Combined analysis of plasma metabolome and intestinal microbiome sequencing to explore jiashen prescription and its potential role in changing intestine-heart axis and effect on chronic heart failure.

Background: Heart failure (HF) is a syndrome with global clinical and socioeconomic burden worldwide owing to its poor prognosis. Jiashen Prescription (JSP), a traditional Chinese medicine (TCM) formula, exhibits unambiguous effects on treating HF. Previously, we have reported that underlying mechanisms of JSP by an untargeted metabolomics approach, but the contribution of gut microbiota and metabolic interaction to the cardioprotective efficacy of JSP remains to be elucidated. Materials and methods: Firstly, the rat model of heart failure was established by the permanent ligation of the left anterior descending coronary artery. The efficacy evaluation of JSP in treating HF rats was per-formed by left ventricular ejection fraction (LVEF). Then, 16S rRNA gene sequencing and LC/MS-based metabolomic analysis were utilized to explore the characteristics of cecal-contents microecology and plasma metabolic profile, respectively. After that, the correlation between intestinal micro-ecological characteristics and plasma metabolic characteristics was analyzed to explore the potential mechanism of the JSP treatment in HF. Results: JSP could improve the cardiac function of heart failure rats and thus ameliorate heart failure via enhancing rat LVEF. Results of intestinal flora analysis revealed that JSP not only adjusted gut microbiota disturbances by enriching species diversity, reducing the abundance of pathogenic bacteria (such as Allobaculum, Brevinema), as well as increasing the abundance of beneficial bacteria (such as Lactobacillus, Lachnospiraceae_NK4A136_group), but also improved metabolic disorders by reversing metabolite plasma levels to normality. Through the conjoint analysis of 8 metabolites and the OTUs relative abundance data in the 16srRNA sequencing results by WGCNA method, 215 floras significantly related to the eight compounds were identified. The results of the correlation analysis demonstrated a significant association between intestinal microbiota and plasma metabolic profile, especially the significant correlation of Ruminococcaceae_UCG-014 and Protoporphyrin IX, Ruminococcaceae_UCG-005, Christensenellaceae_R-7_group and nicotinamide, dihydrofolic acid. Conclusion: The present study illustrated the underlying mechanism of JSP to treat heart failure by affecting intestinal flora and plasma metabolites, provide a potential therapeutic strategy against heart failure.

Plasma metabolomic analysis reveals the therapeutic effects of Jiashen tablets on heart failure.

Background: Heart failure is a chronic progressive condition that significantly affects the quality of life of patients with high hospitalization and mortality rates. Jiashen tablets (JST), a Chinese herbal formula, have been reported to be an effective treatment against heart failure, however the underlying mechanisms remain obscure. This study was designed to determine the effect of JST on the treatment of heart failure and delineate the underlying mechanisms by an untargeted metabolomics approach. Materials and methods: The chronic heart failure model was established by the permanent ligation of the left anterior descending coronary artery in rats. The cardiac functions of rats, including left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), left ventricular internal diameter end diastole (LVIDd) and end systole (LVIDs), and interventricular septum thickness in diastole (IVSd) and in systole (IVSs), were measured by echocardiography. Biochemical analysis and histopathological examination were also performed to evaluate therapeutic effects of JST for treating heart failure. UHPLC-QTOF-MS/MS coupled with multivariate statistical analyses were applied for plasma metabolic profiling to identify biomarkers and potential mechanisms of JST in the treatment of heart failure. Results: Jiashen tablets could improve the cardiac function of heart failure rats and thus ameliorate heart failure via enhancing rat LVEF and LVFS and decreasing LVIDd, LVIDs, IVSd, and IVSs. Results of biochemical analysis and histopathological examination revealed that JST could reduce the serum lactate dehydrogenase (LDH) activity and the level of NT-pro BNP, markers of heart failure and myocardial damage, and inhibit myocardial fibrosis. Furthermore, in metabolomics analysis, a total of 210 metabolites with significant differences were identified between heart failure rats and normal rats, among which 29 metabolites were significantly restored after JST treatment. These metabolites were primarily involved in tryptophan metabolism, branched-chain amino acid metabolism, fatty acids ß-oxidation, and glycerophospholipid metabolism. Conclusion: The present study illustrated the therapeutic effect of JST for the treatment of heart failure and delineated the underlying mechanisms mainly relating to the regulation of amino acid metabolism and lipid metabolism in heart failure rats.

The central-peripheral coupling effect of ocular acupuncture kinesitherapy in post-stroke dyskinesia: A functional neuroimaging and neurotic electrophysiology study protocol.

Background: Dyskinesia is a common manifestation after stroke. Motor functional rehabilitation after stroke is of great significance to the maintenance of national health. Ocular Acupuncture Kinesitherapy (OAKT) can repair nerve injuries, improve motor function, reduce rehabilitation time, and promote dyskinesia recovery after stroke. The mechanism, however, remains a mystery, necessitating urgent research. The M1-thalamus-spinal cord neural signaling pathway is linked to limb motor function. Bold-fMRI can represent the cerebral functional state, and TMS-MEP is of certain practical utility for assessing motor neural function and prognosis. Combining fMRI scanning with TMS-MEP detection is predicted to advance brain-spinal cord regulation and muscle response linkage control mechanism research, as well as completely investigate the central-peripheral coupling effect of Ocular Acupuncture Kinesitherapy on dyskinesia after stroke (PSD). Methods: This is a prospective functional neuroimaging and neurotic electrophysiological study with a case-control design between the PSD with the HC groups and a randomized controlled design within the 3 PSD groups (OAKT group, ocular acupuncture group, and kinesitherapy group). Using fMRI scans and TMS-MEP approach, we will assess the central-peripheral neural function alterations in PSD as well as the coupling effects of OAKT on PSD. We plan to enroll 90 participants at the Hospital of Chengdu University of Traditional Chinese Medicine from Aug 31, 2022, to Dec 31, 2023, including 45 PSD and 45 HC subjects. After enrollment and on the last day after 4-weeks of waiting (HC subjects) or intervention (PSD subjects), all eligible subjects will be evaluated using fMRI scanning, TMS-MEP detection, and the MMT and Fugl-Mayer scales assessment. The MMT and Fugl-Meyer scores will be recorded, and a Pearson correlation analysis will be performed to assess the correlation between clinical and imaging outcomes. Discussion: Findings of this study will help to explain the central-peripheral coupling effect of OAKT on PSD and to further provide the neural processing of acupuncture kinesitherapy covering the entire pathway from peripheral to central nervous system. Clinical trial registration: This study is registered with an identifier (ChiCTR2200060483) at the Chinese Clinical Trial Registry in June 2022. http://www.chictr.org.cn/index.aspx.

Nrf2-mediated liver protection by 18ß-glycyrrhetinic acid against pyrrolizidine alkaloid-induced toxicity through PI3K/Akt/GSK3ß pathway.

BACKGROUND: Misusage of pyrrolizidine alkaloid (PA)-containing plants or unaware intake of PA-contaminated foodstuffs causes thousands of PA poisoning cases in humans. PA intoxication is accompanied by oxidative stress and subsequent extensive hepatocellular damage. Our previous study has demonstrated that 18ß-glycyrrhetinic acid (GA), a bioactive constituent of liquorice, prevented PA-induced hepatotoxicity in rats, however the underlying mechanisms remain unclear. OBJECTIVE: This study aims to explore the mechanisms underlying the hepato-protective effect of GA in combating retrorsine (RTS, a representative toxic PA)-induced liver injury. METHODS: Histological and biochemical assessments were employed to evaluate the protective effect of GA on RTS-induced hepatotoxicity in rats. Sulforhodamine B assay, real-time PCR, western blotting, and immunostaining were used to explore the underlying mechanisms in human hepatocytes and rats. RESULTS: Our findings demonstrated that GA alleviated RTS-induced elevation of serum ALT and bilirubin levels, as well as hepatocytes necrosis and sinusoidal endothelial cells (SECs) damage in rats. GA also enhanced the activities and expressions of several antioxidant enzymes through upregulating nuclear factor-erythroid 2-related factor2 (Nrf2). Moreover, inhibition of Nrf2 blocked the hepatoprotective effect of GA against RTS intoxication. Mechanistically, GA increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and enhanced glycogen synthase kinase 3 beta (GSK3ß) inhibitory phosphorylation at serine 9, thus promoting the nuclear accumulation of Nrf2 and activating its downstream targets. CONCLUSION: This study for the first time demonstrated that GA exerted protective effects against RTS-induced liver injury by potentiating the Nrf2-mediated antioxidant system through PI3K/Akt/GSK3ß pathway. The findings indicated that GA may serve as a potential candidate drug for the treatment of PA intoxication.

Liquorice Extract and 18ß-Glycyrrhetinic Acid Protect Against Experimental Pyrrolizidine Alkaloid-Induced Hepatotoxicity in Rats Through Inhibiting Cytochrome P450-Mediated Metabolic Activation.

Misuse of pyrrolizidine alkaloid (PA)-containing plants or consumption of PA-contaminated foodstuffs causes numerous poisoning cases in humans yearly, while effective therapeutic strategies are still limited. PA-induced liver injury was initiated by cytochrome P450 (CYP)-mediated metabolic activation and subsequent formation of adducts with cellular proteins. Liquorice, a hepato-protective herbal medicine, is commonly used concurrently with PA-containing herbs in many compound traditional Chinese medicine formulas, and no PA-poisoning cases have been reported with this combination. The present study aimed to investigate hepato-protective effects of liquorice aqueous extract (EX) and 18ß-glycyrrhetinic acid (GA, the primary bioactive constituent of liquorice) against PA-induced hepatotoxicity and the underlying mechanism. Histopathological and biochemical analysis demonstrated that both single- and multiple-treatment of EX (500 mg/kg) or GA (50 mg/kg) significantly attenuated liver damage caused by retrorsine (RTS, a representative hepatotoxic PA). The formation of pyrrole-protein adducts was significantly reduced by single- (30.3% reduction in liver; 50.8% reduction in plasma) and multiple- (32.5% reduction in liver; 56.5% reduction in plasma) treatment of GA in rats. Single- and multiple-treatment of EX also decreased the formation of pyrrole-protein adducts, with 30.2 and 31.1% reduction in rat liver and 51.8 and 53.1% reduction in rat plasma, respectively. In addition, in vitro metabolism assay with rat liver microsomes demonstrated that GA reduced the formation of metabolic activation-derived pyrrole-glutathione conjugate in a dose-dependent manner with the estimated IC50 value of 5.07 µM. Further mechanism study showed that GA inhibited activities of CYPs, especially CYP3A1, the major CYP isoform responsible for the metabolic activation of RTS in rats. Enzymatic kinetic study revealed a competitive inhibition of rat CYP3A1 by GA. In conclusion, our findings demonstrated that both EX and GA exhibited significant hepato-protective effects against RTS-induced hepatotoxicity, mainly through the competitive inhibition of CYP-mediated metabolic activation of RTS.

Exploring the synergistic and complementary effects of berberine and paeoniflorin in the treatment of type 2 diabetes mellitus by network pharmacology.

Investigation of the synergistic and complementary effects is vital but difficult for Chinese herbal medicine. We explored the synergistic and complementary mechanisms of berberine (BBR) and paeoniflorin (PF) in the treatment of type 2 diabetes mellitus (T2DM) through network pharmacology and molecular docking. We identified putative targets of BBR, PF, and T2DM, and constructed a protein-protein interaction (PPI) network. Gene ontology and Kyoto encyclopedia of gene and genomes pathway enrichment analysis and molecular docking were used to predict the molecular mechanisms. A diabetes model was induced by a high-fat diet to verify the therapeutic effect. Ninety-two targets of BBR + PF in the treatment of T2DM were identified, which were considered as synergistic targets. Fifty-nine complementary targets of BBR-T2DM and 47 of PF-T2DM were identified. PPI network analysis showed that JAK2, ESR1, IFG1R, STAT3, EGFR, MAPK1, and AKT1 are closely related to T2DM. The enrichment analysis further showed that the synergistic targets mainly involved the AGE-RAGE signaling pathway in diabetic complications, FOXO, AMPK, and VEGF signaling pathways, and glycolysis/gluconeogenesis. AKT1, JAK2, and STAT3, which are common targets of the AGE-RAGE signaling pathway in diabetic complications and the FOXO signaling pathway, were chosen for docking with BBR and PF, respectively, and showed good binding activities. BBR + PF significantly reduced weight and fasting blood glucose, and alleviated insulin resistance. Moreover, BBR + PF promoted the phosphorylation of AKT1, JAK2, and STAT3. This study provides information to understand the synergistic and complementary mechanism of BBR + PF against T2DM, and may facilitate the development of new anti-T2DM drugs.

A Meta-Analysis: Whether Repetitive Transcranial Magnetic Stimulation Improves Dysfunction Caused by Stroke with Lower Limb Spasticity.

OBJECTIVE: To evaluate the efficacy of repetitive transcranial magnetic stimulation (rTMS) in improving lower limb spasticity after stroke. METHODS: The PubMed, Web of Science, Cochrane Library, EMBASE, China National Knowledge Infrastructure (CNKI), China Biology Medicine (CBM) disc, China Science and Technology Journal Database (VIP), and Wanfang databases were searched online from their inception to May 2021 for randomized controlled trials (RCTs) involving repetitive transcranial magnetic stimulation for lower extremity spasticity after stroke. Valid data were extracted from the included literature, and the quality evaluation was conducted with the Cochrane Handbook for Systematic Reviews of Interventions along with the Physiotherapy Evidence Database scale (PE-Dro scale). The data that met the quality requirements were systematically analysed using Review Manager 5.4 software. RESULTS: A total of 554 patients from seven articles (nine studies) were quantitatively analysed. Outcomes included the Modified Ashworth Scale (MAS), Fugl-Meyer Assessment of Lower Extremity (FMA-LE), Modified Barthel Index (MBI), and Timed Up and Go (TUG), measured as the effect of rTMS compared with controls conditions after treatment. The systematic review showed that rTMS reduced MAS and increased MBI scores, respectively (SMD = -0.24, 95% CI [-0.45, -0.03], P = 0.02; MD = 6.14, 95% CI [-3.93,8.35], P ＜ 0.00001), compared with control conditions. Low-frequency rTMS (LF-rTMS) significantly improved FMA-LE scores (SMD = 0.32, 95% CI [0.13, 0.51], P = 0.001). However, there was no significant difference in FMA-LE scores when using high-frequency rTMS (HF-rTMS) (P > 0.1) and in TUG times (P > 0.1) between the treatment and control groups. CONCLUSIONS: rTMS was effective in improving spasticity and activities of daily living. LF-rTMS has positive clinical effects on enhancing motor function in patients who experience lower extremity spasticity after stroke. To better validate the above conclusions, more multicentre, high-quality, and double-blind randomized controlled trials are needed.

Pyrrolizidine Alkaloid-Induced Hepatotoxicity Associated with the Formation of Reactive Metabolite-Derived Pyrrole-Protein Adducts.

Pyrrolizidine alkaloids (PAs) with 1,2-unsaturated necine base are hepatotoxic phytotoxins. Acute PA intoxication is initiated by the formation of adducts between PA-derived reactive pyrrolic metabolites with cellular proteins. The present study aimed to investigate the correlation between the formation of hepatic pyrrole-protein adducts and occurrence of PA-induced liver injury (PA-ILI), and to further explore the use of such adducts for rapidly screening the hepatotoxic potency of natural products which contain PAs. Aqueous extracts of Crotalaria sessiliflora (containing one PA: monocrotaline) and Gynura japonica (containing two PAs: senecionine and seneciphylline) were orally administered to rats at different doses for 24 h to investigate PA-ILI. Serum alanine aminotransferase (ALT) activity, hepatic glutathione (GSH) level, and liver histological changes of the treated rats were evaluated to assess the severity of PA-ILI. The levels of pyrrole-protein adducts formed in the rats' livers were determined by a well-established spectrophotometric method. The biological and histological results showed a dose-dependent hepatotoxicity with significantly different toxic severity among groups of rats treated with herbal extracts containing different PAs. Both serum ALT activity and the amount of hepatic pyrrole-protein adducts increased in a dose-dependent manner. Moreover, the elevation of ALT activity correlated well with the formation of hepatic pyrrole-protein adducts, regardless of the structures of different PAs. The findings revealed that the formation of hepatic pyrrole-protein adducts-which directly correlated with the elevation of serum ALT activity-was a common insult leading to PA-ILI, suggesting a potential for using pyrrole-protein adducts to screen hepatotoxicity and rank PA-containing natural products, which generally contain multiple PAs with different structures.

Characterization of liver injury induced by a pyrrolizidine alkaloid in rats.

BACKGROUND: Pyrrolizidine alkaloids (PAs) are common phytotoxins. PA intoxication is reported to cause severe acute liver damage, typically known as hepatic sinusoidal obstruction syndrome (HSOS), but it remains obscure whether the acute liver damage may progress into chronic liver disease characterized by hepatic fibrosis. PURPOSE: This study aims to characterize the biochemical markers of liver injury and histological features of regressive and progressive liver fibrosis, and to examine changes in hepatic gene expression that may underpin mechanisms of fibrogenesis in rats induced by retrorsine (RTS), a representative toxic PA. STUDY DESIGN/METHODS: Rats were gavaged with RTS via two dosing regimens, i.e. a single dose of 40 mg/kg (Group 1) and two doses of 40 mg/kg and 20 mg/kg on day 0 and day 7 (Group 2), respectively. Rats receiving one (Group 3) or two (Group 4) doses of vehicle served as negative controls. The animals were followed for up to 16 weeks by serum biochemical analyses and histological examination, and gene expression assays of liver tissues. RESULTS: Acute liver injury on day 2 manifested as HSOS, characterized by sinusoidal dilation, endothelial cell damage, and elevated serum alanine aminotransferase activity and bilirubin levels. In Group 1, mild liver fibrosis developed at sinusoids and perisinusoidal space surrounding the central veins at week 1 and 2, and thereafter, all liver injury resolved gradually. In Group 2, liver fibrosis progressed within the 16-week observation period. No apparent liver injury was observed in Groups 3 and 4. Compared with negative control groups, RTS induced myofibroblastic activation, TGF-ß1 signaling, and changes in expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). These dynamic changes differed in Groups 1 and 2, corresponding with the regression and progression of liver fibrosis, respectively, in these groups. CONCLUSION: This study has provided in-vivo proof of concept that "one hit" and "two hits" of RTS lead to acute resolving liver injury and chronic progressive liver fibrosis, respectively. These animal models may serve as powerful tools for studying RTS toxicology and related preventive and therapeutic strategies and as positive controls for studying other PA- and non-PA-induced liver injury.

The dual roles of ginsenosides in improving the anti-tumor efficiency of cyclophosphamide in mammary carcinoma mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyclophosphamide (CTX) is a first line chemotherapeutic agent, but often limited for its unstable therapeutic effect and serious side effects. Ginsenosides could facilitate the anti-tumor efficiency of CTX, including benefiting therapeutic effect and decreasing side effects. AIM OF THE STUDY: To investigate the potential mechanism of ginsenosides on benefiting the anti-tumor efficiency of CTX. MATERIALS AND METHODS: Mammary carcinoma mice were applied to investigate the anti-tumor efficiency and potential mechanism of combinational treatment of ginsenosides and CTX. Therapeutic effect was evaluated based on survival rate, tumor burden, tumor growth inhibition rate, and apoptosis and histological changes of tumor tissues. Anti-tumor immunity was studied by measuring serum level of anti-tumor cytokines. Gut mucositis, one of lethal side effects of CTX, was evaluated by diarrhea degree, gut permeability and tight junction proteins expressions. Gut microbial diversity was analyzed by 16S rRNA gene sequencing, and fecal transplant and antibiotics sterilized animals were performed to evaluate the therapeutic effect of gut microbiota on tumor suppression. RESULTS: Ginsenosides facilitated the therapeutic effect of CTX in mice, which manifested as prolonged survival rate, decreased tumor burden, as well as enhanced tumor growth inhibition rate and apoptosis. The favoring effect was related to elevation of anti-tumor immunity which manifested as the increased anti-tumor cytokines (INF-γ, IL-17, IL-2 and IL-6). Further studies indicated the elevation was ascribed to ginsenosides promoted reproduction of gut probiotics including Akkermansia, Bifidobacterium and Lactobacillus. Moreover, co-administration of ginsenosides in mice alleviated CTX-induced gut mucositis, including lower gut permeability, less diarrhea, less epithelium damage and higher tight junction proteins. Further researches suggested the alleviation was related to ginsenosides activated Nrf2 and inhibited NFκB pathways. CONCLUSION: Ginsenosides show dual roles to facilitate the anti-tumor efficiency of CTX, namely promote the anti-tumor immunity through maintaining gut microflora and ameliorate gut mucositis by modulating Nrf2 and NFκB pathways.

Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China.

Herbs and dietary supplement-induced liver injury (HILI) is the leading cause of drug-induced liver injury in China. Among different hepatotoxic herbs, the pyrrolizidine alkaloid (PA)-producing herb Gynura japonica contributes significantly to HILI by inducing hepatic sinusoidal obstruction syndrome (HSOS), a liver disorder characterized by hepatomegaly, hyperbilirubinemia, and ascites. In China, G. japonica has been used as one of the plant species for Tu-San-Qi and is often misused with non-PA-producing Tu-San-Qi (Sedum aizoon) or even San-Qi (Panax notoginseng) for self-medication. It has been reported that over 50% of HSOS cases are caused by the intake of PA-producing G. japonica. In this review, we provide comprehensive information to distinguish these Tu-San-Qi-related herbal plant species in terms of plant/medicinal part morphologies, medicinal indications, and chemical profiles. Approximately 2156 Tu-San-Qi-associated HSOS cases reported in China from 1980 to 2019 are systematically reviewed in terms of their clinical manifestation, diagnostic workups, therapeutic interventions, and outcomes. In addition, based on the application of our developed mechanism-based biomarker of PA exposure, our clinical findings on the definitive diagnosis of 58 PA-producing Tu-San-Qi-induced HSOS patients are also elaborated. Therefore, this review article provides the first comprehensive report on 2214 PA-producing Tu-San-Qi (G. japonica)-induced HSOS cases in China, and the information presented will improve public awareness of the significant incidence of PA-producing Tu-San-Qi (G. japonica)-induced HSOS and facilitate future prevention and better clinical management of this severe HILI.

Pulmonary toxicity is a common phenomenon of toxic pyrrolizidine alkaloids.

The hepatotoxic pyrrolizidine alkaloids (PAs) are metabolically activated in the liver to form reactive dehydro-PAs, which generate pyrrole-protein adducts leading to hepatotoxicity. Monocrotaline, but not other PAs, is also pneumotoxic, supposedly due to the migration of the liver-generated corresponding dehydro-PA into the lung to form pyrrole-protein adducts to induce pneumotoxicity. The present study investigated whether other PAs are also pneumotoxic. Metabolic activation of four representative hepatotoxic PAs, monocrotaline, retrorsine, riddelliine and clivorine, was investigated using rat liver or lung S9 incubation. All PAs produced pyrrole-protein adducts significantly in rat liver S9 but negligible in lung S9 fraction, revealing that liver is the key organ responsible for metabolic activation generating dehydro-PAs. Furthermore, these four PAs and another two PAs present in the alkaloid extract of Gynura segetum, a widely used PA-producing herb responsible for human PA poisonings in China, were orally administered to rats using the same hepatotoxic dose of 0.2 mmol/kg. All six PAs induced pneumotoxicity in rats within 48 h. The results demonstrated that pneumotoxicity could be a common phenomenon of PAs and the liver-derived dehydro-PAs might move to the lung and form pyrrole-protein adducts, leading to pulmonary toxicity.

Absorption difference between hepatotoxic pyrrolizidine alkaloids and their N-oxides - Mechanism and its potential toxic impact.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are a group of phytotoxins widely present in about 3% of flowering plants. Many PA-containing herbal plants can cause liver injury. Our previous studies demonstrated that PA N-oxides are also hepatotoxic, with toxic potency much lower than the corresponding PAs, due to significant differences in their toxicokinetic fates. AIM OF STUDY: This study aimed to investigate the oral absorption of PAs and PA N-oxides for better understanding of their significant differences in toxicokinetics and toxic potency. MATERIALS AND METHODS: The oral absorption of PAs and PA N-oxides in rats and in rat in situ single pass intestine perfusion model was investigated. The intestinal permeability and absorption mechanisms of five pairs of PAs and PA N-oxides were evaluated by using Caco-2 monolayer model. RESULTS: The plasma concentrations of total PAs and PA N-oxides within 0-60 min were significantly lower in rats orally treated with a PA N-oxide-containing herbal alkaloid extract than with a PA-containing herbal alkaloid extract at the same dose, indicating that the absorption of PA N-oxides was lower than that of PAs. Using the rat in situ single pass intestine perfusion model, less cumulative amounts of retrorsine N-oxide in mesenteric blood were observed compared to that of retrorsine. In Caco-2 monolayer model, all five PAs showed absorption with Papp AtoB values [(1.43-16.26) × 10-6 cm/s] higher than those of corresponding N-oxides with Papp AtoB values lower than 1.35 × 10-6 cm/s. A further mechanistic study demonstrated that except for senecionine N-oxide, retrorsine N-oxide, and lycopsamine N-oxide, all PAs and PA N-oxides investigated were absorbed via passive diffusion. While, for these 3 PA N-oxides, in addition to passive diffusion as their primary transportation, efflux transporter-mediated active transportation was also involved but to a less extent with the efflux ratio of 2.31-3.41. Furthermore, a good correlation between lipophilicity and permeability of retronecine-type PAs and their N-oxides with absorption via passive diffusion was observed, demonstrating that PAs have a better oral absorbability than that of the corresponding PA N-oxides. CONCLUSION: We discovered that among many contributors, the lower intestinal absorption of PA N-oxides was the initiating contributor that caused differences in toxicokinetics and toxic potency between PAs and PA N-oxides.

Investigation of association of chemical profiles with the tracheobronchial relaxant activity of Chinese medicinal herb Beimu derived from various Fritillaria species.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae Bulbus (Beimu in Chinese) is derived from the bulbus of many Fritillaria species (family Liliaceae), which has been used as an antitussive herb in traditional Chinese medicine for more than 2000 years. Due to the complexity of plant origins and significant variations in chemical profiles, the characterization of the profile of the major bioactive constituents and its association with pharmacological activity are important for the quality control of Beimu herbs from different origins. AIM OF THE STUDY: This study aims to investigate the distribution of major bioactive isosteroidal alkaloids in Beimu herbs of different origins and its correlation with the tracheobronchial relaxant activity. METHODS: Quantification of 7 main bioactive 5α-cevanine isosteroidal alkaloids, including ebeiedine, ebeiedinone, hupehenine, isoverticine, verticine, verticinone and imperialine, in 23 Fritillaria species was performed using gas chromatography. The relaxant effect of different extracts of 4 commonly used Beimu herbs, namely Zhe-Beimu (F. thunbergii Miq.), Chuan-Beimu (F. cirrhosa D. Don), Hubei-Beimu (F. hupehensis Hsiao et K. C. Hsia) and Yi-Beimu (F. pallidiflora Schrenk), was evaluated using rat isolated tracheal and bronchial preparations pre-contracted with carbachol, the well established in vitro antitussive model. RESULTS: Amongst 23 Fritillaria species detected, significant variations of the types and quantities of 7 major isosteroidal alkaloids were determined, which served as an important indicator for the classification of different Beimu herbs with distinct geographic distributions. Based on the type and quantity of these alkaloids, different origins of Beimu could be clearly clustered into several subgroups by principal component analysis. Furthermore, both crude alkaloid and water extracts of all 4 Beimu herbs showed a dose-dependent tracheobronchial relaxation with different potencies. The total content of alkaloids (weight adjusted based on the activity of individual alkaloids) in Beimu extracts significantly correlated with their tracheobronchial relaxation effects (r2 > 0.9, p < 0.001). CONCLUSIONS: The results demonstrated that the differences in chemical profile of major bioactive isosteroidal alkaloids and pharmacological activity of Beimu could be incorporated into a simple and unified method for quality control and potential prediction of activity of Beimu herbs from different origins.

Systemic Perturbations of Key Metabolites in Type 2 Diabetic Rats Treated by Polyphenol Extracts from Litchi chinensis Seeds.

Our previous research obtained Litchi chinensis Sonn. seeds extract (LSE) which showed hypoglycaemic effects on type 2 diabetes (T2D) rats. In order to understand the detailed pathogenesis of diabetes intervened by LSE, the metabonomics strategy was used. As a result, LSE decreased the insulin resistance index and the levels of glucose in urine through elevating the mRNA level of insulin, while decreasing the expression of glucagon to enhance the function of the pancreas. Meanwhile, LSE regulated the glucose and fatty acid metabolisms via increasing the expression of glucose transporter (Glu) 2, Glu4, insulin receptor (IR), and IR substrate-2 (IRS2). LSE effectively restored the impairment of the IRS2/PI3K/Akt/mTOR insulin signaling in the livers. All in all, LSE played a pivotal role in the treatment of T2D through regulation of broad-spectrum metabolic changes and inhibition of the glycogenesis, proteolysis, and lipogenesis in T2D rats.

First evidence of pyrrolizidine alkaloid N-oxide-induced hepatic sinusoidal obstruction syndrome in humans.

Pyrrolizidine alkaloids (PAs) are among the most potent phytotoxins widely distributed in plant species around the world. PA is one of the major causes responsible for the development of hepatic sinusoidal obstruction syndrome (HSOS) and exerts hepatotoxicity via metabolic activation to form the reactive metabolites, which bind with cellular proteins to generate pyrrole-protein adducts, leading to hepatotoxicity. PA N-oxides coexist with their corresponding PAs in plants with varied quantities, sometimes even higher than that of PAs, but the toxicity of PA N-oxides remains unclear. The current study unequivocally identified PA N-oxides as the sole or predominant form of PAs in 18 Gynura segetum herbal samples ingested by patients with liver damage. For the first time, PA N-oxides were recorded to induce HSOS in human. PA N-oxide-induced hepatotoxicity was further confirmed on mice orally dosed of herbal extract containing 170 µmol PA N-oxides/kg/day, with its hepatotoxicity similar to but potency much lower than the corresponding PAs. Furthermore, toxicokinetic study after a single oral dose of senecionine N-oxide (55 µmol/kg) on rats revealed the toxic mechanism that PA N-oxides induced hepatotoxicity via their biotransformation to the corresponding PAs followed by the metabolic activation to form pyrrole-protein adducts. The remarkable differences in toxicokinetic profiles of PAs and PA N-oxides were found and attributed to their significantly different hepatotoxic potency. The findings of PA N-oxide-induced hepatotoxicity in humans and rodents suggested that the contents of both PAs and PA N-oxides present in herbs and foods should be regulated and controlled in use.

Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high pressure liquid chromatography coupled with triple quadrupole mass spectrometry.

Polyoxypregnane and its glycosides (POPs) are frequently present in plants of Asclepiadaceae family, and have a variety of biological activities. There is a great need to comprehensively profile these phytochemicals and to quantify them for monitoring their contents in the herbs and the biological samples. However, POPs undergo extensive adduct ion formation in ESI-MS, which has posed a challenge for qualitative and quantitative analysis of POPs. In the present study, we took the advantage of such extensive adduct ion formation to investigate the suitability of adduct ion-targeted analysis of POPs. For the qualitative analysis, we firstly demonstrated that the sodium and ammonium adduct ion-targeted product ion scans (PIS) provided adequate MS/MS fragmentations for structural characterization of POPs. Aided with precursor ion (PI) scans, which showed high selectivity and sensitivity and improved peak assignment confidence in conjunction with full scan (FS), the informative adduct ion-targeted PIS enabled rapid POPs profiling. For the quantification, we used formic acid rather than ammonium acetate as an additive in the mobile phase to avoid simultaneous formation of sodium and ammonium adduct ions, and greatly improved reproducibility of MS response of POPs. By monitoring the solely formed sodium adduct ions [M+Na]+, a method for simultaneous quantification of 25 POPs in the dynamic multiple reaction monitoring mode was then developed and validated. Finally, the aforementioned methods were applied to qualitative and quantitative analysis of POPs in the extract of a traditional Chinses medicinal herb, Marsdenia tenacissima (Roxb.) Wight et Arn., and in the plasma obtained from the rats treated with this herb. The results demonstrated that adduct ion formation could be optimized for the qualitative and quantitative analysis of POPs, and our developed PI/FS-PIS scanning and sole [M+Na]+ ion monitoring significantly improved the analysis of POPs in both herbal and biological samples. This study also provides implications for the analysis of other compounds which undergo extensive adduct ion formation in ESI-MS.

Enhanced absorption and inhibited metabolism of emodin by 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside: Possible mechanisms for Polygoni Multiflori Radix-induced liver injury.

Polygoni Multiflori Radix (PMR) has been commonly used as a tonic in China for centuries. However, PMR-associated hepatotoxicity is becoming a safety issue. In our previous in vivo study, an interaction between stilbenes and anthraquinones has been discovered and a hypothesis is proposed that the interaction between stilbene glucoside-enriching fraction and emodin may contribute to the side effects of PMR. To further support our previous in vivo results in rats, the present in vitro study was designed to evaluate the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG) on the cellular absorption and human liver microsome metabolism of emodin. The obtained results indicated that the absorption of emodin in Caco-2 cells was enhanced and the metabolism of emodin in human liver microsomes was inhibited after TSG treatment. The effects of the transport inhibitors on the cellular emodin accumulation were also examined. Western blot assay suggested that the depressed metabolism of emodin could be attributed to the down-regulation of UDP-glucuronosyltransferases (UGTs) 1A8, 1A10, and 2B7. These findings definitively demonstrated the existence of interaction between TSG and emodin, which provide a basis for a better understanding of the underlying mechanism for PMR-induced liver injury.

(+)/(-)-Phaeocaulin A-D, four pairs of new enantiomeric germacrane-type sesquiterpenes from Curcuma phaeocaulis as natural nitric oxide inhibitors.

Germacrane-type sesquiterpenes, with a flexible 10-membered ring unit as the structural and conformational features, play a central role in the biosynthesis and synthesis of other sesquiterpenes. In this report, two pairs of new sesquiterpene alkaloids, (+)/(-)-phaeocaulin A [(+)-1/(-)-1] and B [(+)-2/(-)-2], and two pairs of new sesquiterpenes, (+)/(-)-phaeocaulin C [(+)-3/(-)-3] and D [(+)-4/(-)-4], along with one related known analog (5), were isolated from the rhizomes of Curcuma phaeocaulis. The absolute configurations of (+)-1/(-)-1, (+)-2/(-)-2, (+)-3/(-)-3 and (+)-4/(-)-4 were unambiguously determined by analysis of single-crystal X-ray diffractions and quantum chemical electronic circular dichroism (ECD) method. It is noteworthy that (+)/(-)-phaeocaulin A [(+)-1/(-)-1] and B [(+)-2/(-)-2] are two pairs of rare N-containing germacrane-type sesquiterpenes. A possible biogenetic pathway for 1-5 was postulated. All of the isolated compounds were tested for their inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages.