Fuzi lizhong pills alter microbial community compositions and metabolite profiles in ulcerative colitis rat with spleen-kidney yang deficiency syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is a chronic inflammatory bowel condition that is frequently related with Spleen-Kidney Yang Deficiency Syndrome (SKYD) in Chinese medicine. Fuzi Lizhong Pill (FLZP), a traditional medicine for SKYD, has been utilized in China for generations, although the exact mechanism by which it treats UC is unknown. AIM OF THE STUDY: The goal of this study is to further understand FLZP's therapeutic mechanism in SKYD-associated UC. MATERIALS AND METHODS: To investigate the impact of FLZP on SKYD-associated UC, we used a comprehensive method that included serum metabolomics and gut microbiota profiling. The chemical composition of FLZP was determined using mass spectrometry. UC rats with SKYD were induced and treated with FLZP. Serum metabolomics and 16S rRNA microbial community analysis were used to evaluate FLZP's effects on endogenous metabolites and gut microbiota, respectively. Correlation analysis investigated the association between metabolites and intestinal flora. A metabolic pathway analysis was undertaken to discover putative FLZP action mechanisms. RESULTS: FLZP contains 109 components, including liquiritin (584.8176 µg/g), benzoylaconine (16.3087 µg/g), benzoylhypaconine (31.9583), and hypaconitine (8.1160 µg/g). FLZP predominantly regulated seven metabolites and eight metabolic pathways involved in amino acid and nucleotide metabolism, with an emphasis on energy metabolism and gastrointestinal digestion. FLZP also influenced intestinal flora variety, increasing probiotic abundance while decreasing pathogenic bacteria prevalence. An integrated investigation identified associations between changes in certain gut flora and energy metabolism, specifically the tricarboxylic acid (TCA) cycle. CONCLUSIONS: FLZP successfully cures UC in SKYD rats by regulating amino acid and energy metabolism. Its positive effects may include altering microbiota composition and metabolite profiles in UC rats with SKYD. These findings shed light on FLZP's mode of action and its implications for UC management.

Structurally diverse phthalides from fibrous roots of Ligusticum chuanxiong Hort. and their biological activities.

Falonolide A (1) and B (2), two novel polyyne hybrid phthalides resulting from unprecedented carbon skeleton polymerized by Z-ligustilide and falcarindiol, along with six new related phthalides (3-8), were isolated from Ligusticum chuanxiong Hort. Their structures were elucidated by spectroscopic analysis, computer-assisted structure elucidation (CASE) analysis, DP4+ probability analysis and electronic circular dichroism (ECD) calculations. A plausible biosynthetic pathway for 1-8 was proposed, and the production mechanism of 2 was revealed by density functional theory (DFT) method. Compounds 4 and 6 exhibited significant vasodilatory activity with EC50 of 8.00 ± 0.86 and 6.92 ± 1.02 µM, respectively. Compound 4 also displayed significant inhibitory effect of NO production with EC50 value of 8.82 ± 0.30 µM. Based on the established compounds library, structure-activity relationship analysis of phthalides was explored to provide insights into the drug development of vasodilators and anti-flammatory.

Unlocking the hidden potential: Enhancing the utilization of stems and leaves through metabolite analysis and toxicity assessment of various parts of Aconitum carmichaelii.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum carmichaelii is widely used in traditional Chinese medicine clinics as a bulk medicinal material. It has been used in China for more than two thousand years. Nevertheless, the stems and leaves of this plant are usually discarded as non-medicinal parts, even though they have a large biomass and exhibit therapeutic properties. Thus, it is crucial to investigate metabolites of different parts of Aconitum carmichaelii and explore the relationship between metabolites and toxicity to unleash the utilization potential of the stems and leaves. AIM OF THE STUDY: Using plant metabolomics, we aim to correlate different metabolites in various parts of Aconitum carmichaelii with toxicity, thereby screening for toxicity markers. This endeavor seeks to offer valuable insights for the development of Aconitum carmichaelii stem and leaf-based applications. MATERIALS AND METHODS: UHPLC-Q-Orbitrap MS/MS-based plant metabolomics was employed to analyze metabolites of the different parts of Aconitum carmichaelii. The cardiotoxicity and hepatotoxicity of the extracts from different parts of Aconitum carmichaelii were also investigated using zebrafish as animal model. Toxicity markers were subsequently identified by correlating toxicity with metabolites. RESULTS: A total of 113 alkaloids were identified from the extracts of various parts of Aconitum carmichaelii, with 64 different metabolites in stems and leaves compared to daughter root (Fuzi), and 21 different metabolites in stems and leaves compared to mother root (Wutou). The content of aporphine alkaloids in the stems and leaves of Aconitum carmichaelii is higher than that in the medicinal parts, while the content of the diester-diterpenoid alkaloids is lower. Additionally, the medicinal parts of Aconitum carmichaelii exhibited cardiotoxicity and hepatotoxicity, while the stems and leaves have no obvious toxicity. Finally, through correlation analysis and animal experimental verification, mesaconitine, deoxyaconitine, and hypaconitine were used as toxicity markers. CONCLUSION: Given the low toxicity of the stems and leaves and the potential efficacy of aporphine alkaloids, the stems and leaves of Aconitum carmichaelii hold promise as a valuable medicinal resource warranting further development.

Therapeutic Effects of Valeriana jatamansi on Ulcerative Colitis: Insights into Mechanisms of Action through Metabolomics and Microbiome Analysis.

Ulcerative colitis (UC), belonging to inflammatory bowel disease (IBD), is a chronic and relapsing inflammatory disorder of the gastrointestinal tract, which has not been completely cured in patients so far. Valeriana jatamansi is a Chinese medicine used clinically to treat "diarrhea," which is closely related to UC. This study was to elucidate the therapeutic effects of V. jatamansi extract (VJE) on dextran sodium sulfate (DSS)-induced UC in mice and its underlying mechanism. In this work, VJE effectively ameliorates the symptoms and histopathological scores and reduces the production of inflammatory factors in UC mice. The colon untargeted metabolomics analysis and 16S rDNA sequencing showed remarkable differences in colon metabolite profiles and intestinal microbiome composition between the control and DSS groups, and VJE intervention can reduce these differences. Thirty-two biomarkers were found and modulated the primary pathways including pyrimidine metabolism, arginine biosynthesis, and glutathione metabolism. Meanwhile, twelve significant taxa of gut microbiota were found. Moreover, there is a close relationship between endogenous metabolites and intestinal flora. These findings suggested that VJE ameliorates UC by inhibiting inflammatory factors, recovering intestinal maladjustment, and regulating the interaction between intestinal microbiota and host metabolites. Therefore, the intervention of V. jatamansi is a potential therapeutic treatment for UC.

Ajuforrestin A, an Abietane Diterpenoid from Ajuga ovalifolia var. calanthe, Induces A549 Cell Apoptosis by Targeting SHP2.

The Src-homology 2 domain-containing phosphatase 2 (SHP2), which is encoded by PTPN11, participates in many cellular signaling pathways and is closely related to various tumorigenesis. Inhibition of the abnormal activity of SHP2 by small molecules is an important part of cancer treatment. Here, three abietane diterpenoids, named compounds 1-3, were isolated from Ajuga ovalifolia var. calantha. Spectroscopic analysis was used to identify the exact structure of the compounds. The enzymatic kinetic experiment and the cellular thermal shift assay showed compound 2 selectively inhibited SHP2 activity in vitro. Molecular docking indicated compound 2 targeted the SHP2 catalytic domain. The predicted pharmacokinetic properties by SwissADME revealed that compound 2 passed the majority of the parameters of common drug discovery rules. Compound 2 restrained A549 proliferation (IC50 = 8.68 ± 0.96 µM), invasion and caused A549 cell apoptosis by inhibiting the SHP2-ERK/AKT signaling pathway. Finally, compound 2 (Ajuforrestin A) is a potent and efficacious SHP2 inhibitor and may be a promising compound for human lung epithelial cancer treatment.

A comprehensive investigation on the chemical diversity and efficacy of different parts of Ligusticum chuanxiong.

Ligusticum chuanxiong Hort. (CX) is a medicinal and edible plant with a wide range of constituents of biological interest. Since the biomass of the non-medicinal parts of CX is huge, discarding them will cause a waste of resources. To expand the medicinal uses of CX, we comprehensively investigated the chemical diversity and efficacy of its different parts (rhizomes, fibrous roots, stems and leaves). 75 compounds in the volatile oil and 243 compounds in the methanol extracts (including 95 phthalides) obtained from CX were characterized by GC-MS and UHPLC/Q-Orbitrap MS analysis, respectively. Of 95 phthalides, 14 potential new compounds and 5 phthalide trimers were identified from CX for the first time. Phthalide monomers were more abundant in rhizomes and fibrous roots, and phthalide dimers or even phthalide trimers mainly in stems and leaves. By multivariate and univariate analyses, 22 and 24 different compounds were found in the volatile oils and the methanol extracts, respectively. In the bioactivity evaluation of different parts, stems and leaves showed the best antioxidant activity, fibrous roots showed the strongest vasodilator activity, and rhizomes showed the most significant anticoagulant activity, which was related to the different metabolites in different parts. Ultimately, this work revealed the similarities and differences of phytochemicals and bioactivities in different anatomical parts of CX. It might provide helpful evidence for the rational application of non-medicinal resources.

[α-lipoic acid ameliorates liver injury in rats with type 2 diabetes mellitus via activating AMPK/mTOR pathway].

OBJECTIVE: To investigate the effect of α-lipoic acid in ameliorating liver injury in rats with type 2 diabetes mellitus via activating adenosine 5'-monophosphate-activate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. METHODS: The T2DM rat models were established by feeding with high-fat, high-sucrose diet and intraperitoneal injection of 27.5 mg/(kg·d) streptozotocin. The 32 rats with T2DM were randomly divided into 4 groups: T2DM group, α-lipoic acid group (LA), Compound C group (Comp C, an inhibitor of AMPK) and LA+Comp C group, with 8 rats in each group. Additionally, 8 Sprague-Dawlay (SD) rats without diabetes were set as normal control. The rats received α-lipoic acid at a dosage of 100 mg/(kg·d) or Compound C at a dosage of 20 mg/(kg·d) by intraperitoneal injection for 8 weeks as needed. The levels of relevant biochemical indexes were detected. The weight of liver was recorded to calculate liver weight index (LWI), and the pathological changes of liver tissues were detected by light and electron microscopy. The levels of AMPK, p-AMPK, mTOR, p-mTOR in rat liver were detected by Western blot. RESULTS: Compared with control group, the levels of LWI, homeostasis model assessment of insulin resistance, fasting blood glucose, alanine transaminase, aspartate transaminase, gamma glutamyl transferase and triglyceride in T2DM group were increased significantly (all P＜0.05). The liver tissue lesions were more serious and hepatic steatosis grade was higher. The expression of p-AMPK was decreased (P＜0.05) and the expression of p-mTOR was increased significantly(P＜0.05). α-lipoic acid could reverse the above-mentioned changes, ameliorate insulin resistance (all P＜0.05), protect the structure and function of liver, and activate the AMPK/mTOR pathway (P＜0.05). The protection of α-lipoic acid was weakened by the inhibition of AMPK with Compound C (P＜0.05). CONCLUSION: α-lipoic acid could protect the liver of rats with T2DM by activating AMPK/mTOR pathway.

Relationship Between Knowledge Base and Innovation-Driven Growth: Moderated by Organizational Character.

Purpose: On the background of innovation-driven growth strategy of the Chinese government, this study aims to explore the impact of the knowledge base on innovation-driven growth of a firm, which is moderated by organizational character. Design/methodology/approach: Based on the data of 965 Chinese listed companies, some hypotheses were tested using the method of hierarchical regression analysis. Findings: Organizational growth relies on both technological and business model innovations and their interactive effect. Knowledge base, both breadth and depth, makes a positive impact on the innovation-driven growth of an enterprise. In the impacting mechanism, an explicit organizational character not only has direct positive effects on business model innovation, it also strengthens the effect of knowledge breadth on business model innovation. On the contrary, an implicit organizational character is not significantly related to innovation. Research limitations/implications: In order to achieve growth, enterprises are suggested to adopt such dual innovation strategy, led by technological innovation and supplemented with business model innovation, which is supported by the integrated management of intangible resources, deep and broad knowledge, and explicit organizational character. Originality/value: A new theoretical framework of organizational innovation-driven growth was proposed. The realization paths of innovation-driven growth were explored. The idea of collaborative governance between the knowledge base and organizational character was raised.

Review of Natural Resources With Vasodilation: Traditional Medicinal Plants, Natural Products, and Their Mechanism and Clinical Efficacy.

For decades, chronic diseases including cardiovascular and cerebrovascular diseases (CCVDs) have plagued the world. Meanwhile, we have noticed a close association between CCVDs and vascular lesions, such as hypertension. More focus has been placed on TMPs and natural products with vasodilation and hypotension. TMPs with vasodilatory and hypotensive activities are mainly from Compositae, Lamiaceae, and Orchidaceae (such as V. amygdalina Del., T. procuinbens L., M. glomerata Spreng., K. galanga L., etc.) whereas natural products eliciting vasorelaxant potentials were primarily from flavonoids, phenolic acids and alkaloids (such as apigenin, puerarin, curcumin, sinomenine, etc.). Furthermore, the data analysis showed that the vasodilatory function of TMPs was mainly concerned with the activation of eNOS, while the natural products were primarily correlated with the blockage of calcium channel. Thus, TMPs will be used as alternative drugs and nutritional supplements, while natural products will be considered as potential therapies for CCVDs in the future. This study provides comprehensive and valuable references for the prevention and treatment of hypertension and CCVDs and sheds light on the further studies in this regard. However, since most studies are in vitro and preclinical, there is a need for more in-depth researches and clinical trials to understand the potential of these substances.

Modulation of the 5-HT3A receptor current by desacylbaldrinal.

The serotonin (5-hydroxytryptamine) type 3 receptor is an important target in the control of digestive dysfunction such as anorexia and bulimia, and 5-HT3 receptor antagonists are effective against eating disorder and the early-phase chemotherapy and radiotherapy evoked vomiting. Our previous research of Valeriana jatamansi revealed the presence of iridoids, which showed potent antitumor activities. Here, we explored the effects of 10π aromatic iridoid desacylbaldrinal isolated from V. jatamansi on the 5-HT3 receptor current. We performed whole cell recordings of 5-HT3A receptor currents in the presence of the compound. The result indicated that desacylbaldrinal inhibited the 5-HT-mediated 5-HT3A receptor current.

Chuanxiongdiolides R4 and R5, phthalide dimers with a complex polycyclic skeleton from the aerial parts of Ligusticum chuanxiong and their vasodilator activity.

Chuanxiongdiolides R4-R6 (1-3), three novel phthalide dimers featuring two classes of unreported monomeric units (ligustilide/senkyunolide A and ligustilide/neocnidilide) with an unprecedented linkage style (3a,7'/7a,7'a), were isolated from the aerial parts of Ligusticum chuanxiong, together with three pairs of enantiomeric phthalide dimers [(-)/(+)-4a/4b, 5a/5b, and 6a/6b]. The bioassays revealed that compounds 1, 3, 4, 5, and 6 showed significant vasodilation effects, and the mechanism may be attributed to Cav1.2 activation blockade. Based on the established compounds library, the structure activity relationship of the phthalides was proposed. Our findings afford possible leads for developing new vasodilator against cardiovascular and cerebrovascular diseases such as hypertension and ischemic stroke.

Effect of Repaglinide on Blood Glucose, Endothelial Function, Lipid Metabolism, and Inflammatory Reaction in a Rat Model of Atherosclerosis.

OBJECTIVE: To investigate the protective effect of repaglinide on rat with atherosclerosis. METHODS: Sprague Dawley (SD) rats were divided into control, model, repaglinide, and metformin groups. In addition to the normal group, rats were given intraperitoneal injection of streptozotocin and high-fat diet (HFD). Meanwhile, repaglinide or metformin was administrated to the treatment rats, respectively, for 4 weeks. Serum, plasma, liver, epididymal fat, and aorta thoracica were obtained to observe the protective effect of repaglinide on rat with atherosclerosis. RESULTS: Compared to the control group, blood glucose was increased in the model group (P < .05), while it was decreased in the drug-administered groups. In addition, the levels of endothelin 1, TG, TC, low-density lipoprotein cholesterol, atherogenic index, liver index, and epididymal fat index were significantly increased, but the levels of high-density lipoprotein cholesterol, plasminogen activator inhibitor 1, and antiatherogenic index were decreased significantly in the model group compared to the control group (P < .05, respectively). And these effects were reversed by treatment with repaglinide (P < .05, respectively). CONCLUSION: Our results suggested that repaglinide may regulate the formation of early atherosclerosis through the abovementioned mechanisms.

A new abietane diterpenoid from Ajuga ovalifolia var. calantha induces human lung epithelial A549 cell apoptosis by inhibiting SHP2.

The Src-homology 2 domain-containing phosphatase 2 (SHP2), encoded by PTPN11, has been reported oncogenic tyrosine phosphatase associated with various tumors and played critical roles in many cell signaling events. Targeting SHP2 by small molecules may be a promising way for cancer therapy. Herein, a new abietane diterpenoid, named 3-acetoxylteuvincenone G (3-AG), was isolated from the whole plants of Ajuga ovalifolia var. calantha. The structure of the new compound was elucidated by means of extensive spectroscopic analyses. Using recombinant enzyme activity assay and cellular thermal shift assay, we found that 3-AG was a selective inhibitor of SHP2. Molecular docking suggested 3-AG displayed an orientation favorable to nucleophilic attack in the catalytic domain of SHP2. 3-AG suppressed A549 cell proliferation (IC50 = 10.79 ± 0.14 µM), invasion and induced cell apoptosis through SHP2/ERK1/2 and SHP2/AKT pathways. In summary, 3-AG, a potent, selective, and efficacious SHP2 inhibitor, may be a promising small molecule to treat human lung epithelial cancer.

miR-346 promotes migration and invasion of nasopharyngeal carcinoma cells via targeting BRMS1.

The aim of this study is to determine the expression and roles of miR-346 in nasopharyngeal carcinoma (NPC). We showed that miR-346 was upregulated in NPC tissues compared with adjacent non-tumorous nasopharyngeal tissues. Inhibition of miR-346 significantly attenuated the migration and invasion of NPC cells. Luciferase reporter assay showed that miR-346 targeted the 3'-untranslated region (3'-UTR) of breast cancer metastasis suppressor 1 (BRMS1). Overexpression of miR-346 suppressed the endogenous expression of BRMS1 in NPC cells. There was a significant negative correlation between miR-346 and BRMS1 protein expression in NPC tissues (r = -0.372, P = 0.008). Rescue experiments demonstrated that overexpression of BRMS1 lacking the 3'-UTR impaired the invasiveness of NPC cells transfected with miR-346 mimic. Taken together, miR-346 shows the ability to promote the migration and invasion of nasopharyngeal cancer cells via targeting BRMS1 and represents a potential therapeutic target for NPC.

Expression of proteinase-activated receptor (PAR)-2 in monocytes from allergic patients and potential molecular mechanism.

Serine proteases play an important role in inflammation via PARs. However, little is known of expression levels of PARs on monocytes of allergic patients, and influence of serine proteases and PARs on TNF-α secretion from monocytes. Using quantitative real-time PCR (qPCR) and flowcytometry techniques, we observed that the expression level of PAR-2 in monocytes of patients with allergic rhinitis and asthma was increased by 42.9 and 38.2 %. It was found that trypsin, thrombin, and tryptase induced up to 200, 320, and 310 % increase in TNF-α release from monocytes at 16 h, respectively. PAR-1 agonist peptide, SFLLR-NH2, and PAR-2 agonist peptide tc-LIGRLO-NH2 provoked up to 210 and 240 % increase in release of TNF-α. Since SCH 79797, a PAR-1 antagonist, and PD98059, an inhibitor of ERK inhibited thrombin- and SFLLR-NH2-induced TNF-α release, the action of thrombin is most likely through a PAR-1- and ERK-mediated signaling mechanism. Similarly, because FSLLRN-NH2, an inhibitor of PAR-2 diminished tryptase- and tc-LIGRLO-NH2-induced TNF-α release, the action of tryptase appears PAR-2 dependent. Moreover, in vivo study showed that both recombinant cockroach major allergens Per a 1 and Per a 7 provoked upregulation of PAR-2 and PAR-1 expression on CD14+ cells in OVA-sensitized mouse peritoneum. In conclusion, increased expression of PAR-2 in monocytes of AR and asthma implicates that PAR-2 likely play a role in allergy. PAR-2- and PAR-1-mediated TNF-α release from monocytes suggests that these unique protease receptors are involved in the pathogenesis of inflammation.

Controlled bile acid exposure to oesophageal mucosa causes up-regulation of nuclear γ-H2AX possibly via iNOS induction.

Using an in vitro model in which flatmounts of oesophagus was periodically exposed to bile acids, we demonstrate, using multiple methods, that the bile acid receptor TGR5, inducible nitric oxide synthase (iNOS) and γ-histone family 2A variant (γ-H2AX) are up-regulated. This indicates that bile acids cause up-regulation of iNOS, which further causes genotoxic stress as evidenced by increase of the highly sensitive marker, phosphorylated histone. In vitro nitric oxide (NO) assays showed increased production of nitric acid in the oesophageal epithelium exposed to the bile acids. This increase was inhibited in the presence of the nonspecific iNOS inhibitor aminoguanidine (AG). Cumulatively, the results of the present study provide suggestion that not only acid reflux, but also non-acid reflux of bile may cause genotoxic stress. These aspects merit to be tested in wide spectrum of Barrett epithelial tissues.

Correlation of IL-18 with Tryptase in Atopic Asthma and Induction of Mast Cell Accumulation by IL-18.

Interleukin- (IL-) 18 and tryptase were previously reported to relate to asthma, but the correlation between these two potent proinflammatory molecules in asthma and their roles in mast cell accumulation remain uninvestigated. Using flow cytometric analysis technique and ovalbumin- (OVA-) sensitized mouse model, it was found that IL-18 and tryptase levels in the plasma of moderate and severe asthma were elevated, and they correlated well with each other. Tryptase and agonist peptides of protease activated receptor- (PAR-) 2 induced substantial quantity of IL-18 release. IL-18 and tryptase provoked mast cell accumulation in peritoneum of OVA-sensitized mice. OVA-sensitization increased number of IL-18 receptor (R)(+) mast cells. IL-18 and tryptase induced dramatic increase in IL-18R(+) mast cells and mean fluorescence intensity (MFI) of IL-18R on mast cells. Moreover, while IL-18 induced an increase in PAR-2(+) mast cells in nonsensitized mice, IL-18 and tryptase provoked increases in IL-4 and thymic stromal lymphopoietin (TSLP) in the peritoneum of OVA-sensitized mice. In summary, the correlation between IL-18 and tryptase in plasma of patients with asthma indicates close interactions between them, which should be considered for development of anti-IL-18 and antitryptase therapies. Interactions between IL-18 and tryptase may contribute to mast cell recruitment in asthma.

Determination of guanine and adenine by high-performance liquid chromatography with a self-fabricated wall-jet/thin-layer electrochemical detector at a glassy carbon electrode.

A sensitive wall-jet/thin-layer amperometric electrochemical detector (ECD) coupled to high-performance liquid chromatography (HPLC) was developed for simultaneous determination of guanine (G) and adenine (A). The analytes were detected at a glassy carbon electrode (GCE) and the HPLC-ECD calibration curves showed good linearity (R(2)>0.997) under optimized conditions. Limits of detection for G and A are 0.6 nM and 1.4 nM (S/N=3), respectively, which are lower than those obtained with an UV-vis detector and a commercial electrochemical detector. We have successfully applied this HPLC-ECD to assess the contents of G and A in hydrochloric acid-digested calf thymus double-stranded DNA. In addition, we compared in detail the analysis of G and A by cyclic voltammetry (CV) and by the HPLC-ECD system on both bare GCE and electroreduced graphene oxide (ERGO) modified GCE. We found that the adsorption of G and A on the electrode surfaces can vary their anodic CV peaks and the competitive adsorption of G and A on the limited sites of the electrode surfaces can cause crosstalk effects on their anodic CV peak signals, but the HPLC-ECD system is insensitive to such electrode-adsorption and can give more reliable analytical results.

Simultaneous analysis of dopamine and homovanillic acid by high-performance liquid chromatography with wall-jet/thin-layer electrochemical detection.

Wall-jet/thin-layer amperometric electrochemical detection (ECD) coupled with high-performance liquid chromatography (HPLC) was used here for the simultaneous analysis of dopamine (DA) and homovanillic acid (HVA) at a glassy carbon electrode. Compared with the conventional thin-layer mode and wall-jet mode, the presented wall-jet/thin-layer ECD has the advantages of enhanced capture of electroactive DA and HVA on the working electrode to give enhanced responses and more convenient washing/refreshment of the working electrode surface. Under optimized conditions, the HPLC-ECD calibration curves show good linearity from 0.01 to 100 µM for DA and HVA, and the limits of detection (LODs) obtained were 1.1 nM for DA and 0.7 nM for HVA which are lower than those obtained with an UV-vis detector and a commercial electrochemical detector. The method was tested on human urine with satisfactory results. The balance of response-signal, signal-background and noise level for our HPLC-ECD system is also discussed. In addition, a demethylation electrooxidation mechanism for HVA is suggested through potentiostatic bulk electrolysis, electrospray ionization-mass spectrometry, fluorescent spectrophotometry and cyclic voltammetry studies.