[Effect of acupuncture on HIF-1α/NLRP3 inflammatory signaling pathway in the rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effects of Xingnao Kaiqiao (regaining consciousness and opening orifices) acupuncture therapy on the expression of hypoxia-inducible factor 1α (HIF-1α) and Nod-like receptor protein 3 (NLRP3) in cerebral ischemia-reperfusion rats, and to explore the mechanism of acupuncture against cerebral ischemia-reperfusion injury. METHODS: Seventy-two male SD rats were randomly divided into a sham-operation group, a model group, an acupuncture group and a non-point acupuncture group, with 18 rats in each one. Using modified Longa thread embolization method, the rat model of acute focal cerebral ischemia was prepared; and after 2 h ischemia, the reperfusion was performed to prepared the model of cerebral ischemia-reperfusion. Immediately after reperfusion, Xingnao Kaiqiao acupuncture method was applied to bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26) in the acupuncture group, while in the non-point acupuncture group, acupuncture was delivered at non-points and all of the needles were retained for 30 min in these two groups. The samples were collected 24 h after reperfusion in the rats of each group. Zea-Longa neurological deficit score was used to evaluate the degree of cerebral neurological impairment, TTC staining was adopted to observe the volume percentage of cerebral infarction, HE staining was provided to observe the morphological changes of brain, and Western blot was applied for detecting the expression of HIF-1α and NLRP3 proteins in the cerebral cortex on the right side. RESULTS: Compared with the sham-operation group, neurological deficit score and volume percentage of cerebral infarction were increased in the model group (P<0.01), and HIF-1α and NLRP3 protein expression was elevated (P<0.01). Compared with the model group, neurological deficit score and volume percentage of cerebral infarction were decreased (P<0.01), and HIF-1α and NLRP3 protein expression was lower (P<0.01) in the acupuncture group. There was no significant difference in above indexes in the non-point acupuncture group compared with the model group (P>0.05). Compared with the sham-operation group, the brain tissue of the rats in the model group and the non-point acupuncture group was loose and edema, and the nuclei were shriveled. The brain tissue morphology in the acupuncture group was similar to that of the sham-operation group. CONCLUSION: Acupuncture can alleviate cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of HIF-1α/NLRP3 signaling pathway to attenuate inflammatory response.

Time-Series-Dependent Global Data Filtering Strategy for Mining and Profiling of Xenobiotic Metabolites in a Dynamic Complex Matrix: Application to Biotransformation of Flavonoids in the Extract of Ginkgo biloba by Gut Microbiota.

Efficient characterization of xenobiotic metabolites and their dynamics in a changing complex matrix remains difficult. Herein, we proposed a time-series-dependent global data filtering strategy for the rapid and comprehensive characterization of xenobiotic metabolites and their dynamic variation based on metabolome data. A set of data preprocessing methods was used to screen potential xenobiotic metabolites, considering the differences between the treated and control groups and the fluctuations over time. To further identify metabolites of the target, an in-house accurate mass database was constructed by potential metabolic pathways and applied. Taking the extract of Ginkgo biloba (EGB) co-incubated with gut microbiota as an example, 107 compounds were identified as flavonoid-derived metabolites (including 67 original from EGB and 40 new) from 7468 ions. Their temporal metabolic profiles and regularities were also investigated. This study provided a systematic and feasible method to elucidate and profile xenobiotic metabolism.

Vascular Protective Effects of Malus toringoides (Rehd.) Hughes Extracts and their Mechanism in Diabetic Rats and HUVECs.

Malus toringoides (Rehd.) Hughes (Rosaceae) is used as a traditional folk medicine in the Tibet autonomous region of China to treat hypertension, hyperglycemia, and hyperlipidemia. However, few modern pharmacological data on the use of this plant against diabetic syndrome are available. In this study, we examined the vascular protection provided by a 70% ethanol extract of M. toringoides (EMT) in human umbilical vein endothelial cells (HUVECs) grown in high-glucose medium and in a high-fat diet/streptozotocin-induced rat diabetes model. EMT significantly suppressed the expression of cell adhesion molecules in both HUVECs and diabetic rats. EMT also inhibited activation of the CX3CL1/CX3CR1 axis and the nuclear factor kappa B (NF-κB) signaling pathway in vivo and in vitro. The results provide a significant information on the vasoprotective properties of M. toringoides that may contribute to the development and application of related herbal medicines.

Quantitative Analysis and Stability Study on Iridoid Glycosides from Seed Meal of Eucommia ulmoides Oliver.

As a traditional Chinese medicine, Eucommia ulmoides Oliver (E. ulmoides Oliv.) is an important medicinal plant, and its barks, male flowers, leaves, and fruits have high value of utilization. The seed meal of E. ulmoides Oliv. is the waste residue produced after oil extraction from seeds of E. ulmoides Oliv. Though the seed meal of E. ulmoides Oliv. is an ideal feed additive, its medicinal value is far from being developed and utilized. We identified six natural iridoid compounds from the seed meal of E. ulmoides Oliv., namely geniposidic acid (GPA), scyphiphin D (SD), ulmoidoside A (UA), ulmoidoside B (UB), ulmoidoside C (UC), and ulmoidoside D (UD). Six natural iridoid compounds were validated to have anti-inflammatory activities. Hence, six compounds were quantified at the optimum extracting conditions in the seed meal of E. ulmoides Oliv. by an established ultra-performance liquid chromatography (UPLC) method. Some interesting conversion phenomena of six tested compounds were uncovered by a systematic study of stability performed under different temperatures and pH levels. GPA was certified to be stable. SD, UA, and UC were only hydrolyzed under strong alkaline solution. UB and UD were affected by high temperature, alkaline, and strong acid conditions. Our findings reveal the active compounds and explore the quantitative analysis of the tested compounds, contributing to rational utilization for the seeds residues of E. ulmoides Oliv.

The qualitative and quantitative profiling for quality assessment of Yinxing Mihuan Oral Solution and the stability study on the focused flavonol glycosides.

Yinxing Mihuan Oral Solution (YMOS) has been widely applied for the treatment of coronary heart disease, angina pectoris, and cerebral ischemic disease in clinical practice. Nonetheless, the limited basic researches on quality analysis of YMOS remain a critical bottleneck that needs to be enhanced for better clinical applications. In this study, a total of 67 chemical components, including flavonoids, terpene lactones, nucleosides, etc., were tentatively characterized by ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap high-resolution mass spectrometry, among which 34 compounds were further identified by comparison with reference substances. By adopting a methodologically validated method, we discovered that the quantitative estimate of multi-compounds in 22 batches of YMOS showed lot-to-lot consistency, and the additives in YMOS also met the corresponding regulations. Furthermore, five flavonol glycosides whose content presented a downward trend in the expired YMOS were focused. A systematic research on stability test focusing on the five targeted flavonol glycosides was performed under different temperatures and pH levels. It was found that ortho-diphenolic hydroxyl group on B-ring and the type of saccharide connected to 3-hydroxyl on C-ring play a pivotal role in the stability of the tested compounds. Subsequently, as the important compounds, ginkgolides A, B, and C in YMOS were simultaneously quantified with ultra performance liquid chromatography coupled with triple quadrupole mass spectrometry. In brief, this study performs a reliable chemical identification and provides a rapid and feasible method for the quality evaluation, which contributes to the in-depth investigation and safe application of YMOS for clinical uses.

From non-targeted to targeted GC-MS metabolomics strategy for identification of TCM preparations containing natural and artificial musk.

BACKGROUND: Moschus is a rare and precious natural medicine. Due to the properties of resources scarcity and expensive price of natural musk, artificial musk has been developed as substitute materials in some prescriptions. Rapid and accurate identification of natural or artificial musk in complex traditional Chinese medicine (TCM) preparations is also a challenge. METHOD: A strategy from non-targeted to targeted gas chromatography-mass spectrometry (GC-MS) metabolomics was developed for discrimination of natural and artificial musk. Firstly, GC-MS-based non-targeted analysis combined with chemometrics was used to find the potential chemical markers to distinguish natural musk and artificial musk. Subsequently, targeted metabolomics was used to analyze musk in preparations with multiple reaction monitoring (MRM) mode by use gas chromatography coupled with triple quadrupole mass spectrometry (GC-QQQ MS). RESULTS: Two chemical markers named prasterone and androsterone have been selected and could be detected in all Compound Pien Tze Huang preparations (CPZHs) containing artificial musk, while the CPZHs containing natural musk did not detect two markers with S/N (signal to noise ratio) less than 3. CONCLUSION: Our work provides an applicable approach to select the practical chemical markers for the assessment of musk in preparations to realize the traceability of musk in TCM and improve the quality control of musk-containing preparations.

Design and semisynthesis of oleanolic acid derivatives as VEGF inhibitors: Inhibition of VEGF-induced proliferation, angiogenesis, and VEGFR2 activation in HUVECs.

Angiogenesis inhibitors targeting the VEGF signaling pathway are developed into drugs for the treatment of vaious diseases, such as cancer, rheumatoid arthritis, and age-related macular degeneration. Recent studies have revealed that oleanolic acid (OA), a natural pentacyclic triterpenoid, inhibited the VEGF/VEGFR2 signaling pathway and angiogenesis in HUVECs, which may represent an attractive VEGF inhibitor. In this paper, rational structural modification towards OA was performed in order to improve its inhibitory effects aganist VEGF and anti-angiogenesis potential. As a result, a series of novel OA derivatives, possessing α,ß-unsaturated ketone system in ring A and amide functional group at C-28, were prepared and evaluated for cytotoxicity and their ability to inhibit VEGF-induced abnormal proliferation of HUVECs. The results showed that two promising derivatives, OA-1 and OA-16, exhibited no in vitro cytotoxicity against HUVECs but showed more potent inhibitory activity against VEGF-induced proliferation and angiogenesis in HUVECs, compared with OA. The results of Western blot indicated that OA-1 and OA-16 inhibited VEGF-induced VEGFR2 activation. Furthermore, small interfering RNA experiments were performed to confirm that both compounds inhibited VEGF-induced angiogenesis via VEGFR2. Thus, the present study resulted in the discovery of new promising OA-inspired VEGF inhibitors, which can serve as potential lead compounds for the treatment of angiogenesis-related diseases.

Dihydrohomoplantagin and Homoplantaginin, Major Flavonoid Glycosides from Salvia plebeia R. Br. Inhibit oxLDL-Induced Endothelial Cell Injury and Restrict Atherosclerosis via Activating Nrf2 Anti-Oxidation Signal Pathway.

Oxidized low-density lipoprotein (oxLDL)-induced endothelium injury promotes the development of atherosclerosis. It has been reported that homoplantaginin, a flavonoid glycoside from the traditional Chinese medicine Salvia plebeia R. Br., protected vascular endothelial cells by inhibiting inflammation. However, it is undetermined whether homoplantaginin affects atherosclerosis. In this study, we evaluated the effect of homoplantaginin and its derivative dihydrohomoplantagin on oxLDL-induced endothelial cell injury and atherosclerosis in apoE-/- mice. Our results showedthat both dihydrohomoplantagin and homoplantaginin inhibited apoptosis and the increased level of ICAM-1 and VCAM-1 in oxLDL-stimulated HUVECs and the plaque endothelium of apoE-/- mice. Additionally, both of them restricted atherosclerosis development of apoE-/- mice. Mechanistic studies showed that oxLDL-induced the increase in ROS production, phosphorylation of ERK and nuclear translocation of NF-κB in HUVECs was significantly inhibited by the compounds. Meanwhile, these two compounds promoted Nrf2 nuclear translocation and increased the anti-oxidation downstream HO-1 protein level in HUVECs and plaque endothelium. Notably, knockdown of Nrf2 by siRNA abolished the cell protective effects of compounds and antagonized the inhibition effects of them on ROS production and NF-κB activation in oxLDL-stimulated HUVECs. Collectively, dihydrohomoplantagin and homoplantaginin protected VECs by activating Nrf2 and thus inhibited atherosclerosis in apoE-/- mice.

[Effect of Wangbuliuxing combined with massage on breastfeeding and lactation function in cesarean section women].

OBJECTIVE: To explore the effect of Wangbuliuxing (semen vaccariae) combined with massage at breast and acupoint on breastfeeding and lactation function in cesarean section women. METHODS: A total of 120 cases of cesarean section women were randomly divided into an observation group and a control group, 60 cases in each group. The control group was treated with routine nursing. On the basis of the control group, the observation group was treated with oral administration of Wangbuliuxing decoction, twice a day for 7 days; in addition, massage at breast and acupoint (Zhongfu [LU 1], Yunmen [LU 2], Danzhong [CV 17], 2 min per acupoint per time) was given, twice a day for 7 days. The onset time of lactation, 48-hour postpartum lactation volume, breast swelling and pain, 42-day postpartum breastfeeding were compared between the two groups; the serum levels of prolactin at 48 and 72 h after delivery were measured, and the body mass of newborns at birth and 42 d after delivery were recorded. RESULTS: The onset time of lactation in the observation group was earlier than that in the control group (P<0.05), the 48-hour postpartum lactation volume was higher than the control group (P<0.05), and the breast swelling pain 48 h after delivery was lighter than the control group (P<0.05). Forty-two days after delivery, the success rate of breastfeeding in the observation group was 91.7% (55/60), which was higher than 76.7% (46/60) in the control group (P<0.05). At 48 and 72 h after delivery, the level of serum prolactin in the observation group was higher than that in the control group (P<0.05). On 42 d after delivery, the body mass of newborns in the two groups was higher than that at birth (P<0.05), and that in the observation group was higher than the control group (P<0.05). CONCLUSION: Wangbuliuxing combined with massage at acupoint and breast could significantly shorten the onset time of lactation, improve the lactation volume, effectively improve breast swelling and pain, increase breastfeeding rate and promote the growth and development of newborns.

An integrated two-step filtering strategy of collision cross-section interval predicting and mass defect filtering for targeted identification of analogues in herbal medicines using liquid chromatography-ion mobility-mass spectrometry.

Rapid identification of chemical analogues in herbal medicines using liquid chromatography-mass spectrometry was an efficient tool for discoveries of potentially active ingredients. Multi-dimensional combination of various separation technologies could significantly enhance the capacities for detection of trace components and discrimination of multiple isomers. In this study, an integrated two-step filtering strategy on liquid chromatography-ion mobility tandem with quadrupole-time-of-flight mass spectrometry (LC-IM-QTOF MS) was developed for identification of analogues in complex matrixes. The extracted raw data were preliminarily filtered by a collision-cross section (CCS) interval generated from power regression with confidence level at 99% for prediction of analogues. Then, the remained ions were further screened using a mass defect filtering (MDF) window based on m/z and decimal m/z of potential skeletons and substituents. By applying this strategy, 86, 102, 73, and 57 isoquinoline alkaloids were identified in herbal materials of Coptis chinensis Franch (CC), C. deltoidea C.Y.Cheng et Hsiao (CD), C. teeta Wall (CT), and Corydalis yanhusuo W.T.Wang (CY). The integrated two-step filtering presented higher efficiencies on exclusion of the background interference and reducing the false-positive rates than previously reported approaches. This study facilitated the application of LC-IM-MS on small molecular analysis and promoted the discoveries of bioactive components of herbal medicines for further pharmacological researches and quality control.

Integrating Study on Qualitative and Quantitative Characterization of the Major Constituents in Shuanghuanglian Injection with UHPLC/Q-Orbitrap-MS and UPLC-PDA.

As a clinically effective traditional Chinese medicine injection (TCMI), Shuanghuanglian injection (SHLI) is widely used in the treatment of upper respiratory tract infection and pneumonia. However, the shortage of quality analysis is a limitation that remains to be improved in the clinical application of SHLI. In this study, taking advantage of ultra-high-performance liquid chromatography tandem Q-Exactive Orbitrap high-resolution mass spectrometry (UHPLC/Q-Orbitrap-MS), 31 chemical components (eight organic acids, eleven flavonoids, five iridoid glycosides, four phenylethanoid glycosides, and three lignans) in SHLI were characterized, among which 22 components were unambiguously identified by reference compounds. The brief prediction results of network pharmacology indicated that the 22 targeted components may have anti-inflammatory, antibacterial, antiviral, and immunomodulatory activities. Using multiwavelength switching method, the 22 targeted components were quantified by ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) after the methodological validation. Based on the successfully established method, the total content of 22 components in 20 batches of SHLIs was efficiently determined with a slight variation between 10.25 and 11.28 mg/mL, which accounted for 38.7% in total solid of SHLI. This study performed a reliable chemical identification and provided a rapid and effective method for quality analysis, which contributed to the in-depth investigation and application of SHLI.

A validated UHPLC-MS/MS method for pharmacokinetic and brain distribution studies of twenty constituents in rat after oral administration of Jia-Wei-Qi-Fu-Yin.

A rapid ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ MS/MS) approach with high sensitivity and selectivity was developed for the quantification of twenty compounds, including 9 saponins, 8 flavonoids, 2 oligosaccharide esters and 1 phenolic acid, in rat plasma and brain, which was administrated intragastrically with Jia-Wei-Qi-Fu-Yin (JWQFY), Mass spectrometric detection was operated under multiple reaction monitoring (MRM) mode. All calibration curves possessed good linearity with correlation coefficients ( r2) higher than 0.9916 in their respective linear ranges. For intra- and inter-day precision, all the relative standard deviations (RSDs) at different levels were less than 14.68 %. Based on the UHPLC-QqQ MS/MS quantitative results, pharmacokinetic study and brain distribution of multiple components in JWQFY was then successfully performed. As a result, constituents with discrepancy structures showed diverse pharmacokinetic and distribution characteristics. For instance, ferulic acid (phenolic acid), 3, 6'-disinapoyl sucrose and tenuifoliside A (oligosaccharide esters) showed short Tmax (< 10 min), whereas the Tmax of ginsenosides Rb1, Rb2 and Rc (ppd-type terpenoid saponins) were much longer (> 4 h). Besides, ferulic acid, epimedin C, icariin, glycyrrhizin, ginsenoside Rb1 and ginsenoside Rg1 were considered as the potential effective ingredients of JWQFY because of their relatively high exposure to blood and brain. Our study would provide relevant information for discovery of pharmacodynamic ingredients, as well as further action mechanisms investigations of JWQFY.

Diagnostic ions guided 2D-locating strategy for characterization of chemical analogues in complex herbal medicines using liquid chromatography-ion mobility-mass spectrometry.

Rapid characterization of chemical analogues in potentially toxic complex matrix was essential for prevention of accidental poisoning. On the basis of the fragment ions possessed not only the same retention times (RT) but the same drift times (DT) on liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS), an alternating frames (AF)-data independent acquisition (DIA) was utilized for simultaneous detection and rapid match of precursor/product ions with a fast switch of low/high collision energy (CE). A diagnostic ions guided 2D-locating strategy was developed for identification of chemical analogues in potentially toxic herbal medicines using LC-IM-MS. Firstly, the 2D-locations (RT, DT) of diagnostic ions were screened from high-fragment IM-MS frames according to their m/z. Then, the correlated precursor ions were extracted from the complex background interference in low-fragment IM-MS frames based on diagnostic ions' 2D-locations. Finally, the remained ions were characterized using double-bond equivalent analysis combined with MS/MS fragment interpretation. Totally, 236 diterpene alkaloids including eight compounds with potential new esterification types were characterized in processed lateral roots of Aconitum carmichaelii Debx. Moreover, the LC-IM-MS distribution regularities of diterpene alkaloids with various chemical structure types were further investigated and discussed. This study presented an innovative idea for revealing the chemical basis related to the toxicities of potentially poisonous herbal medicines to ensure the medication safety.

A modified data filtering strategy for targeted characterization of polymers in complex matrixes using drift tube ion mobility-mass spectrometry: Application to analysis of procyanidins in the grape seed extracts.

BACKGROUNDS: Polymers, widely existing in food or dietary materials, have been attracting researchers, facing challenges, and needing effective strategies on targeted characterization in complex matrixes. METHODS: A modified data filtering strategy (including locating with drift time and m/z ranges, multiple mass defect filtering, validating MS information, and evaluating MS/MS spectra) was developed and applied for procyanidins in the grape seed extracts (GSE) using drift tube ion mobility-mass spectrometry. The procyanidin ions' trendlines were predicted by multi-model regression. Their collision cross-sections (CCSs) were calculated using single-field methods. RESULTS AND DISCUSSION: Totally, 769 CCSs belonging to 686 procyanidins with polymer degrees at 1-15 were characterized. The exponent regression was the most reasonable model (r2 ≥ 0.9379) to reveal the trendlines. The change tendency of CCSs with their polymer degrees, charge states, and linkage types were investigated. CONCLUSION: This study provided an innovative strategy for targeted characterization of polymers in complex matrixes.

A novel curcumin-loaded nanoparticle restricts atherosclerosis development and promotes plaques stability in apolipoprotein E deficient mice.

Although curcumin possesses anti-atherogenic and anti-inflammatory properties, its application is limited because of its low aqueous solubility and poor oral bioavailability. Recently, our group synthesized a novel linear-dendrimer methoxy-poly (ethylene glycol)-b-poly(ε-caprolactone) copolymer nanoparticle loading curcumin (Cur-NPs) which could improve solubility and release property of curcumin. In the present study, we further evaluated its anti-atherosclerotic effect in apolipoprotein E-/- mice. Our results demonstrated that the Cur-NPs significantly decreased atherosclerotic lesion areas and were more effective in stabilizing vulnerable plaques compared with free curcumin. The anti-atherosclerotic mechanisms of Cur-NPs include decreasing the number of introplaque microvessels, inhibiting the matrix metalloproteinase 2 and 9 activity, reducing the inflammatory response and regulating lipoprotein cholesterol metabolism more effectively compared with free curcumin. Furthermore, Cur-NPs could increase the amount of curcumin in the thoracic aorta and no significant toxicity was observed in the blood biochemical parameters in Cur-NPs-treated groups. Overall, our findings suggested that Cur-NPs could be a stabilized aqueous formulation for application with improved curcumin activity, which could be a potential treatment strategy for arteriosclerosis in the future.

Neuroprotective Effects of Taraxacum officinale Wigg. Extract on Glutamate-Induced Oxidative Stress in HT22 Cells via HO-1/Nrf2 Pathways.

Oxidative stress-mediated neuron damage is considered an important contributor to the pathogenesis and development of neurodegenerative diseases. Taraxacum officinale has been reported to possess antioxidant activities. However, whether it can protect neurons against oxidative damage and the underlying molecular mechanisms have not been fully determined. In the present study, we examined the neuroprotective effects of ethanol extracts of this plant (ETOW) on glutamate-induced oxidative stress in HT22 cells. Both cell viability and reactive oxygen species (ROS) assays showed that ETOW effectively attenuated glutamate-induced cytotoxicity and ROS generation. Furthermore, our results revealed that ETOW increased the expression of heme oxygenase-1 (HO-1) and promoted the nuclear translocation of nuclear factor erythroid 2-related factor-2 (Nrf2). The inhibitory effects of ETOW on glutamate-stimulated cell toxicity and ROS production were partially reversed by tin protoporphyrin (SnPP), an HO activity inhibitor. Taken together, these results demonstrate that ETOW can protect HT22 cells against glutamate-induced oxidative damage by inducing the Nrf2/HO-1 pathways. Our study supports the idea that Taraxacum officinale Wigg. is a promising agent for preventing neurodegenerative diseases.

Rapid characterization of chemical markers for discrimination of Moutan Cortex and its processed products by direct injection-based mass spectrometry profiling and metabolomic method.

BACKGROUND: Processing of herbal medicines is a characteristic pharmaceutical technique in Traditional Chinese Medicine, which can reduce toxicity and side effect, improve the flavor and efficacy, and even change the pharmacological action entirely. It is significant and crucial to perform a method to find chemical markers for differentiating herbal medicines in different processed degrees. PURPOSE: The aim of this study was to perform a rapid and reasonable method to discriminate Moutan Cortex and its processed products, and to reveal the characteristics of chemical components depend on chemical markers. METHODS: Thirty batches of Moutan Cortex and its processed products, including 11 batches of Raw Moutan Cortex (RMC), 9 batches of Moutan Cortex Tostus (MCT) and 10 batches of Moutan Cortex Carbonisatus (MCC), were directly injected in electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS) for rapid analysis in positive and negative mode. Without chromatographic separation, each run was completed within 3 min. The raw MS data were automatically extracted by background deduction and molecular feature (MF) extraction algorithm. In negative mode, a total of 452 MFs were obtained and then pretreated by data filtration and differential analysis. After that, the filtered 85 MFs were treated by principal component analysis (PCA) to reduce the dimensions. Subsequently, a partial least squares discrimination analysis (PLS-DA) model was constructed for differentiation and chemical markers detection of Moutan Cortex in different processed degrees. The positive mode data were treated as same as those in negative mode. RESULTS: RMC, MCT and MCC were successfully classified. Moreover, 14 and 3 chemical markers from negative and positive mode respectively, were screened by the combination of their relative peak areas and the parameter variable importance in the projection (VIP) values in PLS-DA model. The content changes of these chemical markers were employed in order to illustrate chemical changes of Moutan Cortex after processed. CONCLUSION: These results showed that the proposed method which combined non-targeted metabolomics analysis with multivariate statistics analysis is reasonable and effective. It could not only be applied to discriminate herbal medicines and their processing products, but also to reveal the characteristics of chemical components during processing.

An UPLC-MS/MS method for simultaneous determination of multiple constituents in Guizhi Fuling capsule with ultrafast positive/negative ionization switching.

Guizhi Fuling capsule (GFC), a traditional Chinese medicine (TCM) with effects of promoting blood circulation and dissipating blood stasis, has been widely used in the clinic. Because of the complex matrix and various chemical structure types, quality control of GFC remains great challenge. In the present study, an ultra performance liquid chromatography hybrid triple-quadrupole mass spectrometry (UPLC-QQQ MS) method with ultrafast positive/negative ionization switching was developed for simultaneous determination of 18 bioactive components in GFC, including methyl gallate, ethyl gallate, oxypaeoniflorin, benzoic acid, albiflorin, paeonolide, paeoniflorin, 1, 2, 3, 4, 6-pentagalloylglucose, mudanpioside C, benzoyloxypaeoniflorin, benzoylpaeoniflorin, pachymic acid, amygdalin, cinnamaldehyde, paeonol, cinnamic acid, 4-hydroxybenzoic acid, and gallic acid. Separation was performed on an Agilent Zorbax Extend-C18 column (2.1 mm × 50 mm, 1.8 µm), using a gradient elution with acetonitrile and water containing 0.1% formic acid. Cholic acid was selected as the internal standard. This newly developed method was fully validated for linearity, precision, accuracy, and stability, and then applied to quality assessment of GFC. Finally, the batch-to-batch reproducibility of GFC samples was evaluated by the cosine ration and Euclidean distance method, which showed high quality consistency. The results demonstrated that the developed method pro vided a reasonable and powerful manner for quality control of GFC.

Anti-Inflammatory Activity of Epimedium brevicornu Maxim Ethanol Extract.

Epimedium brevicornu Maxim has been used as a traditional herbal drug in China. In this study, the anti-inflammatory effects of E. brevicornu Maxim ethanol extract (EBME) were investigated in RAW264.7 macrophages and mice challenged with lipopolysaccharide (LPS). Results showed that EBME attenuated inflammation by decreasing the production of several proinflammatory mediators, such as nitric oxide (NO), prostaglandin (PG) E2, inducible nitric oxide synthase, and cyclooxygenase-2, in LPS-stimulated RAW264.7 macrophages. EBME increased the expression of heme oxygenase-1 (HO-1) and promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2. The inhibitory effects of EBME on LPS-stimulated NO and PGE2 expression were partially reversed by HO-1 inhibitor. EBME also elicited an anti-inflammatory effect by inhibiting the production of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in LPS-induced peritonitis. Therefore, EBME exhibited anti-inflammatory effects in vitro and in vivo.

Fragment-growing guided design of Keap1-Nrf2 protein-protein interaction inhibitors for targeting myocarditis.

Small-molecule inhibitors that block the Keap1-Nrf2 protein-protein interactions are being intensely pursued as a new therapeutic strategy for oxidative stress-related diseases, such as cancer, diabetes, Alzheimer's disease, arteriosclerosis, inflammation and myocarditis. However, there are not enough studies on antioxidant treatments using small molecules in myocarditis. We herein provided a series of novel hydronaphthoquinones as the Keap1-Nrf2 interaction inhibitors targeting LPS-induced myocarditis both in vitro and in vivo. These compounds were designed through an in-silico fragment growing approach based on our previous reported compound, S47 (1). The new compounds were predicted to form additional hydrogen bonds with the S363 residue, leading to higher inhibitory activity. Among these new derivatives, compounds S01 and S05 emerged as inhibitors with significant biochemical potency, as determined by fluorescent anisotropy assay and confirmed by surface plasmon resonance (SPR) and differential scanning fluorimetry (DSF) assays. These inhibitors can dose-dependently protect the H9c2 cardiac cells against LPS-induced injury (100% at 2 µM and 4 µM) and effectively prolong survival or save the life of LPS-injured mice. Mechanistic studies showed that these inhibitors could release Nrf2 in H9c2 cells and LPS-inflammatory mouse models and translocate into the nucleus in a dose-response manner, which significantly increased the downstream genes (HO-1, NQO-1) and the pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), while ROS production dramatically decreased. Their protective effects and the mechanism of action were further confirmed by siNrf2 transfected experiment. Collectively, the novel hydronaphthoquinones can be used as promising lead compounds for the study of Keap1-Nrf2 protein-protein interactions and further anti-myocarditis drug development.