Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo.

Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.

Cerebellar Purkinje cell firing promotes conscious recovery from anesthesia state through coordinating neuronal communications with motor cortex.

Background: The neurobiological basis of gaining consciousness from unconscious state induced by anesthetics remains unknown. This study was designed to investigate the involvement of the cerebello-thalamus-motor cortical loop mediating consciousness transitions from the loss of consciousness (LOC) induced by an inhalational anesthetic sevoflurane in mice. Methods: The neural tracing and fMRI together with opto-chemogenetic manipulation were used to investigate the potential link among cerebello-thalamus-motor cortical brain regions. The fiber photometry of calcium and neurotransmitters, including glutamate (Glu), γ-aminobutyric acid (GABA) and norepinephrine (NE), were monitored from the motor cortex (M1) and the 5th lobule of the cerebellar vermis (5Cb) during unconsciousness induced by sevoflurane and gaining consciousness after sevoflurane exposure. Cerebellar Purkinje cells were optogenetically manipulated to investigate their influence on consciousness transitions during and after sevoflurane exposure. Results: Activation of 5Cb Purkinje cells increased the Ca2+ flux in the M1 CaMKIIα+ neurons, but this increment was significantly reduced by inactivation of posterior and parafascicular thalamic nucleus. The 5Cb and M1 exhibited concerted calcium flux, and glutamate and GABA release during transitions from wakefulness, loss of consciousness, burst suppression to conscious recovery. Ca2+ flux and Glu release in the M1, but not in the 5Cb, showed a strong synchronization with the EEG burst suppression, particularly, in the gamma-band range. In contrast, the Glu, GABA and NE release and Ca2+ oscillations were coherent with the EEG gamma band activity only in the 5Cb during the pre-recovery of consciousness period. The optogenetic activation of Purkinje cells during burst suppression significantly facilitated emergence from anesthesia while the optogenetic inhibition prolonged the time to gaining consciousness. Conclusions: Our data indicate that cerebellar neuronal communication integrated with motor cortex through thalamus promotes consciousness recovery from anesthesia which may likely serve as arousal regulation.

Cerebral Mechanism of Tuina on the Descending Pain Inhibitory System in Knee Osteoarthritis: Protocol for a Randomized Controlled Trial.

BACKGROUND: Knee osteoarthritis (KOA) is reputedly the most common musculoskeletal disease of the lower limbs and the main cause of pain and disability among older individuals. Pain is the most significant and widespread symptom of KOA. The descending pain inhibitory system has a cardinal role in normal pain consciousness, and its malfunction may be one of the pathophysiological mechanisms in KOA. Crucially, the rostral ventromedial medulla (RVM) and periaqueductal gray (PAG), as important components of the descending pain inhibitory system, directly modulate the activity of the spinal neurons involved in pain transmission. Tuina, a manual therapy, is effective and safe for reducing clinical symptoms of KOA; however, the mechanism that influences pain through the descending pain inhibitory system in KOA is unclear. OBJECTIVE: This study aims to investigate the modulatory implications of Tuina on the RVM and PAG, which have critical roles in the descending pain inhibitory system in patients with KOA. METHODS: This randomized controlled parallel trial will be conducted at the Tuina Clinic of the Third Affiliated Hospital of Henan University of Chinese Medicine (Zhengzhou, China). Patients with KOA will be randomly assigned (1:1) to 6 weeks of health education or Tuina. All patients in both groups will accept a resting-state functional magnetic resonance scan at the beginning and end of the experiment, and the resting-state functional connectivity and the voxel-based morphometry analysis will be performed to detect the RVM and PAG function and structure changes. The clinical outcome assessments will be (1) the pressure pain thresholds, (2) the Numerical Rating Scale, (3) the Hamilton Depression Scale (HAMD), and (4) the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Considering that this trial is a study of resting-state functional magnetic resonance imaging technology, resting-state functional connectivity and voxel-based morphometry are the primary outcomes, and clinical outcome assessments are secondary outcomes. Adverse events will be documented and assessed throughout. All main analyses will be carried out on the basis of the intention-to-treat principle. The outcome evaluators and data statisticians will be masked to the treatment group assignment to reduce the risk of bias. RESULTS: This trial was approved by the ethics committee of the Third Affiliated Hospital of Henan University of Chinese Medicine. Enrollment began in December 2023, and the results of this trial are expected to be submitted for publication in May 2025. CONCLUSIONS: This trial will identify a possible relationship between function and structure changes of RVM and PAG and the improvement of clinical variables, elucidating the effect of Tuina on the descending pain inhibitory system of patients with KOA. This trial will provide much-needed knowledge for Tuina for patients with KOA. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2300070289; https://www.chictr.org.cn/showproj.html?proj=182570. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/52820.

IMP: bridging the gap for medicinal plant genomics.

Medicinal plants have garnered significant attention in ethnomedicine and traditional medicine due to their potential antitumor, anti-inflammatory and antioxidant properties. Recent advancements in genome sequencing and synthetic biology have revitalized interest in natural products. Despite the availability of sequenced genomes and transcriptomes of these plants, the absence of publicly accessible gene annotations and tabular formatted gene expression data has hindered their effective utilization. To address this pressing issue, we have developed IMP (Integrated Medicinal Plantomics), a freely accessible platform at https://www.bic.ac.cn/IMP. IMP curated a total of 8 565 672 genes for 84 high-quality genome assemblies, and 2156 transcriptome sequencing samples encompassing various organs, tissues, developmental stages and stimulations. With the integrated 10 analysis modules, users could simply examine gene annotations, sequences, functions, distributions and expressions in IMP in a one-stop mode. We firmly believe that IMP will play a vital role in enhancing the understanding of molecular metabolic pathways in medicinal plants or plants with medicinal benefits, thereby driving advancements in synthetic biology, and facilitating the exploration of natural sources for valuable chemical constituents like drug discovery and drug production.

Miltiradiene Production by Cytoplasmic Metabolic Engineering in Nicotiana benthamiana.

Plant natural products are important sources of innovative drugs, but the extraction and isolation of medicinal natural products from plants is challenging as these compounds have complex structures that are difficult to synthesize chemically. Therefore, utilizing heterologous expression systems to produce medicinal natural products in plants is a novel, environmentally friendly, and sustainable method. In this study, Nicotiana benthamiana was used as the plant platform to successfully produce miltiradiene, the key intermediate of tanshinones, which are the bioactive constituents of the Chinese medicinal plant Salvia miltiorrhiza. The yield of miltiradiene was increased through cytoplasmic engineering strategies combined with the enhancement of isoprenoid precursors. Additionally, we discovered that overexpressing SmHMGR alone accelerated apoptosis in tobacco leaves. Due to the richer membrane systems and cofactors in tobacco compared to yeast, tobacco is more conducive to the expression of plant enzymes. Therefore, this study lays the foundation for dissecting the tanshinone biosynthetic pathway in tobacco, which is essential for subsequent research. Additionally, it highlights the potential of N. benthamiana as an alternative platform for the production of natural products in plants.

Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

Scientific knowledge graph and trend analysis of Tuina: A bibliometric analysis.

OBJECTIVES: Tuina is an effective complementary and alternative therapy. However, no bibliometric analysis has explored the global research status and emerging trends of tuina. Therefore, our study aimed to provide a perspective on the current state and frontier trends in the field. DESIGN: Bibliometric analysis SETTING: Tuina-related publications between January 1, 2003, and December 31, 2022, were obtained from the Web of Science Core Collection database. MAIN OUTCOME MEASURES: The knowledge graph software CiteSpace and VOSViewer were used to quantitatively analyse annual trends in annual publication volume, journals, countries, institutions, authors, cited references, and keywords. RESULTS: Overall, 1877 articles were obtained. Consequently, the number of annual publications in tuina gradually increased. China published the most articles (1402 articles, 58.01%), followed by the Chinese Academy of Sciences (110 articles, 2.57%). Original and review articles were the two main types of publications. Photonics Research ranked first (101 articles, 5.38%) as the most influential affiliate and productive journal. These articles come from 8423 authors, among whom Min Fang published the most publications, and Ernst E was co-cited most often. According to the keyword co-occurrence analysis, the new research frontiers were meta-analyses. CONCLUSION: This comprehensive bibliometric study analysed the publications on tuina and presented them visually, revealing new research trends, pivotal points, research hotspots, and frontiers. Prospective strategies and potential directions for further studies were also provided.

L-glutamine sensitizes Gram-positive-resistant bacteria to gentamicin killing.

IMPORTANCE: Methicillin-resistant Staphylococcus aureus (MRSA) infection severely threatens human health due to high morbidity and mortality; it is urgent to develop novel strategies to tackle this problem. Metabolites belong to antibiotic adjuvants which improve the effect of antibiotics. Despite reports of L-glutamine being applied in antibiotic adjuvant for Gram-negative bacteria, how L-glutamine affects antibiotics against Gram-positive-resistant bacteria is still unclear. In this study, L-glutamine increases the antibacterial effect of gentamicin on MRSA, and it links to membrane permeability and pH gradient (ΔpH), resulting in uptake of more gentamicin. Of great interest, reduced reactive oxygen species (ROS) by glutathione was found under L-glutamine treatment; USA300 becomes sensitive again to gentamicin. This study not only offers deep understanding on ΔpH and ROS on bacterial resistance but also provides potential treatment solutions for targeting MRSA infection.

A Meta-Analysis of Differences in Thyroid and Cardiac Function Between Women with Normal Pregnancies and Gestational Diabetes Mellitus.

Objective: This is a meta-analysis of thyroid function (TF) and cardiac function (CF) differences between women with normal pregnancies and gestational diabetes mellitus (GDM), in order to provide more reliable reference and guidance for the future clinical prevention and treatment of GDM. Methods: Studies on the correlation of GDM with TF and CF were searched in PubMed, Cochrane Library, and other literature databases, and the literature for final analysis was confirmed after screening according to the eligibility criteria. Authors, publication time, research subjects, and endpoints were extracted for meta-analysis using Review 5.3 software. Results: After screening, 10 studies with a total of 2554 subjects were selected, including 1125 GDM patients (GDM group) and 1429 normal pregnant women (control group). All the included papers scored 6-7 points on the Newcastle-Ottawa Scale used for literature quality evaluation, implying high-quality research. In the meta-analysis, the relationship between GDM and TF, TSH, and FT3 increased evidently in the GDM group, while FT4 decreased (P < .05). The meta-analysis of GDM and CF revealed lower LVEF and E/A while higher E/E' in GDM patients compared to the controls (P < .05). The funnel plot showed that the graphs of all the endpoints were basically symmetrical, indicating low publication bias. Conclusion: Given the obvious thyroid dysfunction and cardiac dysfunction in GDM patients, symptomatic intervention measures should be taken actively and timely to improve the safety of GDM patients during pregnancy.

Green Synthesis of Silver Nanoparticles Using Jasminum nudiflorum Flower Extract and Their Antifungal and Antioxidant Activity.

The synthesis of metal nanomaterials is a timely topic due to their widespread use in fields such as crop protection, the environment, medicine, and engineering. Green synthesis of nanoparticles, which uses plant extracts instead of industrial chemical agents to reduce metal ions, has been developed to decrease costs, reduce pollution, and improve environmental and human health safety. In this paper, silver nanoparticles (AgNPs) were synthesized from the flower extract of Jasminum nudiflorum. The green synthesized AgNPs were characterized by UV-Vis, FTIR, XRD, SEM, and other technologies. The antifungal activity of the prepared AgNPs against Alternaria longipes was tested using the plate method, the concentration dilution method, and other methods, and the antioxidant activity of the prepared AgNPs was evaluated by DPPH and hydroxyl free scavenging methods. The results showed that AgNPs synthesized from J. nudiflorum flower extract have a face-centered cubic structure (fcc), and the average grain size of the nanoparticles is 13 nm; they are also mainly spherical in shape. Additionally, the concentration of AgNPs (ranging from 16 to 128 µg/mL) significantly inhibited the mycelial growth of A. longipes in comparison to the control. The inhibitory rate gradually increased with increasing AgNP concentration, ranging from 70.64% to 79.60% at a concentration of 128 µg/mL. The minimum inhibitory concentration was observed at 32 µg/mL. AgNPs induced overaccumulation of MDA in A. longipes, resulting in cell membrane damage and nucleic acid leakage. Moreover, the AgNPs have significant antioxidant properties, which increase with increasing concentration. The clearance rate of DPPH was 25.46 ± 0.90% when the concentration of AgNPs was 8 µg/mL, and the clearance rate of the hydroxyl radical was 28.62 ± 0.59% when the concentration of AgNPs was 128 µg/mL. Thus, the flower extract from J. nudiflorum holds potential as an environmentally friendly and green alternative for the synthesis of AgNPs, which have antifungal and antioxidant potential.

Natural exosomes-like nanoparticles in mung bean sprouts possesses anti-diabetic effects via activation of PI3K/Akt/GLUT4/GSK-3ß signaling pathway.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by hyperglycemia and insulin resistance. Mung bean sprouts are traditionally considered a "folk" hypoglycemic food and their pharmacological effects and underlying mechanisms warrant further investigation. PURPOSE: This study aimed to investigate the anti-diabetic effects of the exosomes-like nanoparticles in mung bean sprouts (MELNs) and explore the related molecular mechanisms. RESULTS: MELNs were isolated using a differential centrifugation-polyethylene glycol (PEG) method, and the identification of MELNs were confirmed by PAGE gel electrophoresis, agarose gel electrophoresis, thin-layer chromatography (TLC), and transmission electron microscopy (TEM). In the high-fat diet/streptozotocin (HFD/STZ) mouse model, MELNs ameliorated the progression of T2DM by increasing oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) results, decreasing the fasting blood glucose level, and reducing the serum triglycerides (TG) and total cholesterol (TC). Histopathological examinations indicated MELNs diminished inflammatory infiltration of hepatocytes and amplified the area of islet B cells. In addition, MELNs decreased the oxidative stress levels in liver tissue and had good biocompatibility. In vitro experiments verified that MELNs improved the viability of glucosamine (GlcN) induced insulin-resistant hepatocytes. Furthermore, this study also revealed that MELNs upregulated GLUT4 & Nrf2 and down-regulated GSK-3ß via activating the PI3K/Akt signaling pathway, promoting the production of antioxidant enzymes, such as HO-1 and SOD, to reduce oxidative stress. CONCLUSION: MELNs mitigated the progression of type 2 diabetes in HFD/STZ mouse model. The underlying molecular mechanism is related to PI3K/Akt/GLUT4/GSK-3ß signaling pathway.

Effect of Three Different Amino Acids Plus Gentamicin Against Methicillin-Resistant Staphylococcus aureus.

Background: The issue of methicillin-resistant Staphylococcus aureus (MRSA) resistant to many antibiotics and causing serious infectious diseases is a growing healthcare concern. Purpose: In recent years, exogenous administration of metabolites in combination with antibiotics can re-sensitize resistant bacteria to antibiotics; however, their effects vary, and their underlying mechanism of action remains elusive. Methods: We assessed the bactericidal effects of the three amino acids in combination with gentamicin in vitro and in vivo. Subsequently, we explored the role of these amino acids on the metabolomics of MRSA using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, we performed the downstream analyses using MetaboAnalyst and Interactive Pathways Explorer. Results: Exogenous threonine showed the best bactericidal efficacy with gentamicin, followed by glycine, wherein serine had no effect. Amino acid treatments mainly up-regulated the metabolites, increased the amino acid abundance, and significantly activated metabolisms; these effects were consistent with the bactericidal efficacy of the three amino acids. Most amino acids participated in the tricarboxylic acid cycle, and threonine supplementation increased the activities of citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, whereas glycine increased activities of citrate synthase and α-ketoglutarate dehydrogenase, and serine did not affect the activities of any of the three key enzymes. We identified 24 biomarkers in the three groups, among which glutamic acid and cysteine showed a gradient decrease and increase, respectively. Subsequent analyses revealed that glutamic acid but not cysteine promoted the bactericidal effect of gentamicin synergistically. Conclusion: Threonine has the best synergistic effect in reversing bacterial resistance compared to glycine and serine. We show that different amino acids combined with an antibiotic mainly affect amino acid metabolism and act via different metabolic regulatory mechanisms, which could help develop effective strategies for tackling MRSA infections.

Functional Study and Efficient Catalytic Element Mining of CYP76AHs in Salvia Plants.

Salvia is a large genus with hundreds of species used in traditional Chinese medicine. Tanshinones are a highly representative class of exclusive compounds found in the Salvia genus that exhibit significant biological activity. Tanshinone components have been identified in 16 Salvia species. The CYP76AH subfamily (P450) is crucial for the synthesis of tanshinone due to its catalytic generation of polyhydroxy structures. In this study, a total of 420 CYP76AH genes were obtained, and phylogenetic analysis showed their clear clustering relationships. Fifteen CYP76AH genes from 10 Salvia species were cloned and studied from the perspectives of evolution and catalytic efficiency. Three CYP76AHs with significantly improved catalytic efficiency compared to SmCYP76AH3 were identified, providing efficient catalytic elements for the synthetic biological production of tanshinones. A structure-function relationship study revealed several conserved residues that might be related to the function of CYP76AHs and provided a new mutation direction for the study of the directed evolution of plant P450.

[Advances in research methods for biosynthetic pathway analysis of active ingredients in traditional Chinese medicine].

The active ingredients in traditional Chinese medicine(TCM)are the foundation for the efficiency of TCM and the key to the formation of Dao-di herbs. It is of great significance to study the biosynthesis and regulation mechanisms of these active ingredients for analyzing the formation mechanism of Daodi herbs and providing components for the production of active ingredients in TCM by synthetic biology. With the advancements in omics technology, molecular biology, synthetic biology, artificial intelligence, etc., the analysis of biosynthetic pathways for active ingredients in TCM is rapidly progressing. New methods and technologies have promoted the analysis of the synthetic pathways of active ingredients in TCM and have also made this area a hot topic in molecular pharmacognosy. Many researchers have made significant progress in analyzing the biosynthetic pathways of active ingredients in TCM such as Panax ginseng, Salvia miltiorrhiza, Glycyrrhiza uralensis, and Tripterygium wilfordii. This paper systematically reviewed current research me-thods for analyzing the biosynthetic functional genes of active ingredients in TCM, elaborated the mining of gene elements based on multiomics technology and the verification of gene functions in plants in vitro and in vivo with candidate genes as objects. Additionally, the paper summarized new technologies and methods that have emerged in recent years, such as high-throughput screening, molecular probes, genome-wide association studies, cell-free systems, and computer simulation screening to provide a comprehensive reference for the analysis of the biosynthetic pathways of active ingredients in TCM.

[Modification of C20 oxidase in tanshinone biosynthesis pathway].

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

[Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora].

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Rapid and Efficient Optimization Method for a Genetic Transformation System of Medicinal Plants Erigeron breviscapus.

Erigeron breviscapus is an important medicinal plant with high medicinal and economic value. It is currently the best natural biological drug for the treatment of obliterative cerebrovascular disease and the sequela of cerebral hemorrhage. Therefore, to solve the contradiction between supply and demand, the study of genetic transformation of E. breviscapus is essential for targeted breeding. However, establishing an efficient genetic transformation system is a lengthy process. In this study, we established a rapid and efficient optimized protocol for genetic transformation of E. breviscapus using the hybrid orthogonal method. The effect of different concentrations of selection pressure (Hygromycin B) on callus induction and the optimal pre-culture time of 7 days were demonstrated. The optimal transformation conditions were as follows: precipitant agents MgCl2 + PEG, target tissue distance 9 cm, helium pressure 650 psi, bombardment once, plasmid DNA concentration 1.0 µg·µL-1, and chamber vacuum pressure 27 mmHg. Integration of the desired genes was verified by amplifying 1.02 kb of htp gene from the T0 transgenic line. Genetic transformation of E. breviscapus was carried out by particle bombardment under the optimized conditions, and a stable transformation efficiency of 36.7% was achieved. This method will also contribute to improving the genetic transformation rate of other medicinal plants.

Research progress of quality control for the seed of Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Suan-Zao-Ren) and its proprietary Chinese medicines.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Ziziphi Spinosae (SZS), the seed of Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Chinese name Suan-Zao-Ren), is widely distributed in China, Laos, Myanmar, and Iran. It is a classic traditional Chinese medicine with sedative and sleeping effects. In clinical practice, there are more than 155 proprietary Chinese medicines containing SZS. However, many commercial SZS products are difficult to qualify using current methods. Moreover, there is a scarcity of quality standards for SZS in proprietary Chinese medicines. AIM OF THE STUDY: The purpose of this study was to clearly reveal the quality indicators during the entire production process of SZS and its products. MATERIALS AND METHODS: This study reviewed more than 230 articles and related books on the quality control of SZS and its proprietary Chinese medicines published over the last 40 years (from January 1979 to October 2022). Moreover, where available, information on the quality of SZS and its proprietary Chinese medicines was also collected from websites for comparison, including online publications (e.g. PubMed, CNKI, Google Scholar, and Web of Science), the information at Yaozhi website and China Medical Information Platform, along with some classic books on Chinese herbal medicine. The literature and information search were conducted using keywords such as "Suan-Zao-Ren", " Ziziphus jujuba" and "quality control", and the latest results from various databases were combined to obtain valid information. The active components, which in vivo exposure, and Q-markers were also summarized. RESULTS: The jujuboside A, jujuboside B, and spinosin were revealed as the key Q-markers for SZS. Moreover, the advancements and prospects of the quality control for SZS and its extract, proprietary Chinese medicines, health foods, and adulterants were comprehensively summarized. The high-performance liquid chromatography-UV/evaporative light scattering detection and fingerprint analysis were found to be the mainstream methods for the SZS quality control. In particular, the novel quality evaluation method based on the unit content was applied for SZS and its proprietary Chinese medicines. Significant fluctuations were found in the contents of Q-markers. Moreover, the mass transfer rule of Q-markers was comprehensively clarified based on the entire production process, including production origins, ripening time, primary process, processing, compatibility decoction/extract, and storage. Ultimately, the crushing and compatibility of SZS were found to be the key steps affecting the active components. CONCLUSIONS: In short, this study provides solid evidences to reveal quality indicators for the entire production process of developing rational quality standards for SZS and its products. Moreover, this study also provides a template quality control overview, which could be extended to other traditional Chinese medicines.

[Modernization of Chinese medicine Salviae Miltiorrhizae Radix et Rhizoma: a review].

Modernization of Chinese medicine is an important development direction of traditional Chinese medical sciences. It is of great significance to understand the mechanism of Chinese medicine with basic research, which can also accelerate the development and utilization of Chinese medicine. Salviae Miltiorrhizae Radix et Rhizoma is one of the most commonly used Chinese medicines in China for the prevention and treatment of cardiovascular and cerebrovascular diseases. It has received key and extensive attention worldwide in the following aspects: main active ingredients and their pharmacological mechanism, function and regulation of their biosynthetic pathway and application of their synthetic biology as well as the clinical preparations. The new and developing chemical analysis, network pharmacology, molecular pharmacognosy and omics make the modernization research of Salviae Miltiorrhizae Radix et Rhizoma comprehensive and in-depth. This study systematically reviewed the modernization research of Salviae Miltiorrhizae Radix et Rhizoma, which focused on its pharmacological effects, preparation research, biosynthesis and regulation mechanism of the active ingredients, and expected to exert the model role of Salviae Miltiorrhizae Radix et Rhizoma in the research of Chinese medicine.