Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.
4-Butyrolactone/analogs & derivatives* ; Apoptosis ; Calcium/pharmacology* ; Glucose/metabolism* ; Humans ; Mitochondrial Proteins ; Mitophagy ; Reactive Oxygen Species/metabolism* ; Reperfusion Injury/genetics*

OBJECTIVES: To find a method to distinguish exogenous gamma-hydroxybutyrate (GHB) from endogenous GHB by establishing ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) based on exosome for quantitative detection of GHB in the rat blood. METHODS: Adult male SD rats were divided into 1 h, 5 h, 10 h administration group and control group. After 1 h, 5 h and 10 h of single precursor of GHB gamma-butyrolactone (GBL) intraperitoneal injection in administration groups, 5 mL blood was collected from the abdominal aorta. Meanwhile, the control group was given a same dose of normal saline, and 5 mL blood was collected at 1 h. Among the 5 mL blood, 0.5 mL was directly detected by HPLC-MS after pretreatment, and exosomes were extracted from the remaining blood by differential centrifugation and detected. RESULTS: The concentration of GHB in the control group was (87.36±33.48) ng/mL, and the concentration with administration at 1 h, 5 h and 10 h was (110 400.00±1 766.35) ng/mL, (1 479.00±687.01) ng/mL and (133.60±12.17) ng/mL, respectively. The results of exosome detection showed that no peak GHB signal was detected in the control group and the 10 h administration group, and the concentrations of GHB at 1 h and 5 h administration groups were (91.47±33.44) ng/mL and (49.43±7.05) ng/mL, respectively. CONCLUSIONS GHB was detected in blood exosome by UPLC-MS, which indicated that exogenous GHB could be detected in plasma exosomes, while endogenous GHB could not be detected, suggesting that this method may be used as a basis to determine whether there is exogenous drug intake.
4-Butyrolactone/chemistry* ; Animals ; Chromatography, Liquid ; Exosomes/chemistry* ; Hydroxybutyrates/chemistry* ; Male ; Rats ; Rats, Sprague-Dawley ; Sodium Oxybate/analysis* ; Tandem Mass Spectrometry/methods*

This study aimed to investigate the effect and mechanism of ligustilide, the main active ingredient in Ligusticum wallichii, on mitochondria fission after PC12 cell injury induced by oxygen and glucose deprivation/reperfusion(OGD/R). In the experiment, an OGD/R model was established in vitro, and PC12 cells were pre-treated with ligustilide for 3 h, and then the cell viability was detected by CCK-8 method. The effect of different concentrations of ligustilide on the morphology of PC12 cells after OGD/R injury was observed under an inverted microscope. Transmission electron microscopy was used to observe the mitochondrial fission of PC12 cells after OGD/R injury. DCFH-DA immunofluorescence staining method was used to detect intracellular reactive oxygen species(ROS) changes. Changes in mitochondria membrane potential(MMP) were detected by flow cytometry. Hochest 33258 was used to observe the apoptosis of PC12 cells. Western blot was used to detect changes in cytochrome C(Cyt C) content in mitochondria and cytoplasm, and mitochondrial fission-related proteins Drp 1 and Fis 1. All results showed that compared with the model group, ligustilide significantly increased the survival rate of PC12 cells and the number of cells. Further experiments showed that ligustilide inhibited the release of ROS and decline of mitochondrial membrane potential in PC12 cells after OGD/R injury. Moreover, ligustilide reduced the release of Cyt C and promoted the expressions of Drp1 and Fis1 in mitochondrial fission proteins. Verification experiments showed that mitochondrial fission inhibitor mdivi-1 decreased cell survival rate and inhibited fission. The results indicated that ligustilide exerted neuro-protective effects by promoting mitochondrial fission and reducing cell damage. It preliminary proves that the mechanism of ligustilide on ischemic brain injury may be related to the promotion of mitochondrial fission and the maintenance of cell homeostasis.
4-Butyrolactone ; analogs & derivatives ; Animals ; Apoptosis ; Cell Survival ; Glucose ; Mitochondria ; Oxygen ; PC12 Cells ; Rats ; Reactive Oxygen Species ; Reperfusion Injury

Ten batches of Angelica sinensis from three producing areas( Tuoxiang,Minxian and Weiyuan of Gansu province) were selected as the research objects,and processed into raw A. sinensis,A. sinensis with alcohol,and A. sinensis with soil respectively through the standard processing methods. Ultra-high performance liquid chromatography( UPLC) was used to establish fingerprint for three processed products of A. sinensis,and determine the contents of 9 phenolic acids and phthalide compounds. The similarity was analyzed with Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine,which showed that the chromatographic peaks of the same processed samples of A. sinensis were basically similar,with all similarities greater than 0. 950. The difference between different processed products and their control spectra was not obvious,with all similarities also higher than 0. 950.On the basis of using principal component analysis( PCA) and OPLS-DA to seek the difference components between groups,the improved distance coefficient method can be used to effectively distinguish the three processed products of A. sinensis by fingerprint similarity. At the same time,the determination method of nine phenolic acids and phthalide in A. sinensis was established by UPLC,and the comparison between different processed products was carried out. The results showed that the content of various components was changed as compared with the raw A. sinensis. The contents of coniferyl ferulate and ligustilide in the A. sinensis with alcohol were increased significantly,and the content of coniferyl ferulate was obviously increased in A. sinensis with soil. The method established in this paper can effectively distinguish different processed products of A. sinensis and determine the content of the main components in them.
4-Butyrolactone ; analogs & derivatives ; analysis ; Angelica sinensis ; chemistry ; Benzofurans ; analysis ; Chromatography, High Pressure Liquid ; Coumaric Acids ; analysis ; Drugs, Chinese Herbal ; analysis ; Hydroxybenzoates ; analysis ; Medicine, Chinese Traditional ; Principal Component Analysis

Two new isomeric modified tripeptides, aspergillamides C and D (compounds 1 and 2), together with fifteen known compounds (compounds 3-17), were obtained from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008. The structures of the new compounds, including absolute configurations, were determined by extensive analyses of spectroscopic data (NMR, MS, UV, and IR) and comparisons between the calculated and experimental electronic circular dichroism (ECD) spectra. Butyrolactone I (compound 11) exhibited strong inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with the IC being 5.11 ± 0.53 μmol·L, and acted as a noncompetitive inhibitor based on kinetic analysis.
4-Butyrolactone ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Animals ; Aspergillus ; chemistry ; Chemistry Techniques, Analytical ; Dipeptides ; chemistry ; isolation & purification ; pharmacology ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; Indoles ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Mycobacterium tuberculosis ; drug effects ; Peptides ; chemistry ; isolation & purification ; pharmacology ; Polyketides ; chemistry ; isolation & purification ; pharmacology ; Porifera ; microbiology ; Protein Tyrosine Phosphatases ; chemistry

The present study carried out a phytochemical investigation on the root barks of Dictamnus dasycarpus Turcz, leading to the isolation and characterization of two new aromatic ring butyrolactone derivatives, dasycarpusphenol acid A (1) and dasycarpusphenol acid B (2). Their structures were elucidated by using spectroscopic techniques and HR-FAB-MS. Compounds 1 and 2 exhibited antioxidant activity, with their IC values being 28.95 and 41.76 mg·mL, respectively.
4-Butyrolactone ; chemistry ; isolation & purification ; Antioxidants ; chemistry ; isolation & purification ; Dictamnus ; chemistry ; Molecular Structure ; Plant Bark ; chemistry ; Plant Extracts ; chemistry ; isolation & purification

To investigate the neuroprotective of ligustilide (LIG) against glutamate-induced apoptosis of PC12 cells, cell viability were examined by MTT assay. Flow cytometry was applied to assay cell apoptosis rate. Intracellular calcium concentration was measured by using fluorescent dye Fluo-3/AM. Cytochrome C (Cyt C), Caspase-3, Bax and Bcl-2 protein expression were assayed by western blot. The results showed that glutamate is cytotoxic with an inhibitory concentration 50 (ID50) of 15 mmol · L(-1). Pretreatment with LIG (1, 5, 15 μmol · L(-1)) significantly improved cell viability. The apoptosis rate in glutamate-induced PC12 cells was 13.39%, and decreased in the presence of LIG (1, 5, 15 μmol · L(-1)) by 9.06%, 6.48%, 3.82%, separately. Extracellular accumulation of Ca2+ induced by glutamate were significantly reduced by LIG. The results of western blot manifested that pretreatment LIG could decrease the release of Cyt C from mitochondria, down-regulate Caspase-3 protein expression and up-regulate Bcl-2/Bax ratio, thereby protects PC12 cells from apoptosis. In summary, LIG had protective effect on glutamate-induced apoptosis in PC12 cells through attenuating the increase in intracellular Ca2+ concentration, and inhibiting the release of Cyt C from mitochondria to cytoplasm.
4-Butyrolactone ; analogs & derivatives ; pharmacology ; Aniline Compounds ; Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cell Survival ; Cytochromes c ; metabolism ; Glutamic Acid ; adverse effects ; Mitochondria ; metabolism ; PC12 Cells ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Xanthenes ; bcl-2-Associated X Protein ; metabolism

A quantitative analysis method of multi-components with a single marker (QAMS) for simultaneous determination of six marker compounds (one from phenolic acids and five from phthalides) in Chuanxiong Rhizoma was established by applying HPLC and using butylidenephthalide as the internal reference substance. And also the feasibility and accuracy of the established method for quality evaluation and application of Chuanxiong Rhizoma were investigated and validated. The analysis was performed with the mobile phase consisting of acetonitrile - 0.2% aqueous formic acid. The flow rate was 1.0 mL . min-1 and the column temperature was maintained at 30 °C. The detection wavelengths were set at 252 nm (for ferulic acid, Z-ligustilide, and butylidenephthalide) and 266 nm (for senkyunolide I, senkyunolide A, and coniferyl ferulate), separately, and 20 µL was injected for analysis with gradient elution. The results showed that there were no significant differences observed between the HPLC-QAMS method and the external standard method (RSD <5%). The relative correction factors were credible (RSD < 5%) in changed chromatographic conditions. The established HPLC-QAMS method can be accurately used for simultaneously evaluating and controlling the quality of Chuanxiong Rhizoma with multi-components.
4-Butyrolactone ; analogs & derivatives ; Acetonitriles ; Benzofurans ; Chromatography, High Pressure Liquid ; Coumaric Acids ; Drugs, Chinese Herbal ; analysis ; standards ; Hydroxybenzoates ; Quality Control ; Rhizome ; chemistry

Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradicate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing (QS), a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacterial population changes but also could react to environmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.
4-Butyrolactone ; analogs & derivatives ; chemistry ; metabolism ; Bacterial Proteins ; metabolism ; Iron ; chemistry ; metabolism ; Pseudomonas aeruginosa ; physiology ; Quorum Sensing ; physiology ; Signal Transduction ; Trans-Activators ; metabolism ; Virulence

A new γ-alkylated-γ-butyrolactone, named melongenolide A (1), along with nine known compounds were obtained from the roots of Solanum melongena, and their structures were identified as melongenolide A (1), (+)-syringaresinol (2), (+)-lyoniresinol (3), 5,5'-dimethoxy lariciresinol (4), (+)-(7R,8R)-4-hydroxy-3,3',5'-trimethoxy-8',9'-dinor-8,4'-oxyneoligna-7, 9-diol-7'-aldehyde (5), kaempferol-3-O-(2″,6″-di-O-p-trans-coumaroyl)-β-glucoside (6), arjunolic acid (7), vanillic acid (8), scoparone (9), and β-sitosterol (10). Compounds 2, 6, and 7 showed potent inhibitory effects on nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages, with IC50 values being 5.62 ± 0.86, 11.47 ± 0.98, and 27.75 ± 1.26 μmol·L(-1), respectively.
4-Butyrolactone ; analogs & derivatives ; isolation & purification ; Animals ; Furans ; isolation & purification ; pharmacology ; Inflammation ; drug therapy ; metabolism ; Inhibitory Concentration 50 ; Kaempferols ; isolation & purification ; pharmacology ; Lignans ; isolation & purification ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; Nitric Oxide ; metabolism ; Plant Extracts ; chemistry ; pharmacology ; therapeutic use ; Plant Roots ; chemistry ; RAW 264.7 Cells ; Solanum melongena ; chemistry ; Triterpenes ; isolation & purification ; pharmacology