The chemical components of Carthami Flos were investigated by using macroporous resin, silica gel column chromatography, reversed-phase octadecylsilane(ODS) column chromatography, Sephadex LH-20, and semi-preparative high-performance liquid chromatography(HPLC). The planar structures of the compounds were established based on their physicochemical properties and ultraviolet-visible(UV-Vis), infrared(IR), high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), and nuclear magnetic resonance(NMR) spectroscopic technology. The absolute configurations were determined by comparing the calculated and experimental electronic circular dichroism(ECD). Six flavonoid C-glycosides were isolated from the 30% ethanol elution fraction of macroporous resin obtained from the 95% ethanol extract of Carthami Flos, and identified as saffloquinoside F(1), 5-hydroxysaffloneoside(2), iso-5-hydroxysaffloneoside(3), isosafflomin C(4), safflomin C(5), and vicenin 2(6). Among these, the compounds 1 to 3 were new chalcone C-glycosides. The compounds 1, 2, 4, and 5 could significantly increase the viability of H9c2 cardiomyocytes damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) at a concentration of 50 μmol·L~(-1), showing their good cardioprotective activity.
Glycosides/pharmacology* ; Flowers/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Carthamus tinctorius/chemistry* ; Chalcones/pharmacology* ; Animals

Glaucocalyxin A （GLA） is a natural diterpenoid extracted from Isodon amethystoides belonging to Labiatae. Modern pharmacological research has shown that GLA has anti-inflammatory， anti-tumor， anti-fibrotic， osteoporosis-ameliorating， and cardiovascular system-protecting activities and good biosafety. However， the low content in plants， poor solubility， high metabolic rate， and low bioavailability limit the application of GLA. To address these issues， researchers have studied the total synthesis， structural modification， and nanomedicine development of GLA. By reviewing the available studies about GLA in the past five years， we summarize the research progress in the total synthesis， pharmacological activities and mechanisms， and in vivo metabolic transformation， aiming to provide a theoretical basis for clarifying the specific mechanisms underlying the pharmacological activities of GLA and for further research， development， and clinical applications of GLA.

Sucrose synthase plays a crucial role in the plant sugar metabolism pathway by catalyzing the production of uridine diphosphate (UDP)-glucose, which serves as a bioactive glycosyl donor for various metabolic processes. In this study, a sucrose synthase gene named CtSus was cloned from Cistanche tubulosa, a traditional Chinese medicine known for its rich content of diverse glycosides, based on transcriptome analysis results. The open reading frame of CtSus was 2 418 bp long encoding 805 amino acids. Sequence analysis revealed conserved domains associated with the plant sucrose synthase family at both the N-terminal and C-terminal regions of CtSus protein. Phylogenetic analysis demonstrated that CtSus shares a close evolutionary relationship with sucrose synthases from other plants within the same order. Functional identification of CtSus was performed through whole-cell catalysis coupling with UGT71BD1, a characterized glycosyltransferase enzyme. The results showed that the introduction of CtSus significantly enhanced the conversion rate of glycosylation catalyzed by UGT71BD1. The soluble expression of CtSus in Escherichia coli was further achieved using the pCold^TM TF expression vector. In vitro enzymatic assay indicated the activity of CtSus to catalyze the formation of UDP-glucose in the presence of sucrose and UDP. Real-time quantitative-polymerase chain reaction results showed that the expression patterns of CtSus gene in different parts of C. tubulosa and the suspension cell cultures of C. tubulosa under drought stress correlated with the accumulation patterns observed for phenylethanol glycosides, respectively. Furthermore, key amino acids and their interactions between enzyme and substrate were explored based on protein structure prediction and molecular docking results. Overall, our findings identify a sucrose synthase CtSus responsible for the supply of the active glycosyl donor UDP-glucose during the biosynthesis of glycoside products in C. tubulosa and have also provided a gene element that can be utilized in engineering strain construction for glycoside products production.

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.
Biological Products/chemistry* ; Catalysis ; Alkaloids ; Biocatalysis

OBJECTIVE: To investigate the anti-angiogenic activity of Kunxian Capsule (KX) extract and explore the underlying molecular mechanism using zebrafish. METHODS: The KX extract was prepared with 5.0 g in 100 mL of 40% methanol followed by ultrasonication and freeze drying. Freeze dried KX extract of 10.00 mg was used as test stock solution. Triptolide and icariin, the key bioactive compounds of KX were analyzed using ultra-high performance liquid chromatography. The transgenic zebrafish Tg(flk1:GFP) embryos were dechorionated at 20-h post fertilization (hpf) and treated with PTK 787, and 3.5, 7, 14 and 21 µg/mL of KX extract, respectively. After 24-h post exposure (hpe), mortality and malformation (%), intersegmental vessels (ISV) formation, and mRNA expression level of angiogenic pathway genes including phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular signal-regulated kinases (ERKs), mitogen-activated protein kinase (MAPK), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) were determined. Further, the embryos at 72 hpf were treated with KX extract to observe the development of sub-intestinal vein (SIV) after 24 hpe. RESULTS: The chromatographic analysis of test stock solution of KX extract showed that triptolide and icariin was found as 0.089 mg/g and 48.74 mg/g, respectively, which met the requirements of the national drug standards. In zebrafish larvae experiment, KX extract significantly inhibited the ISV (P<0.01) and SIV formation (P<0.05). Besides, the mRNA expression analysis showed that KX extract could significantly suppress the expressions of PI3K and AKT, thereby inhibiting the mRNA levels of ERKs and MAPK. Moreover, the downstream signaling cascade affected the expression of VEGF and its receptors (VEGFR and VEGFR-2). FGF-2, a strong angiogenic factor, was also down-regulated by KX treatment in zebrafish larvae. CONCLUSION KX extract exhibited anti-angiogenic effects in zebrafish embryos by regulating PI3K/AKT-MAPK-VEGF pathway and showed promising potential for RA treatment.
Animals ; Fibroblast Growth Factor 2 ; Human Umbilical Vein Endothelial Cells ; Mitogen-Activated Protein Kinases/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Zebrafish

In light of related methods in Chinese Pharmacopoeia(2020 edition), this study established the quality standard for Lobeliae Chinensis Herba. The TLC identification method was established with silica gel GF_(254) thin layer plate, diosmin standard, linarin standard, and the reference material of Lobeliae Chinensis Herba. The loss on drying, total ash, acid-insoluble ash, and ethanol-soluble extracts of 18 batches of Lobeliae Chinensis Herba samples were determined according to the general principles in Chinese Pharmacopoeia. Then, HPLC was adopted in the establishment of characteristic chromatogram and content determination. The results showed that the established method can achieve good separation for diosmin, linarin, and lobetyolin. Based on the results of detection for 18 batches of Lobeliae Chinensis Herba samples, the draft quality standard was established, which was expected to provide reference for the revision of this medicinal herb in Chinese Pharmacopoeia.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/standards* ; Lobelia/chemistry* ; Plants, Medicinal/chemistry*

Eight sesquiterpenoids were isolated from petroleum ether extract of Aquilariae Lignum Resinatum by various column chromatography techniques including silica gel, ODS, and semi-preparative HPLC. Their structures were identified on the basis of physicochemical properties, UV, IR, MS, and NMR spectroscopic data as(4S,5S,7R,10S)-5,7-dihydroxy-11-en-eudesmane(1),(7R,10S)-eudesma-4-en-11,15-diol(2),(2R,4S,5R,7R)-2-hydroxyeremophila-9,11-dien-8-one(3), 7α-H-9(10)-ene-11,12-epoxy-8-oxoeremophilane(4),(+)-9β,10β-epoxyeremophila-11(13)-en(5), 4(14)-eudesmene-8α,11-diol(6), 12,15-dioxo-selina-4,11-dien(7), and 2β,8 aα-dihydroxy-11-en-eremophilane(8). Compounds 1 and 2 are new compounds, and their absolute configurations were determined by calculating ECD. Compounds 1, 4, and 6-8 could significantly improve taurocholic acid(TCA)-induced gastric mucosal GES-1 cell injury at a concentration of 20 μmol·L~(-1), and the cell protection rates were 23.51%±2.79%, 16.10%±1.25%, 24.45%±4.89%, 17.48%±2.93%, and 21.44%±2.39%, respectively.
Chromatography, High Pressure Liquid ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Sesquiterpenes/chemistry*

Qijiao Shengbai Capsules(QJ) are a common Miao medicine serving as an adjuvant cancer therapy in clinical practice.QJ consists of seven medicinal materials such as Astragalus membranaceus and Lespedeza buergeri.Its chemical components have not been clarified and the quality control needs to be improved.In this study, LC-IT-TOF-MS was used to comprehensively collect MS~1 and MS~2 fragment information of QJ and rapidly identify the chemical compositions.The chromatographic separation was performed on the Capcell core ADME column(2.1 mm×150 mm, 2.7 μm) with 0.1% formic acid aqueous solution(A) and acetonitrile(B) as mobile phases for gradient elution.High-resolution mass spectrometric information was obtained by scanning in the positive and negative ion ESI modes.A total of 107 compounds were structurally identified according to the deduced MS fragmentation patterns and comparison with standards and data reported in the literature, including 54 flavonoids, 16 phthalides, 13 alkaloids, 12 phenolic acids, 7 saponins, 2 coumarins, 2 condensed tannins, and 1 purine.This study clarified the chemical composition of QJ and provided references for the improvement of its quality standards and the elucidation of its medicinal substances.
Acetonitriles ; Alkaloids ; Capsules ; Chromatography, High Pressure Liquid ; Coumarins/analysis* ; Drugs, Chinese Herbal/chemistry* ; Flavonoids/analysis* ; Formates ; Proanthocyanidins/analysis* ; Purines ; Saponins ; Tandem Mass Spectrometry

Eleven condensed tannins were isolated from the roots of Indigofera stachyodes by various column chromatography techniques including silica gel, octadecyl silica(ODS), Sephadex LH-20, and semi-preparative high performance liquid chromatography(HPLC). These compounds were identified on the basis of physicochemical properties, nuclear magnetic resonance(NMR) and mass spectrometry(MS) data as stachyotannin A(1), epicatechin-(2β→O→7,4β→8)-epiafzelechin-(4β→8)-catechin(2), cinnamtannin D1(3), cinnamtannin B1(4), epicatechin-(2β→O→7,4β→8)-epiafzelechin-(4α→8)-epicatechin(5), gambiriin C(6), proanthocyanidin A1(7), proanthocyanidin A2(8), aesculitannin B(9), proanthocyanidin A4(10), and procyanidin B5(11). Compound 1 is a new compound. Compounds 2-11 were isolated from Indigofera for the first time. Furthermore, compounds 1, 2, and 4-11 showed inhibitory effects on thrombin-induced ATP release in platelets.
Chromatography, High Pressure Liquid ; Indigofera ; Magnetic Resonance Spectroscopy ; Plant Extracts ; Proanthocyanidins

Longxue Tongluo Capsules(LTC) has good efficacy against blood stasis syndrome during the recovery period of ischemic stroke. Its main active ingredient is the phenolic extract of Chinese dragon's blood. In our previous study, the primary mass fragmentation pathways of phenolic derivatives from LTC were clarified. Herein, the metabolites in rat plasma were characterized following the oral administration of loureirin A and loureirin C using liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectro-metry(LC-IT-TOF-MS), with 18 and 55 metabolites identified, respectively. On this basis, with the help of the obtained accurate molecular weight, characteristic fragment ions, reference comparison, combined with LTC database and natural products database self-created in our group, 18 prototypes and 106 metabolites were tentatively identified in rat plasma after oral gavage of LTC at a dose of 500 mg·kg~(-1). Glucuronidation, sulfonation, and methylation were major biotransformation pathways of LTC. This study preliminarily clarified the LTC constituents absorbed into blood and laid the foundation for clarifying the effective substances of LTC.
Administration, Oral ; Animals ; Capsules ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Drugs, Chinese Herbal ; Gas Chromatography-Mass Spectrometry ; Rats