A Novel R2R3-MYB Transcription Factor SbMYB12 Positively Regulates Baicalin Biosynthesis in Scutellaria baicalensis Georgi.

Scutellaria baicalensis Georgi is an annual herb from the Scutellaria genus that has been extensively used as a traditional medicine for over 2000 years in China. Baicalin and other flavonoids have been identified as the principal bioactive ingredients. The biosynthetic pathway of baicalin in S. baicalensis has been elucidated; however, the specific functions of R2R3-MYB TF, which regulates baicalin synthesis, has not been well characterized in S. baicalensis to date. Here, a S20 R2R3-MYB TF (SbMYB12), which encodes 263 amino acids with a length of 792 bp, was expressed in all tested tissues (mainly in leaves) and responded to exogenous hormone methyl jasmonate (MeJA) treatment. The overexpression of SbMYB12 significantly promoted the accumulation of flavonoids such as baicalin and wogonoside in S. baicalensis hairy roots. Furthermore, biochemical experiments revealed that SbMYB12 is a nuclear-localized transcription activator that binds to the SbCCL7-4, SbCHI-2, and SbF6H-1 promoters to activate their expression. These results illustrate that SbMYB12 positively regulates the generation of baicalin and wogonoside. In summary, this work revealed a novel S20 R2R3-MYB regulator and enhances our understanding of the transcriptional and regulatory mechanisms of baicalin biosynthesis, as well as sheds new light on metabolic engineering in S. baicalensis.

Whole genome and transcriptome reveal flavone accumulation in Scutellaria baicalensis roots.

Scutellaria baicalensis Georgi is a medicinal plant in the Lamiaceae family that contains high levels of 4'-deoxyflavone and other flavonoids in its roots. Therefore, it has strong potential as a plant resource for researching the biosynthesis of specific flavonoids. In this study, we report on a chromosome-level S. baicalensis genome assembled to nine chromosomes (376.81M) using PacBio, HiSeq XTen, and Hi-C assisted assembly. The assembly ratio was 99.22%, the contig N50 was 1.80 million bases, and the scaffold N50 was 40.57 million bases, with 31896 genes being annotated. Comparative genome analysis revealed that S. baicalensis and Salvia miltiorrhiza belonged to the same branch, and diverged 36.3 million years ago. Other typically correlated species were Boea hygrometrica and Sesamum indicum. We investigated the structural genes involved in flavonoid synthesis in combination with transcriptome sequencing analysis for different tissues (roots, stems, flowers, leaves) of purple, pink, and white flowers. The results revealed that S.baiF6H is involved in the accumulation of baicalein and was significantly increased in both purple roots vs. pink roots and white roots vs. pink roots. S.baiMYB gene family expression pattern analysis and co-expression network analysis revealed that S.baiMYB transcription factors primarily regulated the production of flavonoids in S. baicalensis. S.baiMYB serves as a major factor regulating flavonoid synthesis in the roots, where yeast one-hybrid assays revealed that these transcription factors could bind to the promoter regions of structural genes to control the accumulation of flavonoids. Genome and transcriptome sequencing, co-expression analysis, and yeast one-hybrid experiments provided valuable genetic resources for understanding flavonoid biosynthesis in S. baicalensis. These findings contribute to a better understanding of the accumulation of metabolites in Lamiaceae.

SmMYB4 Is a R2R3-MYB Transcriptional Repressor Regulating the Biosynthesis of Phenolic Acids and Tanshinones in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge is one of the most famous traditional Chinese medicinal plants. The two most important classes of pharmaceutically relevant compounds in S. miltiorrhiza are phenolic acids and tanshinones. The MYB family of transcription factors may efficiently regulate the secondary metabolism in plants. In this study, a subgroup 4 R2R3MYB transcription factor gene, SmMYB4, was isolated from S. miltiorrhiza and functionally characterized using overexpression and a RNAi-mediated silencing. We achieved a total of six overexpressions and eight RNAi transgenic lines from the Agrobacterium leaf disc method. The content of the total phenolics, rosmarinic acid, and salvianolic acid B markedly decreased in the SmMYB4-overexpressing lines but increased in the SmMYB4-RNAi lines. The content of the total tanshinones, cryptotanshinone, and tanshinone IIA decreased in the SmMYB4-overexpressing transgenic lines but increased in the SmMYB4-RNAi lines. A gene expression analysis demonstrated that SmMYB4 negatively regulated the transcription of the critical enzyme genes involved in the phenolic acid and tanshinone biosynthesis. The genetic control of this transcriptional repressor may be used to improve the content of these bioactive compounds in the cultivated S. miltiorrhiza.

Systematic Analysis and Functional Characterization of R2R3-MYB Genes in Scutellaria baicalensis Georgi.

R2R3-MYB transcription factors participate in multiple critical biological processes, particularly as relates to the regulation of secondary metabolites. The dried root of Scutellaria baicalensis Georgi is a traditional Chinese medicine and possesses various bioactive attributes including anti-inflammation, anti-HIV, and anti-COVID-19 properties due to its flavonoids. In the current study, a total of 95 R2R3-MYB genes were identified in S. baicalensis and classified into 34 subgroups, as supported by similar exon-intron structures and conserved motifs. Among them, 93 R2R3-SbMYBs were mapped onto nine chromosomes. Collinear analysis revealed that segmental duplications were primarily responsible for driving the evolution and expansion of the R2R3-SbMYB gene family. Synteny analyses showed that the ortholog numbers of the R2R3-MYB genes between S. baicalensis and other dicotyledons had a higher proportion compared to that which is found from the monocotyledons. RNA-seq data indicated that the expression patterns of R2R3-SbMYBs in different tissues were different. Quantitative reverse transcriptase-PCR (qRT-PCR) analysis showed that 36 R2R3-SbMYBs from different subgroups exhibited specific expression profiles under various conditions, including hormone stimuli treatments (methyl jasmonate and abscisic acid) and abiotic stresses (drought and cold shock treatments). Further investigation revealed that SbMYB18/32/46/60/70/74 localized in the nucleus, and SbMYB18/32/60/70 possessed transcriptional activation activity, implying their potential roles in the regulatory mechanisms of various biological processes. This study provides a comprehensive understanding of the R2R3-SbMYBs gene family and lays the foundation for further investigation of their biological function.

Identification and Characterization of Jasmonic Acid Biosynthetic Genes in Salvia miltiorrhiza Bunge.

Jasmonic acid (JA) is a vital plant hormone that performs a variety of critical functions for plants. Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as Danshen, is a renowned traditional Chinese medicinal herb. However, no thorough and systematic analysis of JA biosynthesis genes in S. miltiorrhiza exists. Through genome-wide prediction and molecular cloning, 23 candidate genes related to JA biosynthesis were identified in S. miltiorrhiza. These genes belong to four families that encode lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-OPDA reductase3 (OPR3). It was discovered that the candidate genes for JA synthesis of S. miltiorrhiza were distinct and conserved, in contrast to related genes in other plants, by evaluating their genetic structures, protein characteristics, and phylogenetic trees. These genes displayed tissue-specific expression patterns concerning to methyl jasmonate (MeJA) and wound tests. Overall, the results of this study provide valuable information for elucidating the JA biosynthesis pathway in S. miltiorrhiza by comprehensive and methodical examination.

Genome-Wide Identification and Characterization of the WRKY Gene Family in Scutellaria baicalensis Georgi under Diverse Abiotic Stress.

The WRKY gene family is an important inducible regulatory factor in plants, which has been extensively studied in many model plants. It has progressively become the focus of investigation for the secondary metabolites of medicinal plants. Currently, there is no systematic analysis of the WRKY gene family in Scutellaria baicalensis Georgi. For this study, a systematic and comprehensive bioinformatics analysis of the WRKY gene family was conducted based on the genomic data of S. baicalensis. A total of 77 WRKY members were identified and 75 were mapped onto nine chromosomes, respectively. Their encoded WRKY proteins could be classified into three subfamilies: Group I, Group II (II-a, II-b, II-c, II-d, II-e), and Group III, based on the characteristics of the amino acid sequences of the WRKY domain and genetic structure. Syntenic analysis revealed that there were 35 pairs of repetitive fragments. Furthermore, the transcriptome data of roots, stems, leaves, and flowers showed that the spatial expression profiles of WRKYs were different. qRT-PCR analysis revealed that 11 stress-related WRKYs exhibited specific expression patterns under diverse treatments. In addition, sub cellular localization analysis indicated that SbWRKY26 and SbWRKY41 were localized in nucleus. This study is the first to report the identification and characterization of the WRKY gene family in S. baicalensis, which is valuable for the further exploration of the biological function of SbWRKYs. It also provides valuable bioinformatics data for S. baicalensis and provides a reference for assessing the medicinal properties of the genus.

[Study on serum pharmacodynamic material basis of Gegen Qinlian Decoction in treatment of ulcerative colitis based on UHPLC-Q-Orbitrap-MS].

This study established a mouse model of ulcerative colitis and explored the serum transitional components of Gegen Qinlian Decoction by UHPLC-Q-Orbitrap-MS. Based on the exact relative molecular weight and MS/MS spectrum, 55 prototype components and 59 metabolites were identified from the model group, while 18 prototype components and 35 metabolites from the control group. The prototype components in serum were mainly flavonoids and the characteristic components of the model group were alkaloids. Glucuronidation, sulfonation, and glycosylation have been confirmed to be the main metabolic types in vivo. The results of comparative analysis of differences indicated that puerarin, baicalin, wogonoside, wogonin, chrysin, oroxylin A, berberine, berberrubine, and palmatine were the characteristic components in model state, which at the same time, were confirmed by pharmacological studies to be the serum pharmacodynamic material basis of Gegen Qinlian Decoction in the treatment of ulcerative colitis. This study has provided reference for explaining the metabolic transformation pattern and mechanism of action of Gegen Qinlian Decoction in vivo.

Primary and secondary metabolites produced in Salvia miltiorrhiza hairy roots by an endophytic fungal elicitor from Mucor fragilis.

Salvia miltiorrhiza is one of the most commonly used medicinal materials in China. In recent years, the quality of S. miltiorrhiza has attracted much attention. Biotic and abiotic elicitors are widely used in cultivation to improve the quality of medicinal plants. We isolated an endophytic fungus, Mucor fragilis, from S. miltiorrhiza. We compared the effects of endophytic fungal elicitors with those of yeast extract together with silver ion, widely used together as effective elicitors, on S. miltiorrhiza hairy roots. Seventeen primary metabolites (amino acids and fatty acids) and five secondary metabolites (diterpenoids and phenolic acids) were analyzed after elicitor treatment. The mycelium extract promoted the accumulation of salvianolic acid B, rosmarinic acid, stearic acid, and oleic acid in S. miltiorrhiza hairy roots. Additionally, qPCR revealed that elicitors affect the accumulation of primary and secondary metabolites by regulating the expression of key genes (SmAACT, SmGGPPS, and SmPAL). This is the first detection of both the primary and secondary metabolites of S. miltiorrhiza hairy roots, and the results of this work should help guide the quality control of S. miltiorrhiza. In addition, the findings confirm that Mucor fragilis functions as an effective endophytic fungal elicitor with excellent application prospect for cultivation of medicinal plants.

A new insulin-sensitive enhancer from Silene viscidula, WPTS, treats type 2 diabetes by ameliorating insulin resistance, reducing dyslipidemia, and promoting proliferation of islet ß cells.

Wacao pentacyclic triterpenoid saponins (WPTS) is a newly discovered insulin sensitivity enhancer. It is a powerful hypoglycemic compound derived from Silene viscidula, which has a hypoglycemic effect similar to that of insulin. It can rapidly reduce blood glucose levels, normalizing them within 3 days of administration. However, its mechanism of action is completely different from that of insulin. Thus, we aimed to determine the pharmacological effects and mechanism of activity of WPTS on type 2 diabetes to elucidate the main reasons for its rapid effects. The results showed that WPTS could effectively improve insulin resistance in KKAy diabetic mice. Comparative transcriptomics showed that WPTS could upregulate the expression of insulin resistance-related genes such as glucose transporter type 4 (Glut4), insulin receptor substrate 1 (Irs1), Akt, and phosphoinositide 3-kinase (PI3K), and downregulate the expression of lipid metabolism-related genes such as monoacylglycerol O-acyltransferase 1 (Moat1), lipase C (Lipc), and sphingomyelin phosphodiesterase 4 (Smpd4). The results indicated that the differentially expressed genes could regulate lipid metabolism via the PI3K/AKT metabolic pathway, and it is noteworthy that WPTS was found to upregulate Glut4 expression, decrease blood glucose levels, and attenuate insulin resistance via the PI3K/AKT pathway. Q-PCR and western blotting further validated the transcriptomics findings at the mRNA and protein levels, respectively. We believe that WPTS can achieve a rapid hypoglycemic effect by improving the lipid metabolism and insulin resistance of the diabetic KKAy mice. WPTS could be a very promising candidate drug for the treatment of diabetes and deserves further research.

Cell metabolomics to study the function mechanism of Cyperus rotundus L. on triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is a kind of malignant tumor with higher recurrence and metastasis rate. According to historical records, the dry rhizomes Cyperus rotundus L. could be ground into powder and mixed with ginger juice and wine for external application for breast cancer. We studied the effect of the ethanol extract of Cyperus rotundus L. (EECR) on TNBC cells and found its' apoptosis-inducing effect with a dose-relationship. But the function mechanism of EECR on TNBC is still mysterious. Hence, the present research aimed to detect its function mechanism at the small molecule level through ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) metabolomics. METHODS: The CCK-8 assay and the Annexin V-FITC/PI assay were applied to test the effect of EECR on MDA-MB-231 cells and MDA-MB 468 cells at various concentrations of 0, 200, 400, and 600 µg/ml. UPLC-Q-TOF-MS/MS based metabolomics was used between the control group and the EECR treatment groups. Multivariate statistical analysis was used to visualize the apoptosis-inducing action of EECR and filtrate significantly changed metabolites. RESULTS: The apoptosis-inducing action was confirmed and forty-nine significantly changed metabolites (VIP > 1, p < 0.05, and FC > 1.2 or FC < 0.8) were identified after the interference of EECR. The level of significant differential metabolites between control group, middle dose group, and high dose group were compared and found that which supported the apoptosis-inducing action with dose-dependence. CONCLUSION: By means of metabolism, we have detected the mechanism of EECR inducing apoptosis of TNBC cells at the level of small molecule metabolites and found that EECR impacted the energy metabolism of TNBC cells. In addition, we concluded that EECR induced apoptosis by breaking the balance between ATP-production and ATP-consumption: arresting the pathways of Carbohydrate metabolism such as Central carbon metabolism in cancer, aerobic glycolysis, and Amino sugar and nucleotide sugar metabolism, whereas accelerating the pathways of ATP-consumption including Amino Acids metabolism, Fatty acid metabolism, Riboflavin metabolism and Purine metabolism. Although further study is still needed, EECR has great potential in the clinical treatment of TNBC with fewer toxic and side effects.

[Research and development thought on biopharmaceutics classification system of Chinese materia medica].

The biopharmaceutics classification system( BCS) is a scientific framework or method for classifying drugs based on drug solubility and permeability,which can be used to provide drug bioavailability-absorption correlation analysis. Based on the characteristics of multi-component and multi-target of traditional Chinese medicine( TCM) as well as the concept,method and technology of BCS,the research group proposed biopharmaceutics classification system of Chinese materia medica( CMMBCS) and carried out research and data accumulation of classical prescriptions. Based on the previous research results,further development ideas under the CMMBCS concept and framework were further proposed in this study. In the course of research,the influence of the intermediate links of the complex interactions of the multi-component environment was omitted,and the component absorption studies on the main clinical effects of prescription ingredients were directly concerned,or the components and data were reversely extracted from the aspects of metabolism,pharmacodynamic pathways and absorption principles. Studies were conducted from two aspects( single component and compound prescription) to comprehensively evaluate the absorption properties of TCM compound. In the research path,the different ways in which Chinese medicine could exert its efficacy were fully considered,and CMMBCS classification and establishment rules were clarified mainly by focusing on the absorption pathway into the blood. Specifically,the network pharmacology and molecular docking technology were used to screen the compound index components of TCM; the absorption rules were studied by the physiologically based pharmacokinetic models and the absorption parameters of CMMBCS were calculated by reverse reasoning. Then the CMMBCS classification of TCM prescription was corrected by studying the efficacy or absorption pathway. In this paper,the theoretical framework and research methodology of CMMBCS were systematically improved based on the establishment of CMMBCS basic theory,the supplementary of drug-oriented research ideas and the application of modern mature Chinese medicine methodology.