Rapid identification of chemical constituents of Rhodiola crenulata using liquid chromatography-mass spectrometry pseudotargeted analysis.

Rhodiola crenulata (R. crenulata), is a famous traditional Chinese medicine, with observable effects such as anti-high-altitude illness and fatigue resistance. Nevertheless, comprehensive and systematic structural identification of its components remains a challenge. In this study, a pseudotargeted analytical method, involving key fragment filtering by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry, was developed for rapid detection and identification of the chemical constituents of R. crenulata. The process consists of three steps: (i) acquiring sufficient mass spectral data, (ii) constructing a key fragments schedule and discovering the substructures rapidly by pseudotargeted key fragment filtering, and (iii) further identification of the compound structures based on accurate masses, fragment ions, related literatures, and authentic standards. As a result, 104 compounds were identified and divided into five categories, among which three potentially new and 59 ones were reported for the first time in R. crenulata. These results indicated that the major types of components are flavanols and gallic acid derivatives, organic acids, alcohols and their glycosides, flavonoids and their glycosides. This study enhances the understanding of R. crenulata and provides a reference for rapid and comprehensive identification of constituents in other herbal medicines.

Preparing the key metabolite of Z-ligustilide in vivo by a specific electrochemical reaction.

The key in vivo metabolites of a drug play an important role in its efficacy and toxicity. However, due to the low content and instability of these metabolites, they are hard to obtain through in vivo methods. Electrochemical reactions can be an efficient alternative to biotransformation in vivo for the preparation of metabolites. Accordingly, in this study, the metabolism of Z-ligustilide was investigated in vitro by electrochemistry coupled online to mass spectrometry. This work showed that five oxidation products of the electrochemical reaction were detected and that two of the oxidation products (senkyunolide I and senkyunolide H) were identified from liver microsomal incubation as well. Furthermore, after intragastric administration of Z-ligustilide in rats, senkyunolide I and senkyunolide H were detected in the rat plasma and liver, while 6,7-epoxyligustilide, a key intermediate metabolite of Z-ligustilide, was difficult to detect in vivo. By contrast, 6,7-epoxyligustilide was obtained from the electrochemical reaction. In addition, for the first time, 6 mg of 6,7-epoxyligustilide was prepared from 120 mg of Z-ligustilide. Therefore, electrochemical reactions represent an efficient laboratory method for preparing key drug metabolites.

[Studies on constituents of Polygonum multiflorum extract in vivo and in vitro based on UPLC-Q-TOF-MS].

To explore the drug-induced constituents in vivo of Polygonum multiflorum extract (PM). This study was the first to study the drug-induced constituents in target organ liver. Agilent MassHunter qualitative analysis software and Metabolite ID software were applied for the analysis of retention time, exact relative molecular mass, primary and secondary mass spectrum information based on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and targeted-MS/MS. By comparison with literature and standards, a total of 5 prototypes and 6 metabolites were identified or tentatively elucidated from the liver samples. In addition, the drug-induced constituents in plasma and PM were also analyzed in this study and 8 prototypes and 19 metabolites were detected from the plasma samples, while 30 compounds were detected from the extract of PM. Emodin oxidative acetylation metabolites, hydroxyl methylation metabolites, carboxylation glucuronidation metabolites and ketone glucuronidation metabolites in this study were first reported. Through the comparative analysis between the in vivo and in vitro constituents of PM, the study preliminarily revealed the drug-induced constituents (prototypes and metabolites) in liver and clarified the transfer process and transmutation rules of constituents in PM, blood and liver, which would further deepen our understanding on constituents of PM in vivo.

The Anti-hyperlipidemia Effects of Raw Polygonum multiflorum Extract in Vivo.

Polygonum multiflorum is widely used in the prevention and treatment of hyperlipidemia in traditional Chinese Medicine. In this study, the effects and relevant mechanisms of lipid-regulation by raw Polygonum multiflorum (RPM) were investigated. The results indicated that the basal plasma lipids, such as low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG), were significantly decreased in RPM treatment groups compared with the model group, especially in the RPM high dose group. The key enzymes involved in lipid metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in plasma were generally reduced after oral administration, which was consistent with the transcription levels of their target genes. In addition, the hepatotoxicity of RPM was investigated, and RPM showed slightly less liver injury than that induced by simvastatin. Histological analysis indicated that the fat vacuoles and steatosis in hepatocytes were relieved after oral administration of RPM extract at a high dose of 16.2 g/kg, which was more obvious than that induced by simvastatin. These results revealed that RPM exerted its lipid-lowering effect by regulating the expression of related genes, and performed better than simvastatin in the treatment of hyperlipidemia.

Analysis of Potential Amino Acid Biomarkers in Brain Tissue and the Effect of Galangin on Cerebral Ischemia.

Galangin, a potent scavenger of free radicals, has been used as an herbal medicine for various ailments for centuries in Asia. With complex pathophysiology, ischemic stroke is one of the most frequent causes of death and disability worldwide. We have reported that galangin provides direct protection against ischemic injury as a potential neuroprotective agent and has potential therapeutic effects on the changes of serum amino acids in ischemic stroke; however, the mechanism of the changes of amino acids in the ischemic brain tissue has not yet been clarified. In this paper, we explored brain tissue amino acid biomarkers in the acute phase of cerebral ischemia and the effect of galangin on those potential biomarkers. Finally, we identified that glutamic acid, alanine and aspartic acid showed significant changes (p < 0.05 or p < 0.01) in galangin-treated groups compared with vehicle-treated rats and the four enzymes associated with these three AAs' metabolic pathways; GLUD1, SLC16A10, SLC1A1 and GPT were identified by multiplex interactions with the three amino acids. By metabolite-protein network analysis and molecular docking, six of 28 proteins were identified and might become potential galangin biomarkers for acute ischemic stroke. The data in our study provides thoughts for exploring the mechanism of disease, discovering new targets for drug candidates and elucidating the related regulatory signal network.

Erjingpill bionic cerebrospinal fluid alleviates LPS-induced inflammatory response in BV2 cells by inhibiting glycolysis via mTOR.

ETHNOPHARMACOLOGICAL RELEVANCE: Erjingpill, a well-known prescription documented in the classic Chinese medical text "Shengji Zonglu," has been proven to have effective alleviating effects on neuroinflammation in Alzheimer's disease (AD). Although the alterations in microglial cell glycolysis are known to play a crucial role in the development of neuroinflammation, it remains unclear whether the anti-neuroinflammatory effects of Erjingpill are associated with its impact on microglial cell glycolysis. AIM OF THE STUDY: This study aims to determine whether Erjingpill exerts anti-neuroinflammatory effects by influencing microglial cell glycolysis. MATERIALS AND METHODS: Firstly, Erjingpill decoction was prepared into an Erjingpill bionic cerebrospinal fluid (EBCF) through a process of in vitro intestinal absorption, hepatocyte incubation, and blood-brain barrier (BBB) transcytosis. Subsequently, UPLC/Q-TOF-MS/MS technology was used to analyze the compounds in Erjingpill and EBCF. Next, an in vitro neuroinflammation model was established by LPS-induced BV2 cells. The impact of EBCF on BV2 cell proliferation activity was evaluated using the CCK-8 assay, while the NO release was assessed using the Griess assay. Additionally, mRNA levels of pro-inflammatory factors (IL-1ß, IL-6, TNF-α, and COX-2), anti-inflammatory factors (IL-10, IL-4, Arg-1, and TGF-ß), M1 microglial markers (iNOS, CD86), M2 microglial markers (CD36, CD206), and glycolytic enzymes (HK2, GLUT1, PKM, and LDHA) were measured using qPCR. Furthermore, protein expression of microglial activation marker Iba-1, M1 marker iNOS, and M2 marker CD206 were identified through immunofluorescence, while concentrations of pro-inflammatory cytokines IL-1ß and TNF-α were measured using ELISA. Enzymatic activity of glycolytic enzymes (HK, PK, and LDH) was assessed using assay kits, and the protein levels of pro-inflammatory factors (IL-1ß, iNOS, and COX-2), anti-inflammatory factors (IL-10 and Arg-1), and key glycolytic proteins GLUT1 and PI3K/AKT/mTOR were detected by Western blot. RESULTS: Through the analysis of Erjingpill and EBCF, 144 compounds were identified in Erjingpill and 40 compounds were identified in EBCF. The results demonstrated that EBCF effectively inhibited the elevation of inflammatory factors and glycolysis levels in LPS-induced BV2 cells, promoted polarization of M1 microglial cells towards the M2 phenotype, and suppressed the PI3K/AKT/mTOR inflammatory pathway. Moreover, EBCF alleviated LPS-induced BV2 cell inflammatory response by modulating mTOR to inhibit glycolysis. CONCLUSIONS: EBCF exhibits significant anti-neuroinflammatory effects, likely attributed to its modulation of mTOR to inhibit microglial cell glycolysis. This study furnishes experimental evidence supporting the clinical utilization of Erjingpill for preventing and treating AD.

Extract of Tinospora sinensis alleviates LPS-induced neuroinflammation in mice by regulating TLR4/NF-κB/NLRP3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried rattan stem of Tinospora sinensis (Lour.) Merr. is valued for its efficacy of clearing heat and removing toxicity, calming and soothing the nerves. It is widely used in Tibetan medicine for the treatment of rheumatic and aging diseases. Studies have confirmed its anti-inflammatory and ameliorating effects on Alzheimer's disease; however, the anti-neuroinflammation efficacy and mechanism remain unclear. AIM: This study aimed to explore the anti-neuroinflammation efficacy, major effective ingredients, and potential mechanism of extract of Tinosporae sinenisis (TIS). METHODS: UPLC-Q-TOF/MS was used to identify the compounds of TIS and the plasma components of rats after gastric administration of TIS. C57BL/6J mice were continuously intraperitoneally injected with lipopolysaccharide (LPS) (250 µg/kg) for 14 d to establish a neuroinflammation model. The effects of TIS (4.5 g/kg, 9 g/kg) on the learning and memory abilities in mice with neuroinflammation was evaluated using spontaneous activity, novel object recognition, and Morris water maze tests. Pathological changes in the hippocampus were observed using hematoxylin and eosin staining. Gene and protein levels of inflammatory factors in the brain were detected using qRT-PCR and ELISA kits. Iba-1 levels in the brain were detected using immunofluorescence to assess the degree of microglial activation. Network pharmacology, based on the components absorbed into plasma of TIS, was used to predict potential targets and pathways. Proteomics was used to study the differentially expressed proteins and related pathways in the brain tissue of mice with neuroinflammation. Finally, correlation analysis was performed on the results of network pharmacology and proteomics, and proteins related the anti-neuroinflammatory effect of TIS were detected by western blot. RESULTS: A total of 39 compounds were identified in TIS: genipingentiobioside, isocorydin, reticuline, (-)-argemonine, tinosineside A, tinosinenside A, and costunolide were absorbed into the plasma. After continuous intraperitoneal injection of LPS into C57BL/6J mice, microglia in the brain tissue were activated and the gene and protein levels of IL-1ß, TNF-α, IL-6, and iNOS were increased in the brain tissue, suggesting that the neuroinflammation model was successfully established. TIS reduced Iba-1 levels and gene expression and protein levels of inflammatory factors in the brain of mice with neuroinflammation. Furthermore, TIS improved the pathological changes in the hippocampus and learning and memory abilities caused by neuroinflammation. Network pharmacology has predicted that TNF, IL-1ß, and IκBKB are closely related to neuroinflammation. Proteomics identified key differentially expressed proteins, including TNF, NF-κB2, NF-κBIA, and TLR4. Toll-like receptor (TLR), NF-κB, and NOD-like receptor (NLR) signaling pathways are involved in neuroinflammation-related pathways. Correlation analysis revealed TLR, TNF and NLR signaling pathways were closely related to the anti-neuroinflammatory effects of TIS. We observed that TIS alleviated neuroinflammation by inhibiting the TLR4/NF-κB/NLRP3 pathway. CONCLUSION: Thirty-nine compounds were identified from TIS, among which seven were absorbed into the plasma as prototype components. TIS alleviated LPS-induced neuroinflammation in mice, and its mechanism was related to inhibition of TLR4/NF-κB/NLRP3 signaling pathway.

[Preparation of Kushen-Dilong nanoemulsion gel and transdermal characterization in vitro].

OBJECTIVE: To prepare Kushen-Dilong nanoemulsion and nanoemuls-ion gel, and investigate its content, physical and chemical properties. Their transdermal properties in vitro were studied as well. METHOD: IPM acted as oil phase, EL35 as surfactant, EtOH as cosurfactant, Pheretima aqueous solution was added dropwise to the oil phase to prepare Kushen-Dilong nanoemulsion at room temperature using magnetic stirring. HPLC was used to determine the content of matrine and oxymatrine in the nanoemulsion. Transmission electron microscopy and laser particle size analyzer was used to determine the shape and size of the nanoemulsion. NP700 was used as substrate to prepare Kushen-Dilong nanoemulsion gel. Franz diffusion cell was used for the nanoemulsion and gel transdermal characteristics in vitro. RESULT: The Kushen-Dilong nanoemulsion was O/W nanoemulsion, its uniform particle size was 20.6 nm with roundness appearance and stable content. The steady-state permeation rate of Kushen-Dilong nanoemulsion, nanoemulsion gel, saturated aqueous solution, hydro gel were 0.1484, 0.1183, 0.0306, 0.0321 mg x cm(-2) x h(-1), respectively. CONCLUSION: The 24 h cumulative infiltration and infiltration rate of Kushen-Dilong nanoemulsion and nanoemulsion gel were better than the saturated aqueous solution and hydro gel, which could provide a new dosage form for Kushen-Dilong transdermal drug delivery.

[Physicochemical properties and skin penetration in vitro of total alkaloids of Sophora flavescens nanoemulsion].

The research aimed at investigating the physicochemical properties, stability and skin penetration in vitro of total alkaloids of Sophora flavescens nanoemulsion. Prepare total alkaloids of S. flavescens nanoemulsion and detect the determination of matrine and oxymatrine in the nanoemulsion using HPLC method. Transmission electron microscopy and laser particle size analyzer were utilized to detect the shape and size of the nanoemulsion respectively. And also the stability of nanoemulsion was studied under the conditions of low temperature (4 degrees C), normal temperature (25 degrees C) and high temperature (60 degrees C). Franz diffusion cell was used to research the transdermal absorption of nanoemulsion in vitro. The results found that the nanoemulsion we prepared presented appearance of rounded, uniform; its average diameter was (15.55 +/- 2.24) nm, and particle size distribution value was 0. 161; the appearance, diameter and percentage determination of total alkaloids of S. flavescens had no variations after 15 d under 4, 25, 60 degrees C respectively. The steady-state permeation rate was 4.564 1 microg x cm(-2) x h(-1), 24 h cumulative amount of penetration was 110.7 microg x cm(-2), which was 1.86 fold of 24 h cumulative amount of aqueous solution (59.41 microg x cm(-2)). All the results demonstrated total alkaloids of S. flavescens nanoemulsion had good permeability, and could provide a new preparation for its clinical application.

15, 16-Dihydrotanshinone I protects against ischemic stroke by inhibiting ferroptosis via the activation of nuclear factor erythroid 2-related factor 2.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of antioxidative stress responses, which are associated with ferroptosis inhibition. Ferroptosis is closely related to the pathophysiological process of ischemic stroke. 15, 16-Dihydrotanshinone I (DHT), a lipophilic tanshinone extracted from the root of Salvia miltiorrhiza Bunge (Danshen), has various pharmacological effects. However, its effect against ischemic stroke remains to be examined. PURPOSE: This study aimed to investigate the protective effect of DHT against ischemic stroke and its underlying mechanism. METHODS: Rats with permanent middle cerebral artery occlusion (pMCAO)-induced cerebral ischemia rats and tert-butyl hydroperoxide (t-BHP)-injured PC12 cells were used to investigate the protective effect of DHT against ischemic stroke effect and the potential mechanism. RESULTS: The results showed that DHT decreased ferroptosis in-vitro experiment, as indicated by decreased lipid ROS generation, increased Gpx4 expression and the ratio of GSH/GSSG, and improved mitochondrial function. The inhibitory effect of DHT on ferroptosis was decreased after Nrf2 silencing. Furthermore, DHT decreased the neurological score, infarct volume, and cerebral edema, increased regional cerebral blood flow, and improved the microstructure of white-grey matter in pMCAO rats. In addition, DHT activated Nrf2 signaling and inhibited ferroptosis marker events. Nrf2 activator and ferroptosis inhibitor also exerted protective effects on pMCAO rats. CONCLUSIONS: These data demonstrated that DHT might have therapeutic potential for ischemic stroke and protects against ferroptosis via the activation of Nrf2. This study provides new insight into DHT-mediated prevention of ferroptosis in ischemic stroke.

Nuciferine attenuates acute ischemic stroke in a rat model: a metabolomic approach for the mechanistic study.

Lotus leaves have the dual identity of medicine and food homology as included in the Chinese Pharmacopoeia. Nuciferine is the major bioactive component which is highly abundant in the leaves of Nelumbo nucifera Gaertn. Nuciferine has been shown to potentially improve energy metabolism and protect neurons in cerebral ischemia. However, the mechanisms underlying the protective effects of nuciferine on acute ischemic stroke (AIS) are still unclear. Metabolomics was used for uncovering the underlying therapeutic mechanism of nuciferine in AIS with the help of 1H NMR. The rat model of AIS was generated by the occlusion of the middle cerebral artery (MCAO). After treatment with nuciferine, several indexes of oxidative stress and inflammation, such as total antioxidant capacity (T-AOC), total glutathione/oxidized glutathione (GSH/GSSG), malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were significantly improved, and some metabolic biomarkers (histidine, glycine, glycerol, serine, tyrosine, lysine, choline, etc.) were significantly regulated. Bioinformatic analysis demonstrated that these biomarkers and the derived genes (myeloperoxidase, catalase, etc.), fatty acid and amino acid metabolisms and 9 key metabolic pathways were involved in the nuciferine activity, which indicated the potential therapeutic mechanisms of nuciferine in AIS.

Dendrobium nobile protects against ovalbumin-induced allergic rhinitis by regulating intestinal flora and suppressing lung inflammation.

Antibiotic exposure-induced dysbiosis of the intestinal flora increases the risk of developing allergic rhinitis. Hence, regulating the balance of intestinal flora may be useful for preventing and treating allergic rhinitis. However, the underlying mechanism is unclear. Dendrobium nobile (Shihu) exhibits anti-inflammatory and immune activities. Hence, in this study, we investigated the mechanism via which Shihu may improve allergic rhinitis. Mouse models of allergic rhinitis with intestinal flora dysbiosis (Model-D, antibiotics induce intestinal flora dysbiosis with ovalbumin-induced allergy) and normal intestinal flora with allergic rhinitis (Model-N, ovalbumin-induced allergy) were established. The effect of Shihu on intestinal flora and inflammation caused during allergic rhinitis were analyzed. Allergic symptoms, infiltration of hematoxylin and eosin in the lungs and nose, and the release of various factors [interleukin (IL)-2, IL-4, IFN-γ, IL-6, IL-10, and IL-17] in the lungs were evaluated. The results indicate that intestinal flora dysbiosis exacerbated lung and nose inflammation in allergic rhinitis. However, treatment with the Shihu extract effectively reversed these symptoms. Besides, the Shihu extract inhibited the PI3K/AKT/mTOR pathway and increased the level of Forkhead box protein in the lungs. Additionally, the Shihu extract reversed intestinal flora dysbiosis at the phylum and genus levels and improved regulator T cell differentiation. Furthermore, in the Model-D group, the Shihu extract inhibited the decrease in the diversity and abundance of the intestinal flora. Screening was performed to determine which intestinal flora was positively correlated with Treg differentiation using Spearman's correlation analysis. In conclusion, we showed that Shihu extract restored the balance in intestinal flora and ameliorated inflammation in the lungs of allergic rhinitis mice and predicted a therapeutic new approach using Traditional Chinese Medicine to improve allergic rhinitis.

Screening of Anti-Inflammatory Components of Qin Jin Hua Tan Tang by a Multivariate Statistical Analysis Approach for Spectrum-Effect Relationships.

Qing Jin Hua Tan Tang (QJHTT) exerts therapeutic effects in patients with chronic obstructive pulmonary disease (COPD) by alleviating inflammation. However, the anti-inflammatory components of QJHTT have not yet been reported. Our study aimed to screen the active anti-inflammatory components of QJHTT using a multivariate statistical analysis approach for spectrum-effect relationships. Different polar fractions of QJHTT were prepared using ethanol, ethyl acetate, and n-butanol to analyze the phytochemical components. Phytochemical fingerprints were generated using ultrahigh-performance liquid chromatography. In total, 24 peaks were observed in ten batches of QJHTT extracts. The anti-inflammatory activity was evaluated using a xylene-induced ear-swelling mouse model. Additionally, the spectrum-effect relationship between the relative areas of the 24 peaks and pharmacological activity was investigated using multivariate statistical analysis. The potential anti-inflammatory ingredients obtained from the screening (multivariate statistical analysis) will be validated for their anti-inflammatory effects and mechanisms utilizing a lipopolysaccharide-induced macrophage inflammation model. QJHTT ethanol extract 1 exhibited good anti-inflammatory activity. Peaks 11, 12, 13, 14, and 16, which were closely correlated with anti-inflammatory activity, were identified as meranzin, baicalin, baicalein, chrysin-7-O-ß-D-glucuronide, and wogonoside, respectively. The anti-inflammatory activities of meranzin, baicalin, baicalein, and wogonoside were verified in vitro. These four bioactive components significantly inhibited the secretion of inflammatory factors in the lipopolysaccharide-stimulated macrophage cell line. This research successfully screened the QJHTT anti-inflammatory active ingredient group. Meranzin, baicalin, baicalein, chrysin-7-O-ß-D-glucuronide, and wogonoside were predicted to be the anti-inflammatory active ingredient groups of QJHTT.

Prebiotics enhance the biotransformation and bioavailability of ginsenosides in rats by modulating gut microbiota.

BACKGROUND: Gut microbiota mainly function in the biotransformation of primary ginsenosides into bioactive metabolites. Herein, we investigated the effects of three prebiotic fibers by targeting gut microbiota on the metabolism of ginsenoside Rb1 in vivo. METHODS: Sprague Dawley rats were administered with ginsenoside Rb1 after a two-week prebiotic intervention of fructooligosaccharide, galactooligosaccharide, and fibersol-2, respectively. Pharmacokinetic analysis of ginsenoside Rb1 and its metabolites was performed, whilst the microbial composition and metabolic function of gut microbiota were examined by 16S rRNA gene amplicon and metagenomic shotgun sequencing. RESULTS: The results showed that peak plasma concentration and area under concentration time curve of ginsenoside Rb1 and its intermediate metabolites, ginsenoside Rd, F2, and compound K (CK), in the prebiotic intervention groups were increased at various degrees compared with those in the control group. Gut microbiota dramatically responded to the prebiotic treatment at both taxonomical and functional levels. The abundance of Prevotella, which possesses potential function to hydrolyze ginsenoside Rb1 into CK, was significantly elevated in the three prebiotic groups (P < 0.05). The gut metagenomic analysis also revealed the functional gene enrichment for terpenoid/polyketide metabolism, glycolysis, gluconeogenesis, propanoate metabolism, etc. CONCLUSION: These findings imply that prebiotics may selectively promote the proliferation of certain bacterial stains with glycoside hydrolysis capacity, thereby, subsequently improving the biotransformation and bioavailability of primary ginsenosides in vivo.

Mitochondrial protective effect of neferine through the modulation of nuclear factor erythroid 2-related factor 2 signalling in ischaemic stroke.

BACKGROUND AND PURPOSE: Ischaemic stroke is a leading cause of death and long-term disability. Promising neuroprotective compounds are urgently needed to overcome clinical therapeutic limitations. Neuroprotective agents are limited to single-target agents, which further limit their clinical effectiveness. Due to the brain's particular energy requirements, the energy micro-environment, centred in mitochondria, is a new research hotspot in the complex pathology of ischaemic stroke. Here, we studied the effects of neferine (Nef), a bis-benzylisoquinoline alkaloid extracted from the seed embryo of Nelumbo nucifera Gaertn, on ischaemic stroke and its underlying mitochondrial protective mechanisms. EXPERIMENTAL APPROACH: Rats with permanent middle cerebral artery occlusion (pMCAO)-induced focal cerebral ischaemia and tert-butyl hydroperoxide (t-BHP)-injured PC12 cells were used to investigate the neuroprotective effects of Nef, particularly with regard to energy micro-environment regulation by mitochondria and its mechanism in vivo and in vitro. KEY RESULTS: Nef protected t-BHP-injured PC12 cells in vitro and ameliorated neurological score, infarct volume, regional cerebral blood flow, cerebral microstructure and oxidant-related enzyme deficits in pMCAO rats in vivo. Nef also prevented mitochondrial dysfunction both in vivo and in vitro. The underlying mechanism of the mitochondrial protective effect of Nef might be attributed to the increased translocation of Nrf2 to the nucleus. Furthermore, the translocation of Nrf2 to nucleus was also decreased by sequestosome 1 (p62) knockdown. CONCLUSIONS AND IMPLICATIONS: Our results demonstrated that Nef might have therapeutic potential for ischaemic stroke and may exert its protective role through mitochondrial protection. This protection might be attributed to the modulation of Nrf2 signalling.

Global Metabolomics Reveals the Metabolic Dysfunction in Ox-LDL Induced Macrophage-Derived Foam Cells.

Atherosclerosis (AS) is a chronic disorder of large arteries that is a major risk factors of high morbidity and mortality. Oxidative modification LDL is one of the important contributors to atherogenesis. Macrophages take up ox-LDL and convert into foam cells, which is the hallmark of AS. To advance the understanding of the metabolic perturbation involved in ox-LDL induced macrophage-derived foam cells and discover the potential biomarkers of early AS, a global metabolomics approach was applied based on UHPLC-QTOF/MS. Multivariate statistical analyses identified five metabolites (25-azacholesterol, anandamide, glycocholate, oleoyl ethanolamide, and 3-oxo-4, 6-choladienoate) for distinguishing foamy macrophages from controls. Among the six main metabolic pathways, the unsaturated fatty acid, especially arachidonic acid metabolism, contributed importantly to early AS. A new biomarker, anandamide (AEA), whose synthesis and metabolism in macrophages are disturbed by overloaded ox-LDL, results in metabolic obstruction. This study is the first to investigate the metabolic disturbance in macrophage-derived foam cells induced by ox-LDL and screen potential biomarkers and metabolic pathways associated with early AS. Our findings provide a new insight in the underlying pathophysiological mechanisms and also help to identify novel targets for the intervention of AS.

Studies of the microbial metabolism of flavonoids extracted from the leaves of Diospyros kaki by intestinal bacteria.

Flavonoid glycosides are metabolized by intestinal bacteria, giving rise to a wide range of phenolic acids that may exert systemic effects in the body. The microbial metabolism of flavonoids extracted from the leaves of Diospyros kaki (FLDK) by intestinal bacteria was investigated in vitro. High-performance liquid chromatography/linear trap quadrupole orbitrap mass spectrometry was performed to analyze the metabolites of flavonoids in vivo using Xcalibur2.1 software. The results showed that the levels of flavonoid glycosides and flavonoid aglycones decreased rapidly in the process of microbial metabolism by intestinal bacteria in vitro, and the metabolic rate may be related to the concentration of intestinal bacteria in the culture solution. In vivo metabolites of FLDK were detected in rat plasma and urine after oral administration of FLDK. Eight flavonoids were identified in the urine, and three were identified in the plasma; however, flavonoid aglycones were not found in the plasma.

Synergism and rules of the new combination drug Yiqijiedu formulae (YQJD) on ischemic stroke based on amino acids (AAs) metabolism.

The use of combination drugs is considered to be a promising strategy to control complex diseases such as ischemic stroke. The detection of metabolites has been used as a versatile tool to reveal the potential mechanism of diverse diseases. In this study, the levels of 12 endogenous AAs were simultaneously determined quantitatively in the MCAO rat brain using RRLC-QQQ method. Seven AAs were chosen as the potential biomarkers, and using PLS-DA analysis, the effects of the new combination drug YQJD, which is composed of ginsenosides, berberine, and jasminoidin, on those 7 AAs were evaluated. Four AAs, glutamic acid, homocysteine, methionine, and tryptophan, which changed significantly in the YQJD-treated groups compared to the vehicle groups (P < 0.05), were identified and designated as the AAs to use to further explore the synergism of YQJD. The result of a PCA showed that the combination of these three drugs exhibits the strongest synergistic effect compared to other combination groups and that ginsenosides might play a pivotal role, especially when combined with jasminoidin. We successfully explored the synergetic mechanism of multi-component and provided a new method for evaluating the integrated effects of combination drugs in the treatment of complex diseases.