Unveiling allelopathic dynamics and impacts of invasive Erigeron bonariensis and Bidens pilosa on plant communities and soil parameters.

Invasive alien species are becoming more and more prevalent worldwide, Erigeron bonariensis and Bidens pilosa are two invasive species of Asteraceae in Egypt. To mitigate their detrimental effects and understand their differences in invasiveness, we compared the allelopathic potentials of E. bonariensis and B. pilosa using leachates, decaying residues, and volatilization processes. Notably, the allelopathic variances in leachates were significant, influenced by plant types, concentrations, and response patterns of target plant traits, as indicated by EC50. The relative phytotoxicity of the invasive species decayed residues peaked between 20 and 25 days in the soil, with a positive correlation with concentrations and soil properties. The highest quantities of phenolic acids were chlorogenic acid and caffeic acid reaching (5.41 and 4.39 µg g-1) E. bonariensis and (4.53 and 4.46 µg g-1) B. pilosa, in leachates extracts respectively, while in the soil extract of decayed residues were coumaric acid and ferulic acid measuring (1.66 and 1.67 µg g-1) E. bonariensis and (1.47 and 1.57 µg g-1) B. pilosa, respectively. Using GC/MS analysis, the main volatile components in E. bonariensis were 1, 8 cineole (5.62%), and α-terpinene (5.43%) and iso-Caryophyllene (5.2%) which showed the greatest inhibitory effects. While B. pilosa main constituents were trans-sabinene (5.39%) and Camphene (5.11%), respectively. Finally, the high invasion level displayed from E. bonariensis (0.221) compared with B. pilosa (0.094) which correlated with the stronger allelopathic activities against plant species, and soil properties. Therefore, the allelopathic potentialities of these species are critically relevant to their invasion success.

Caffeoylquinic acids protect against alcoholic liver injury and rare C10 acetylenic acids from Erigeron breviscapus.

Three new compounds (1-2, 4) along with ten known ones (3, 5-13), were isolated from the whole plant of Erigeron breviscapus. Compounds 1 and 2, two novel C10 acetylenic acids and compound 4, a jasmone glucoside were elucidated by the detailed analysis of 1D and 2D NMR, HRESIMS spectra, and experimental and calculated electronic circular dichroism (ECD). Compounds 1-3 represent the first example of acetylenic acids incorporating C10 skeleton from E. breviscapus. In addition, the antioxidant effects of all compounds were evaluated by ferric reducing power, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate acid) (ABTS) and 2.2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. Our results indicated the significant antioxidant activity of caffeoylquinic acids. Additionally, compounds 10-11 and 13 played protective role on alcoholic liver injury cells in a dose-dependent manner.

Study on the anti-mitochondrial apoptosis mechanism of Erigeron breviscapus injection based on UPLC-Q-TOF-MS metabolomics and molecular docking in rats with cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Erigeron breviscapus is a common medicine of eight ethnic minorities, including Miao, Naxi, and Yi. As early as the Ming Dynasty (AD 1368-1644), Lanmao's Materia Medica of Southern Yunnan (AD 1436) recorded that the medicine is used for the treatment of "Zuo tan you huan." In modern pharmacological research, Erigeron breviscapus injection is the most commonly used preparation in the treatment of ischemic stroke caused by acute cerebral infarction, but its mechanism of action in the treatment of ischemic stroke is not well understood. AIM OF THE STUDY: In this study, a metabonomics study based on ultraperformance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) was used in investigating the effect of a traditional Chinese medicine preparation Erigeron breviscapus injection on the rat model of focal cerebral ischemia-reperfusion and the affinity of its main components with the targets of mitochondrial apoptotic pathways. MATERIALS AND METHODS: This study used molecular docking technology to verify the effective binding ability of main effective components of Erigeron breviscapus injection to target proteins related to mitochondrial apoptosis pathway. This study developed a metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of focal cerebral ischemia-reperfusion model group and healthy control group was achieved. RESULTS: Erigeron breviscapus injection can significantly reduce the area of cerebral infarction, improve tissue morphological lesion in rats, and can increase the number of Nissl bodies. It may be a promoting factor by inhibiting hippocampal nerve cell apoptosis and Bax protein expression and by exerting effects against ischemia reperfusion after the induction of apoptosis. Thus, it plays a role in brain protection. Moreover, it can considerably promote the recovery of neurological deficiency signs in advance. Meanwhile, Erigeron breviscapus decreased malondialdehyde content and T-NOS activity. Its curative effect from strong to weak order: low dose > high dose > medium dose. The representative components of Erigeron breviscapus have good affinity with the active sites of mitochondrial apoptosis-related proteins. Metabolomics found that the potential biomarkers regulated by breviscapine are kynurequinolinic acid, succinylornithine, and leucine proline. It is speculated that it may participate in TRP-kynurequinolinic acid and succinylornithine-urea cycle-NO metabolic pathways. CONCLUSIONS: This paper revealed the potential biomarkers and metabolic pathways regulated by Erigeron breviscapus. It was speculated that the mechanism is related to its inhibition of mitochondrion-mediated apoptosis. Erigeron breviscapus could restore the metabolic profiles of the model animals to normal animal levels. The mechanism may be related to the potential biomarkers of quinolinic acid, succinylornithine, and leucine proline and the metabolic pathways involved. However, the exact mechanism by which Erigeron breviscapus inhibits mitochondrion-mediated apoptosis remains to be further explored.

Dengzhan Xixin injection derived from a traditional Chinese herb Erigeron breviscapus ameliorates cerebral ischemia/reperfusion injury in rats via modulation of mitophagy and mitochondrial apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengzhan Xixin injection (DX), a preparation of extracts from traditional Chinese medicine Erigeron breviscapus (Vaniot) Hand.-Mazz., has been widely used in clinical treatment of cerebral ischemia sequelae in China for a long history. However, its underlying mechanisms remain unclear. AIM OF THE STUDY: The objective of this present study aimed to investigate the therapeutic effects of DX on cerebral ischemia/reperfusion (I/R) injury in a rat model. Meanwhile, its underlying molecular mechanisms on mitochondrial protection were further interpreted. MATERIALS AND METHODS: The major components of DX were detected by high-performance liquid chromatography analysis. The model of cerebral I/R injury was established by middle cerebral artery occlusion (MCAO) in SD rats. We firstly performed neurobehavioral score, the regional cerebral blood flow (rCBF) assay, and TTC, HE and Nissl staining for evaluating the effects of DX on I/R injury. And then, the cortical levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) were determined by commercial kits. Whereafter, real time-PCR and transmission electron microscopy were employed to investigate the relative copy number of mitochondrial DNA (mtDNA) and neuronal ultrastructure changes, respectively. Further, the potential interactions of major components in DX with mitophagy/apoptosis-related proteins were predicted by Schrodinger molecular docking. The expression of mitophagy-related proteins LC3, p62, TOM20, PINK1 and Parkin was estimated by western blot and immunofluorescence analyses. Furthermore, TUNEL staining and western blot were used to detect the apoptotic phenomenon and the protein expression of Bax, Bcl-2, Cytochrome c (Cyto-c) and cleaved Caspase-3. RESULTS: DX mainly contains scutellarin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, caffeic acid and 5-O-caffeoylquinic acid. Compared with the model group, DX could remarkably relieve ischemia-provoked neurological deficit, rCBF deficiency and cerebral infarction. Pathological changes and neuronal loss in a MCAO model of rats were memorably ameliorated by DX administration. Meanwhile, DX reduced the surged ROS and MDA, while increased the level of SOD. Notably, DX treatment conversed the collapse of ATP and MMP, along with decreased in the relative copy number of mtDNA, contributing to the maintaining of mitochondrial ultrastructure via the increased number of autophagy lysosomes. The representative ingredients in DX had a potential bind with the active sites of mitophagy/apoptosis-related proteins. DX stimulated the protein expression of LC3, PINK1 and Parkin, while reduced the levels of p62 and TOM20. In addition, DX confined TUNEL-positive cell rate with the decreased expressions of Bax, Cyto-c and cleaved Caspase-3 as well as the increased Bcl-2 level. CONCLUSIONS: We demonstrated that the protection of DX against brain ischemia could attribute to alleviating mitochondrial damage by upregulating mitophagy and inhibiting mitochondria-mediated apoptosis.

Chemical characteristics and significant antitussive effect of the Erigeron canadensis polyphenolic polysaccharide-protein complex.

ETHNOPHARMACOLOGICAL RELEVANCE: Erigeron canadensis has been used in traditional medicine to treat a variety of respiratory diseases, including acute upper and lower respiratory tract infections and cough-related asthma. There is as yet no relevant experimental or clinical study in the scientific literature evaluating the efficacy of plants in these disorders. AIM OF THE STUDY: To investigate the active ingredients in Erigeron canadensis, a complex isolated from flowering parts of a plant was tested for airway defense reflexes, in particular for cough reflexes and airway reactivity. Both were experimentally induced by a chemical irritant that simulated the inflammatory conditions of their formation. MATERIAL AND METHODS: The polyphenolic polysaccharide-protein (PPP) complex was isolated from the flowering parts of Erigeron canadensis by hot alkaline extraction and a multi-stage purification process. The antitussive activity was confirmed as a decrease in the number of citric acid-induced coughs and the bronchodilator effect was verified as a decrease in specific airway resistance (sRaw) in conscious guinea pigs. RESULTS: The dark brown Erigeron complex with a molecular weight of 38,000 g/mol contained phenolics (13.2% wt%), proteins (16.3% wt%), and uronic acids (6.3% wt%). The neutral carbohydrate part of Erigeron consisted mainly of xylose (12.1 wt%), glucose (13.3 wt%), arabinose (24.1 wt%), and galactose (41.0 wt%) residues. Arabinogalactan and 4-OMe-glucuronoxylan have been found to be the major polysaccharides in the Erigeron complex. Using a method of chemically-induced cough reflex and guinea pigs test system the Erigeron complex exhibited statistically significant, the dose-dependent antitussive activity, which was similar to that of the centrally-acting opioid agonist codeine. CONCLUSION: Pharmacological tests have revealed a new pharmacodynamic effect of the Erigeron complex, namely an antitussive effect. Its activity was most pronounced in comparison with all previously tested compounds from other medicinal plants and approached the effect of codeine, the most potent antitussive used in clinical practice. The results provide the scientific basis for the application of this herb in traditional medicine.

Comparative genomics reveal the convergent evolution of CYP82D and CYP706X members related to flavone biosynthesis in Lamiaceae and Asteraceae.

Distant species producing the same secondary metabolites is an interesting and common phenomenon in nature. A classic example of this is scutellarein whose derivatives have been used clinically for more than 30 years. Scutellarein occurs in significant amounts in species of two different orders, Scutellaria baicalensis and Erigeron breviscapus, which diverged more than 100 million years ago. Here, according to the genome-wide selection and functional identification of 39 CYP450 genes from various angiosperms, we confirmed that only seven Scutellaria-specific CYP82D genes and one Erigeron CYP706X gene could perform the catalytic activity of flavone 6-hydroxylase (F6H), suggesting that the convergent evolution of scutellarein production in these two distant species was caused by two independently evolved CYP450 families. We also identified seven Scutellaria-specific CYP82D genes encoding flavone 8-hydroxylase (F8H). The evolutionary patterns of CYP82 and CYP706 families via kingdom-wide comparative genomics highlighted the evolutionary diversity of CYP82D and the specificity of CYP706X in angiosperms. Multi-collinearity and phylogenetic analysis of CYP82D in Scutellaria confirmed that the function of F6H evolved from F8H. Furthermore, the SbaiCYP82D1A319D , EbreCYP706XR130A , EbreCYP706XF312D and EbreCYP706XA318D mutants can significantly decrease the catalytic activity of F6H, revealing the contribution of crucial F6H amino acids to the scutellarein biosynthesis of distant species. This study provides important insights into the multi-origin evolution of the same secondary metabolite biosynthesis in the plant kingdom.

Protective mechanism of Erigeron breviscapus injection on blood-brain barrier injury induced by cerebral ischemia in rats.

This study investigates the protective effect of Erigeron breviscapus injection, a classic traditional Chinese medicine most typically used by Chinese minority to treat stroke, on cerebral ischemia-reperfusion injury and the related signaling pathways. Use network pharmacology methods to study the relationship between E. breviscapus (Vant.) Hand-Mazz. and ischemic stroke, predict the mechanism and active ingredients of E. breviscapus (Vant.) Hand-Mazz. in improving ischemic stroke disease. We study the protective effect of E. breviscapus injection on blood-brain barrier (BBB) injuries induced by cerebral ischemia in rats by regulating the ROS/RNS-MMPs-TJs signaling pathway. The rat model of focal cerebral ischemia-reperfusion injury has been prepared using the wire-suppository method. Firstly, the efficacy of E. breviscapus injection, Scutellarin and 3,5-dicaffeoylquinic acid in protecting BBB injury caused by cerebral ischemia has been evaluated. Secondly, the following two methods have been used to study the mechanism of E. breviscapus injection in regulating the ROS/RNS-MMPS-TJS signaling pathway: real-time PCR and western blot for the determination of iNOS, MMP-9, claudin-5, occludin, ZO-1 mRNA and protein expression in brain tissue. We find that PI3K-Akt signaling pathway predicted by network pharmaology affects the blood-brain barrier function, so we chose the blood-brain barrier-related MMP-9, claudin-5, iNOS, occludin and ZO-1 proteins are used for research. The results of our research show that 3 drugs can reduce the rate of cerebral infarction in rats, relieve the abnormal neuroethology of rats, reduce the degree of brain tissue lesion, increase the number of the Nissl corpuscle cells and repair the neuron ultrastructure in injured rats. At the same time, it can obviously reduce the ultrastructure damage of the BBB in rats. All three drugs significantly reduced the content of Evans blue in the ischemic brain tissue caused by cerebral ischemia in rats with BBB injury. In addition, E. breviscapus injection, Scutellarin and 3,5-dicaffeoylquinic acid can decrease the protein expression of iNOS and MMP-9 in rat ischemic brain tissue. In addition, 3,5-dicaffeoylquinic acid can increase the protein expression of claudin-5. We conclude that E. breviscapus injection, Scutellarin and 3,5-dicaffeoylquinic acid have obvious therapeutic effects on BBB and neuron injury induced by cerebral ischemia in rats. Our results from studying the mechanism of action show that E. breviscapus injection and Scutellarin inhibited the activation of MMP-9 by inhibiting the synthesis of iNOS, 3,5-dicaffeoylquinic acid inhibits the expression and activation of MMP-9 by inhibiting the activation of iNOS and reducing the generation of free radicals, thus reducing the degradation of important cytoskeleton connexin claudin-5 in the tight junction (TJ) structure by inhibiting the expression and activation of MMP-9. Finally BBB structure integrity was protected.

Metabolism and Pharmacological Mechanisms of Active Ingredients in Erigeron breviscapus.

BACKGROUND: Erigeron breviscapus (Vant.) Hand-Mazz. is a plant species in the Compositae family. More than ten types of compounds-such as flavonoids, caffeinate esters, and volatile oils-have been identified in Erigeron breviscapus; however, it remains unknown as to which compounds are associated with clinical efficacy. In recent years, flavonoids and phenolic acids have been considered as the main effective components of Erigeron breviscapus. The metabolism and mechanisms of these compounds in vivo have been extensively studied to improve our understanding of the drug. METHODS: In the present review, we summarize the relationships among these compounds, their metabolites, and their pharmacodynamics. Many methods have been implemented to improve the separation and bioavailability of these compounds from Erigeron breviscapus. RESULTS: In China, Erigeron breviscapus has been used for many years. In recent years, through the study of its metabolism and the mechanisms of its effective components, the effects of Erigeron breviscapus in the treatment of various diseases have been extensively studied. Findings have indicated that Erigeron breviscapus improves cardiovascular and cerebrovascular function and that one of its ingredients, scutellarin, has potential value in the treatment of Alzheimer's disease, cancer, diabetic vascular complications, and other conditions. In addition, phenolic acid compounds and their metabolites also play an important role in anti-oxidation, anti-inflammation, and improving blood lipids. CONCLUSION: Erigeron breviscapus plays an important role in the prevention and treatment of cardiovascular/ cerebrovascular diseases, neuroprotection, and cancer through many different mechanisms of action. Further investigation of its efficacious components and metabolites may provide more possibilities for the clinical application of traditional Chinese medicine and the development of novel drugs.

Scutellarin Suppresses RPMI7951 Melanoma Cell Proliferation by Targeting TOPK.

BACKGROUND: T-LAK cell-Originated Protein Kinase (TOPK) belongs to the serine/threonine protein kinase family. It is highly expressed in RPMI7951 melanoma cells. Scutellarin (SCU) is an active ingredient extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. Its main physiological functions are related to its anti-inflammatory and antitumour activities. METHODS: The relationship between SCU and TOPK was assessed by molecular docking, an in vitro binding assay and an in vitro kinase assay. The effect of SCU on RPMI7951 cells was detected by MTS and soft agar assays. TOPK knockdown was induced by lentiviral infection. The TOPK downstream signalling pathway was detected by western blot and immunohistochemical analyses in vitro and in vivo. RESULTS: SCU was found to directly bind with TOPK and inhibit TOPK activity in vitro. SCU inhibited the proliferation and colony formation of RPMI7951 cells in a dose-dependent manner. Silencing TOPK decreased the sensitivity of colon cancer cells to SCU. SCU inhibited the phosphorylation levels of Extracellular Regulated protein Kinases 1/2 (ERK1/2) and histone H3 in a time- and dose-dependent manner in RPMI7951 cells. In addition, SCU inhibited the growth of xenograft tumours of RPMI7951 cells and decreased the phosphorylation levels of extracellular regulated protein kinases 1/2 and histone H3 in vivo. CONCLUSION: The results showed that SCU exerts promising antitumour effects on human RPMI7951 cells by inhibiting the activity of TOPK.

PK-PD Correlation of Erigeron Breviscapus Injection in the Treatment of Cerebral Ischemia-Reperfusion Injury Model Rats.

By measuring the cerebral infarction rate and neurological behavioral score of rats in a sham operation group, an MCAO model control group and an Erigeron breviscapus injection treatment group, we explored the therapeutic effects of Erigeron breviscapus injection on brain tissue and neuroethological injury in rats. Plasma samples were collected at 18 time points after intravenous injection of Erigeron breviscapus. The levels of scutellarin, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, chlorogenic acid and isochlorogenic acid B in rat plasma at the various time points were determined by an HPLC method, and drug concentration versus time plots were constructed to estimate the pharmacokinetic parameters. Finally, a PK-PD combined model was used to analyze the relationship between the blood concentration, time and therapeutic effects of the seven active components. The results of the pharmacodynamics studies showed that the cerebral infarction rate of rats in the Erigeron breviscapus injection group decreased significantly at 5 min, 10 min, 20 min, 6 h, 8 h, 18 h, 24 h, 32 h, 40 h and 48 h after cerebral ischemia. Abnormal neurological behavior scores were significantly reduced after 4 h of cerebral ischemia. The pharmacokinetics results showed that the seven chemical constituents in Erigeron breviscapus injection reached their highest detection value after 5 min of cerebral ischemia. The lowest detection values of scutellarin and isochlorogenic acid B appeared after 6 h of cerebral ischemia but could not be detected after 8 h. The lowest detection values of 5-caffeoylquinic acid and 4,5-dicaffeoylquinic acid were found in the third hour of cerebral ischemia but not after 4 h. The lowest detection values of 4-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and chlorogenic acid were found during the second hour of cerebral ischemia but not at the third hour. However, at 18 h, 24 h, 32 h and 40 h of cerebral ischemia, the cerebral infarction rates of rats in the Erigeron breviscapus injection group were significantly reduced, with decreased values of 6.22%, 11.71%, 6.92% and 4.96%, respectively, and the effects were stronger than those after 5-20 min of cerebral ischemia. The decreased values reached their highest value after 24 h of cerebral ischemia. Our results show that the effects of Erigeron breviscapus injection on reducing the cerebral infarct rate in MCAO model rats are characterized by a fast onset and long maintenance time. The 5-min blood concentration in cerebral ischemia was the highest test value, and after this time, the cerebral infarction rate of MCAO rats began to decrease. However, the peak value of the effects lagged behind that of the plasma concentration. The maximum effective time for Erigeron breviscapus injection appeared 24 h after cerebral ischemia, which provides a reference for the screening of specific drugs for ischemic stroke, optimal dosing regimens and rational clinical drug use. Graphical Abstract.

Systems Pharmacology Dissection of Mechanisms of Dengzhan Xixin Injection against Cardiovascular Diseases.

Dengzhan Xixin injection (DZXXI), a herbal product prepared from a Chinese herb called Erigeron breviscapus, is a classical and traditional therapeutic for cadiovascular diseases (CVDs), including coronary heart disease (CHD), angina, and stroke, etc. However, its potential pharmacology mechanism against CVDs remains unclear. In this paper, a systems pharmacology-based strategy is presented for predicting drug targets and understanding therapeutic mechanisms of DZXXI against CVDs. The main ingredients were identified by HPLC-diode array detector (DAD). The target fishing was performed on the PharmMapper Server (http://lilab-ecust.cn/pharmmapper/). Potential targets were confirmed by two molecular docking tools, Sybyl-X 1.3 and Ledock to ensure the accuracy. The resulting target proteins were applied as baits to fish their related diseases and pathways from the molecular annotation system (MAS 3.0, http://bioinfo.capitalbio.com/mas3/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/). Network generation and topological analysis were performed in Cytoscape 3.6.0. 15 main ingredients from DZXXI were identified. Forty five putative drug targets and 50 KEGG pathways, which have highly relevance to the therapeutic effects of DZXXI against CVDs, were then obtained. The systems analysis suggested that DZXXI could attenuate cardiac fibrosis, regulate cardiac contractility, and preserve heart function in adverse cardiac remodeling; meanwhile DZXXI also could have the function of activating blood circulation and dilating blood vessels. DZXXI exerts its therapeutic effects on CVDs possibly through multi-targets including CMA1, epidermal growth factor receptor (EGFR), phenylalanine-4-hydroxylase (PAH), SRC, F7, etc., and multi-pathways including Focal adhesion, mitogen-activated protein kinase (MAPK) signaling pathway, complement and coagulation cascades, Wnt signaling pathway, vascular endothelial growth factor (VEGF) signaling pathway, Renin-angiotensin system, etc.

Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4'-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups.

A series of novel scutellarin methyl ester-4'-dipeptide conjugates exhibiting active transport characteristics and protection against pathological damage caused by hypoxic-ischemic encephalopathy (HIE) were successfully designed and synthesized. The physiochemical properties of the obtained compounds, as well as the Caco-2 cell-based permeability and uptake into hPepT1-MDCK cells were evaluated using various analytical methods. Scutellarin methyl ester-4'-Val-homo-Leu dipeptide (5k) was determined as the optimal candidate with a high apparent permeability coefficient (Papp A to B) of 1.95 ± 0.24 × 10-6 cm/s, low ER (Papp BL to AP/Papp AP to BL) of 0.52 in Caco-2 cells, and high uptake of 25.47 µmol/mg/min in hPepT1-MDCK cells. Comprehensive mechanistic studies demonstrated that pre-treatment of PC12 cells with 5k resulted in more potent anti-oxidative activity, which was manifested by a significant decrease in the malondialdehyde (MDA) and reactive oxygen species (ROS) levels, attenuation of the H2O2-induced apoptotic cell accumulation in the sub-G1 peak, and improvement in the expression of the relevant apoptotic proteins (Bcl-2, Bax, and cleave-caspase-3). Moreover, evaluation of in vivo neuroprotective characteristics in hypoxic-ischemic rat pups revealed that 5k significantly reduced infarction and alleviated the related pathomorphological damage. The compound was also shown to ameliorate the neurological deficit at 48 h as well as to decrease the brain tissue loss at 4 weeks. Conjugate 5k was demonstrated to reduce the amyloid precursor protein (APP) and ß-site APP-converting enzyme-1 (BACE-1) expression. Pharmacokinetic characterization of 5k indicated favorable druggability and pharmacokinetic properties. The conducted docking studies revealed optimal binding of 5k to PepT1. Hydrogen bonding as well as cation-π interactions with the corresponding amino acid residues in the target active site were clearly observed. The obtained results suggest 5k as a potential candidate for anti-HIE therapy, which merits further investigation.

[Research advance in chemical compounds,pharmacological action and clinical application of Dengzhan Shengmai Capsules].

Dengzhan Shengmai Capsules( DZSMC),a well-known traditional Chinese medicine( TCM) formula,is comprised of the main drug of Erigeron breviscapus,and supplemented with Panax ginseng,Ophiopogon japonicus and Schisandra chinensis,with functions of supplementing Qi and nourishing Yin,promoting blood circulation and strengthening brain. DZSMC is the only Chinese patent drug with A-level evidence-based medicine in secondary prevention for stroke and ranks first among TCMs for neurological treatment. Modern studies indicate that the chemical constituents of DZSMC mainly include flavonoids,phenolic acids,lignans,saponins and so on. Pharmacological experimental studies have shown that DZSMC has such pharmacological effects as anti-oxidation,anti-inflammatory and anti-myocardial ischemia. DZSMC is mainly used in the convalescent care of ischemic cardiovascular and cerebrovascular diseases,and is often used in combination with various conventional therapeutic drugs to exert clinical efficacy through brain protection,neuroprotection,etc.,and improve clinical symptoms in patients. In this review,according to domestic and international related literature combined with research results obtained by our project,the research advances in the chemical constituents,pharmacological effects and clinical application of DZSMC have been systematically reviewed and summarized,providing reference and support for further study and secondary development of the formula.

Sesquiterpenoids and γ-pyranone derivatives from the whole plant of Erigeron breviscapus and their neuroprotective effects.

Four new sesquiterpenes (1-2, 6-7), a new pyranone glycoside (10) along with six known compounds, were isolated from the whole plant of Erigeron breviscapus. Their planar structures were elucidated using extensive spectroscopic analyses. Brevisterpene A (1) and brevisterpene B (2) were proved to be a pair of diastereomer followed by mixtures resolution using chiral HPLC. Their absolute configurations were determined by ECD calculation. The relative configuration of brevisnoside B (7) was elucidated by a combined analysis of NOESY spectrum and computation of 13C NMR chemical shifts, and determination of the absolute configurations of 6 and 7 assisted by optical rotation calculations. Compounds 1 and 2 displayed moderate neuroprotective effects against H2O2-induced damage in SH-SY5Y cells.

Identification and Characterization of Two New 1- O-Acyl-glucose-ester Forming Glucosyltransferases from Erigeron breviscapus.

Two versatile UDP-glucosyltransferases, UGT75L25 and UGT75X1, were isolated from Erigeron breviscapus. The enzymes display high sequence identity to flavonoid 7- O-glucosyltransferase from Malus species and cluster to the phylogenetic group L of plant glucosyltransferases, also involved in the formation of hydroxycinnamoyl glucose esters, which are used as bifunctional donors in the glucosylation or acylation of anthocyanins. The enzymes, functionally expressed in Escherichia coli, exhibit broad substrate specificity toward 21 structurally diverse types of phenolic acids, including (hydroxy)cinnamates, vanillic acid, 3-hydroxycoumarin, and 7-hydroxyflavonoids. The catalytic characteristics of UGT75L25 and UGT75X1 were exploited to generate the corresponding acyl-glucose-esters or glucosides with high efficiency. These findings demonstrate the significant potential of acyl-glucose-esters in the further enzymatic synthesis of bioactive anthocyanins.

Research advance in chemical compounds,pharmacological action and clinical application of Dengzhan Shengmai Capsules / 中国中药杂志

Dengzhan Shengmai Capsules( DZSMC),a well-known traditional Chinese medicine( TCM) formula,is comprised of the main drug of Erigeron breviscapus,and supplemented with Panax ginseng,Ophiopogon japonicus and Schisandra chinensis,with functions of supplementing Qi and nourishing Yin,promoting blood circulation and strengthening brain. DZSMC is the only Chinese patent drug with A-level evidence-based medicine in secondary prevention for stroke and ranks first among TCMs for neurological treatment. Modern studies indicate that the chemical constituents of DZSMC mainly include flavonoids,phenolic acids,lignans,saponins and so on. Pharmacological experimental studies have shown that DZSMC has such pharmacological effects as anti-oxidation,anti-inflammatory and anti-myocardial ischemia. DZSMC is mainly used in the convalescent care of ischemic cardiovascular and cerebrovascular diseases,and is often used in combination with various conventional therapeutic drugs to exert clinical efficacy through brain protection,neuroprotection,etc.,and improve clinical symptoms in patients. In this review,according to domestic and international related literature combined with research results obtained by our project,the research advances in the chemical constituents,pharmacological effects and clinical application of DZSMC have been systematically reviewed and summarized,providing reference and support for further study and secondary development of the formula.

Rapid characterization and identification of the chemical constituents and rat metabolites of Deng-Zhan-Xi-Xin injection using ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Deng-Zhan-Xi-Xin injection is a well-known traditional Chinese medicine prescription for the treatment of cardiovascular and cerebral vessel diseases. However, there have been few reports on its chemical constituents and metabolic pathway, which has blocked its further quality control and studies on its pharmacology and mechanism of action. In this study, an integrative method was established to rapidly explore the chemical constituents and metabolites of Deng-Zhan-Xi-Xin injection using ultra high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and the UNIFI™ software combined with multiple data processing approaches. As a result, a total of 40 compounds, including 9 flavonoids and 31 phenolic acids were identified or tentatively characterized, and five compounds were first reported in Deng-Zhan-Xi-Xin injection. Under the same analysis conditions, 70 compounds have been detected in rats, including 25 prototypes and 45 metabolites. This was the first systematic research study on the metabolic profiling of Deng-Zhan-Xi-Xin injection. This study provides valuable chemical information for the quality control and research on pharmacology and mechanism of action of Deng-Zhan-Xi-Xin injection. Moreover, it provides a valuable strategy for analyzing the chemical components and metabolites of other traditional Chinese medicine prescriptions.

Scutellarin Ameliorates Learning and Memory Deficit via Suppressing ß-Amyloid Formation and Microglial Activation in Rats with Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion is considered as a pivotal factor of cognitive impairment that occurs in cerebrovascular diseases. This study investigated the ameliorating effect of scutellarin (SCT) on spatial cognitive impairment and ß-amyloid (Aß) formation in rats with chronic cerebral hypoperfusion induced by permanent bilateral common carotid artery occlusion (pBCAO). SCT is a flavonoid in medicinal herb of Erigeron breviscapus (vant.) Hand. Mazz. known to have neuroprotective, antioxidative and anti-inflammatory effects. However, the beneficial effect and pivotal mechanism of SCT on cognitive impairment are still unclear. SCT was treated orally with two doses (10 or 30 mg/kg) for 4 weeks. Results of Morris water maze test performed on the ninth week after pBCAO revealed that SCT (30 mg/kg)-treated rats had significantly shortened escape latencies in acquisition training trials, significantly prolonged swimming time at the platform and its surrounding zone, significant increase in memory score, significant reduction in the number of target heading, and significant reduction in the time required for the first target heading during the retention trial compared to rats in the sham-control group. SCT significantly inhibited the production of Aß(1-40) and Aß(1­42) in brain tissues. However, SCT significantly upregulated the expression levels of amyloid precursor protein and ß-site APP-converting enzyme-1 in the hippocampus. In addition, SCT significantly inhibited the activation of Iba1-expressing microglia in brain tissues. The results suggest that SCT can exert ameliorating effect on spatial cognitive impairment caused by chronic cerebral hypoperfusion through suppressing Aß formation and microglial activation in brain tissues. Therefore, SCT can be used as a beneficial drug for vascular dementia and Alzheimer's disease.

A UPLC-ESI-MS/MS Method for Simultaneous Quantitation of Chlorogenic Acid, Scutellarin, and Scutellarein in Rat Plasma: Application to a Comparative Pharmacokinetic Study in Sham-Operated and MCAO Rats after Oral Administration of Erigeron breviscapus Extract.

Erigeron breviscapus, a traditional Chinese medicine, is clinically used for the treatment of occlusive cerebral vascular diseases. We developed a sensitive and reliable ultra-performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS) method for simultaneous quantitation of chlorogenic acid, scutellarin, and scutellarein, the main active constituents in Erigeron breviscapus, and compared the pharmacokinetics of these active ingredients in sham-operated and middle cerebral artery occlusion (MCAO) rats orally administrated with Erigeron breviscapus extract. Plasma samples were collected at 15 time points after oral administration of the Erigeron breviscapus extract. The levels of chlorogenic acid, scutellarin, and scutellarein in rat plasma at various time points were determined by a UPLC-ESI-MS/MS method, and the drug concentration versus time plots were constructed to estimate pharmacokinetic parameters. The concentration of chlorogenic acid in the plasma reached the maximum plasma drug concentration in about 15 min and was below the limit of detection after 4 h. Scutellarin and scutellarein showed the phenomenon of multiple absorption peaks in sham-operated and MCAO rats, respectively. Compared with the sham-operated rats, the terminal elimination half-life of scutellarein in the MCAO rats was prolonged by more than two times and the area under the curve of each component in the MCAO rats was significantly increased. The results showed chlorogenic acid, scutellarin, and scutellarein in MCAO rats had higher drug exposure than that in sham-operated rats, which provided a reference for the development of innovative drugs, optimal dosing regimens, and clinical rational drug use.

Protective effects of Erigeron breviscapus Hand.- Mazz. (EBHM) extract in retinal neurodegeneration models.

Purpose: To investigate the neuroprotective effects of scutellarin, an active component of the multifunctional traditional Chinese herb Erigeron breviscapus (vant.) Hand.-Mazz. (EBHM), which has been used as a neuroprotective therapy for cerebrovascular diseases. We performed the experiments using in vitro and in vivo models of retinal neurodegeneration. Methods: In the in vitro experiments, we exposed BV-2 cells to low oxygen levels in an incubator for 24 and 48 h to generate hypoxia models. We then treated these cells with scutellarin at concentrations of 2, 10, and 50 µM. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA). The levels of the components of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome signaling pathway, including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved caspase-1, interleukin-18 (IL-18), and IL-1ß were analyzed using western blots and ELISAs. In the in vivo study, we raised the intraocular pressure of Brown Norway rats to 60 mmHg for 30 min to generate a high intraocular pressure (HIOP) model, that is, an acute glaucoma model. The rats were then treated with scutellarin via oral gavage for 2 consecutive weeks. The relevant components of the NLRP3 inflammasome signaling pathway were analyzed with western blots and ELISAs. Retinal ganglion cells (RGCs) were retrogradely labeled using 4% Fluoro-Gold, and then the numbers of cells were calculated. Retinal microglial cells were labeled using immunofluorescence, and then the morphological changes were observed. Results: In the in vitro cell viability experiments, 50 µM scutellarin statistically significantly enhanced the viability rate when compared to 2 µM and 10 µM scutellarin (hypoxia + 50 µM EBHM group: 94.01±2.130% and 86.02±2.520% after 24 and 48 h, respectively; hypoxia model group: 74.98±3.860% and 64.41±4.890% after 24 and 48 h, respectively; for all when compared to normal control, p<0.001). Scutellarin inhibited the expression of NLRP3 in vitro (the hypoxia + EBHM group/normal control group ratio versus the hypoxia model group/normal control group ratio: 2.30±0.12 versus 4.06±0.19, p<0.01) and in vivo (the HIOP + EBHM group/normal control group ratio versus the HIOP model group/normal control ratio: 3.39±0.42 versus 6.07±0.22, p<0.01). Scutellarin administration also reduced the upregulation of ASC, cleaved caspase-1, IL-18, and IL-1ß in vitro and in vivo. In the in vivo study, the RGC survival rate was statistically significantly improved following scutellarin administration (p<0.001 versus the HIOP group), and the number of impaired retinal microglial cells was statistically significantly reduced following scutellarin treatment when compared with the HIOP model group. Conclusions: EBHM extract scutellarin exhibits protective effects in retinal hypoxia models by inhibiting NLRP3 inflammasome-mediated inflammatory reactions. Thus, EBHM extract scutellarin may be an appropriate therapeutic option for disorders related to retinal neurodegeneration, such as glaucoma.