promoting effect on follicular growth and improving effect on ovarian function, also to further explore the molecular mechanism of kidney-nourishing medicine in promoting follicular development, this paper would explain the microcosmic relationship between autophagy and follicular development based on the theory of "kidney governing reproduction". All of these would be of great significance to prevent and intervene the diseases of reproductive system timely and effectively.

Unusual microbial lactonization and hydroxylation of asiatic acid by Umbelopsis isabellina.

Asiatic acid (1) is a natural triterpenoid isolated from Centella asiatica. This paper reports the microbial transformation of asiatic acid by an endophytic fungus Umbelopsis isabellina to obtain derivatives potentially useful for further studies. Incubation of asiatic acid with U. isabellina afforded two derivatives 2α,3ß,7ß, 23-tetrahydroxyurs-12-ene-28-oic acid (2) and 2α,3ß,7ß,23-tetrahydroxyurs-11-ene-28,13-lactone (3). The structures of these compounds were elucidated by spectral data. Compound 3 has formed an unusual lactone. These two products are new compounds. The possible transformation passway was also discussed.

Yi-Shen-Hua-Shi granule ameliorates diabetic kidney disease by the "gut-kidney axis".

ETHNOPHARMACOLOGICAL RELEVANCE: Yi-Shen-Hua-Shi (YSHS) granule is an effective prescription widely used in traditional Chinese medicine to treat diabetic kidney disease (DKD), its exact efficacy in treating DKD has been confirmed but the underlying regulatory mechanism has not been fully elucidated. AIM OF THE STUDY: To explore the mechanism by which YSHS granule regulates intestinal flora and serum metabolites and then regulates renal mRNA expression through the "gut-kidney axis", so as to improve DKD. MATERIALS AND METHODS: 40 rats were divided into five groups: Normal group (N) (normal saline), model group (M) (STZ + normal saline), YSHS granule low-dose group (YL) (STZ + 2.27 g kg-1 d-1), YSHS granule high-dose group (YH) (STZ + 5.54g kg-1 d-1) and valsartan group (V) (STZ + 7.38mg kg-1 d-1). After 6 weeks, changes in blood glucose, blood lipids, and renal function related indexes were observed, as well as pathological changes in the kidney and colon. Intestinal microbiota was sequenced by 16S rDNA, serum differential metabolites were identified by LC-MS/MS, and renal differences in mRNA expression were observed by RNA-seq. Further, through the association analysis of intestinal differential microbiota, serum differential metabolites and kidney differential mRNAs, the target flora, target metabolites and target genes of YSHS granule were screened and verified, and the "gut-metabolism-transcription" co-expression network was constructed. RESULTS: In group M, blood glucose, blood lipid and proteinuria were increased, inflammation, oxidative stress and renal function were aggravated, with the proliferation of mesangial matrix, vacuolar degeneration of renal tubules, accumulation of collagen and lipid, and increased intestinal permeability, and YSHS granule and valsartan improved these disorders to varying degrees. High dose of YSHS granule improved the diversity and abundance of flora, decreased the F/B value, greatly increased the abundance of Lactobacillus and Lactobacillus_murinus, and decreased the abundance of Prevoella UCG_001. 14 target metabolites of YSHS granule were identified, which were mainly enriched in 20 KEGG pathways, such as Glycerophospholipid metabolism, Sphingolipid metabolism and Phenylalanine, tyrosine and tryptophan biosynthesis. 96 target mRNAs of YSHS granule were also identified. The enriched top 20 pathways were closely related to glucose and lipid metabolism, of which a total of 21 differential mRNAs were expressed. Further correlation analysis revealed that Lactobacillus, Lactobacillus_murinus and Prevotella UCG_001 were highly correlated with Glycerophospholipid metabolism, Sphingolipid metabolism and Phenylalanine, tyrosine and tryptophan biosynthesis pathways. At the same time, 6 pathways including Glycerophospholipid metabolism, Arachidonic acid metabolism, Purine metabolism, Primary bile acid biosynthesis, Ascorbate and aldarate metabolism and Galactose metabolism were co-enriched by the target metabolites and the target mRNAs of YSHS granule, including 7 differential metabolites such as phosphatidylethanolamine and 7 differential genes such as Adcy3. The 7 differential metabolites had high predictive value of AUC, and the validation of 7 differential genes were highly consistent with the sequencing results. CONCLUSION: YSHS granule could improve DKD through the "gut-kidney axis". Lactobacillus and Lactobacillus_murinus were the main driving forces. 6 pathways related to glucose and lipid metabolism, especially Glycerophospholipid metabolism, may be an important follow-up response and regulatory mechanism.

[Chemical Reaction Activity and Source Apportionment of Atmospheric VOCs in Summer in Dalian].

To investigate the characteristics and source apportionment of atmospheric volatile organic compounds (VOCs) in Dalian, the concentration level, composition characteristics, reaction activity, and source apportionment of atmospheric VOCs in Dalian were analyzed based on high-resolution online observation VOCs data from June to August 2020. The results showed that the average φ(VOCs) was (10.21±5.71)×10-9, in which alkanes accounted for 66.35%, alkenes for 11.89%, alkynes for 7.75%, and aromatics for 14.01%. VOCs and NOx were high at night and low during the day, whereas the change trend of O3 was opposite. Considering the species activity, it was determined that toluene, ethylene, m/p-xylene, 1-hexene, n-butane, isopentane, n-pentane, and isoprene were the key species affecting atmospheric VOCs in Dalian. Priority control of olefin and aromatic hydrocarbon emissions is the key to improve O3 pollution in summer in Dalian. By applying the positive matrix factorization (PMF) model, six major VOCs sources were extracted, namely traffic sources (26.38%), combustion sources (22.75%), industrial emission sources (17.09%), solvent usage sources (14.59%), natural sources (11.72%), and others (7.47%). The emissions of traffic sources and combustion sources are the key pollution sources for O3 prevention and control in Dalian in summer.

Comparative Genomic and Phylogenetic Analysis of Forty Gentiana Chloroplast Genomes.

BACKGROUND: Gentiana plants, which have great medicinal and ornamental value, are widely distributed in diverse habitats and have complex taxonomy. Here 40 Gentiana chloroplast genomes were used for comparative genomic analysis and divergence time estimation. METHODS: The complete chloroplast genome of G. rhodantha was sequenced, assembled, and annotated. Comparative genomic and phylogenetic analysis were provided for variation analysis of Gentiana. RESULTS: Gentiana species satisfy the characteristics of intra-Sect conservation and inter-Sect variation in chloroplast genome structure and IR boundaries. All Gentiana Sects can be clustered into a single one and separated from each other; however, Ser. Apteroideae and Ser. Confertifoliae in Sect. Monopodiae are more closely related to Sect. Frigida and Sect. Cruciata, respectively. Gentiana has experienced two large gene loss events; the first, the collective loss of the rps16 gene at genus formation and the second, the collective loss of the ndh gene when Ser. Ornatae and Ser. Verticillatae completed their differentiation. Comparative genomic analysis support that Sect. Stenogyne and Sect. Otophora became the independent genera Metagentiana and Kuepferia. Seven divergence hotspot regions were screened based on Pi values, and could serve as DNA-specific barcodes for Gentiana. CONCLUSIONS: This study provides a further theoretical basis for taxonomic analysis, genetic diversity, evolutionary mechanism and molecular identification in Gentiana.

Chinese herbal medicine resources: Where we stand.

In recent years, the development of Chinese herbal medicine (CHM) has been challenged by shortages of CHM resources and drug safety concerns related to end products. There have been significant efforts by Chinese scholars to tackle these challenges, which are revealed by analyzing the research trend of CHM resources via surveying Chinese Traditional and Herbal Drugs (Zhong Cao Yao), a representative journal in CHM. Our study focused on 781 articles in CHM resources from 2013 to 2018 and included four subject areas: germplasm resources, quality analysis and evaluation, cultivation, and bioengineering of CHM. Discussion and prospective for future investigations were also presented, including: construct the core germplasm of medicinal plants and expand germplasms; combine molecular research with field experiments and promote the deeper study of cultivation of CHM plants; improve the quality evaluation method of CHM and strengthen the identification of Chinese patented medicines; promote the sustainable development of CHM resources by utilizing bioengineering and synthetic biology. This study helps international scholars understand the status quo of CHM research and provides theoretical support for the healthy, modern, and international development of CHM, and it will facilitate the sustainable development of the traditional Chinese medicine industry.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata.

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

Neurogenic hypothesis of cardiac ischemic pain.

Coronary heart disease is the major cause of death worldwide and is affecting millions of people in both developed and developing countries. Patients with myocardial ischemia typically experience chest pain (angina pectoris). Traditional viewpoint of ischemic cardiac pain might be related to "mechanical hypothesis" in early time and "chemical hypothesis" in modern time. However, perception of cardiac ischemic pain is still not well understood. The previous studies suggested that neurogenic mechanisms including neurogenic inflammation and neurogenic activity might participate in the pathophysiological processes following myocardial ischemia. Therefore, we raise "neurogenic hypothesis", that is, neurogenic mechanisms might play a pivotal role in myocardial ischemic injury. Analgesia intervention, rivalry of neurogenic inflammatory reactions and electrostimulatory therapy, etc. could not only relieve the pain symptoms, but also block nociception of body and neurogenic reaction induced by ischemia. Thereby ischemic myocardial injury would be extenuated and myocardial protection be produced. Attempts to confirm this hypothesis may lead to new theory of pathophysiologic mechanisms and provide potential intervention strategy for cardiac ischemic pain.

Acculturation and changes in dietary behavior and anthropometric measures among Chinese international students in South Korea.

BACKGROUND/OBJECTIVES: International students face dissimilar food environments, which could lead to changes in dietary behaviors and anthropometric characteristics between before and after migration. We sought to examine the risk factors, including dietary behaviors, acculturation, and demographic characteristics, related to overweight subjects residing in South Korea. SUBJECTS/METHODS: We conducted a cross-sectional study, collecting data from 142 Chinese international students (63 males, 79 females) in 2013. RESULTS: The mean age of the subjects was 25.4 years, and almost half of them immigrated to South Korea to earn a master's degree or doctoral degree (n = 70, 49.3%). Chinese international students showed an increase in skipping meals and eating speed, but a decrease in the frequency of fruit and vegetable consumption in South Korea compared to when they lived in China. We found a statistically significant increase in weight (69.4 → 73.9 kg) and BMI (22.4 → 23.8 kg/m(2)) for male subjects (P < 0.001) but no change for female subjects. We also found that overweight subjects were more likely to be highly acculturated and male compared with normal-weight subjects. CONCLUSION: Among Chinese international students living in South Korea, male and more highly acculturated subjects are more vulnerable to weight gain. This study provides useful information to design tailored nutrition intervention programs for Chinese international students.

Long-time course of protease-activated receptor-1 expression after intracerebral hemorrhage in rats.

Thrombin plays an important role in brain injuries associated with intracerebral hemorrhage (ICH). The protease-activated receptor (PAR)-1 is responsible for the vast majority of the thrombin's cellular activation functions. We tested the hypothesis that thrombin-induced brain damage after ICH, at least in part, is mediated by PAR-1. We report that there are significant differences between PAR-1 positive cell number and PAR-1 mRNA absorbance ratio between ICH model group (at 6h, 24h, 3 d, 7 d and 14 d) and normal group (P<0.05). These results suggest that the long-time course of PAR-1 expression may be partly involved in the mechanism of thrombin-induced brain damage after ICH.