Aim: To develop and compare the ultra-performance liquid chromatography( UPLC) and HPLC methods for the determination of dihydroflavonoids in Smilacis glabrae Rhizoma, and establish the quality evaluation system of the above-mentioned crude drug. Methods: Four dihydroflavonoids in the crude drugs collected from 15 localities were determined using the UPLC and HPLC methods, respectively. The resolution, sensitivity, precision, accuracy and the content determination results of the four compounds were compared between the two methods. Results: The UPLC method was more fast and sensitive than the HPLC method with no significant differences among the linearity range, precision, accuracy and the content determination results between the two methods. Conclusion: The developed HPLC method was proved practicable and reliable for the quality control of Smilacis glabrae Rhizoma. The UPLC method was provided to be a more sensitive, fast and solvent-saving method compared to HPLC and can be applied in the quality evaluation of Chinese medicines.

Investigation of the pharmacokinetics of paeonol microemulsion, microemulsion-based gels and marketed paeonol ointments by the skin-blood synchronous microdialysis coupled with LC/MS is reported in this study. The microdialysis systems were established by linear probes and concentric circles probes. In vivo recovery of paeonol in skin is (69.7 +/- 4.8) % and in blood is (51.6 +/- 7.2)%. The paeonol microemulsion, microemulsion-based gels and marketed paeonol ointments were administered to rats. PBS (pH 7.4) served as perfused solution. The perfusion rate was 5 microL x mL(-1) and the microdialysis samples were collected every 20 min intervals. The paeonol concentration in perfused solution was determined by LC/MS. The results showed that paeonol microemulsion and microemulsion-based gels significantly raised the drug concentrations in skin more than that of paeonol ointments. The paeonol microemulsion-based gels has similar bioavailability as the paeonol ointments in blood, but its blood drug concentrations were steadier. The paeonol microemulsion-based gels may be developed into a new preparation for dermis eczema. The skin-blood synchronous microdialysis technique proved to be a new method for the pharmacokinetics study of transdermal delivery systems.

Objective To observe the effect of heat stress-induced reactive oxygen species (ROS) burst on the regulation of expression of Bcl-2 and Bax in human umbilical vein endothelial cell (HUVEC) apoptosis induced by heat stress,and explore the pathogenesis of vascular endothelial damage caused by severe heat stroke.Methods HUVEC heat stress model was reproduced.Cells of heat stress group were incubated at either 39,41,or 43 ℃ for 2 hours,then all the cells were further incubated at 37 ℃ and 5％ CO2 for 24 hours.Before heat stress,cells of 43 ℃ heat stress group were pretreated with 10 μmol/L MnTMPyP,which was a specific scavenger of ROS,for 1 hour.Cells of control group were incubated at 37 ℃ and 5％ CO2.The amount of ROS was assayed with 2',7'-dichlorofluorescin diacetate (DCFH-DA) and dihydroethidium (DHE) staining.Apoptosis was determined by using staining with Hoechst33258.The mRNA expressions of Bcl-2 and Bax were determined by reverse transcription-polymerase chain reaction (RT-PCR).The protein levels of Bcl-2,Bax,caspase-3 were analyzed by Western Blot.In addition,the effect of MnTMPyP on heat stress-induced apoptosis was also studied.Results Compared with control group,there was no obvious change in cells after 39 ℃ heat stress.With the increase in heat stress temperature up to 41 ℃ and 43 ℃,viability of cells showed a lowering trend,with a burst of ROS,and an increase of mRNA and protein of Bax,and the protein of caspase-3 was significantly increased,the mRNA and protein of Bcl-2 were significantly decreased in a temperature-dependent manner.These changes were marked in 43 ℃ heat stress group as compared with those of the control group [cell viability:(46.00 ±4.00)％ vs.(96.33 ± 1.53)％,t=20.164,P=0.001; ROS (fluorescence relative value):400.67 ± 12.10 vs.99.33 ±4.04,t=32.909,P=0.001; Bax mRNA (A value):3.03 ±0.15 vs.1.00 ± 0.00,t=23.056,P=0.001; Bax protein (gray value):3.97 ±0.21 vs.1.00 ± 0.00,t=24.684,P=0.001; caspase-3 protein (gray value):4.80 ± 0.20 vs.1.00 ± 0.00,t=32.909,P=0.001 ; Bcl-2 mRNA(A value):0.42 ± 0.30 vs.1.00 ± 0.00,t=33.072,P=0.001 ; Bcl-2 protein (gray value):0.39 ± 0.25 vs.1.00 ± 0.00,t=42.212,P=0.001].It was shown that pre-condition with the antioxidant MnTMPyP significantly decreased the heat stress-induced expression of Bax,caspase-3,and apoptosis,and the expression of Bcl-2 was elevated [Bax mRNA (A value):2.00 ± 0.20 vs.3.33 ±0.25,t=7.184,P=0.002; Bax protein (gray value):2.03 ±0.25 vs.3.23 ±0.25,t=5.840,P=0.004; caspase-3 protein (gray value):2.07 ± 0.21 vs.5.00 ± 0.20,t=17.600,P=0.001 ; Bcl-2 mRNA(A value):0.71 ± 0.40 vs.0.42 ± 0.26,t=8.126,P=0.002; Bcl-2 protein (gray value):0.57 ± 0.31 vs.0.40 ± 0.06,t=5.091,P=0.007].Conclusions A burst in an increase of ROS plays an important role on heat stress-induced HUVEC apoptosis,and the mechanism is probably related to the expressions of Bcl-2 and Bax.The vascular endothelial cells apoptosis may be one of the pathogenetic factor in severe heat stroke.

Results With the prolonged exposure to heat , the mice exhibited swollen and disorderly arranged neurons , shrunken cells , and contracted and deeply stained nuclei , with significantly higher scores on nerve pathological injury evaluation at 6, 12, and 24 h (2.78 ± 0.71, 3.21 ±0.56, and 3.36 ±0.63) than the control mice (0.43 ±0.10) (P<0.05).ELISA showed remarkably elevated levels of UCH-L1 in the serum (F=147.7, P=0.05) and brain tissue (F=145.7, P=0.05) in the heat stress group as compared with the con-trol, and Western blot also revealed a markedly higher expression of UCH-L1 in the brain tissue in the former group than in the latterObjective The abnormal expression of ubiquitin C-terminal hydrolase-L1 ( UCH-L1 ) has an important role in the diagnosis and prognosis of brain damage .This study was to investigate the changes of UCH-L1 in the serum and brain tissue in the mouse model of heat stress . Methods Twelve BALB/c mice were randomly divided into a control and a heat stress group of equal number, the former placed at a temperature of (25.0 ±0.5)℃and a relative humidity of (35 ±5)%and the latter in a simulated in-cubator at (35.5 ±0.5)℃and a relative humidity of (60 ±5)%.When the rectal temperature reached 42℃, the animals were re-moved from the incubator and cooled at an ambient temperature of (25.0 ±0.5)℃and a humidity of (35 ±5)%for 0, 6, 12, and 24 h.Then the brain tissues of all the animals were harvested for HE staining , evaluation of neuronal injury under the light microscope , measurement of the UCH-L1 levels in the serum and brain tissue by ELISA , Western blot, and immunohistochemistry , respectively. (F=261.2, P=0.01).Immunohistochemistry manifested that , with the prolonged exposure to heat , the UCH-L1 expression in the brain tissue was characterized by gradually increased light brown of the neurons at staining . Conclusion Severe heatstroke causes brain injury in a time-dependent manner , and the abnormally elevated levels of UCH-L1 in the serum and brain tissue can be a marker of heatstroke-induced brain injury .

Objective To observe the expressions of cytochrome C,Caspase-9,Caspase-3 and their relationships,and investigate apoptosis signal transduction mechanism after heat stress-induction in human umbilical vein endothelial cell (HUVEC),and explore pathogenesis of vascular endothelial damage in the wake of severe heat stroke.Methods Human umbilical vein endothelial cell heat stress model was set up.Control group were incubated at 37℃,while heat stress group of cells were incubated at 39℃,41℃,and 43℃ for 2h,then all the cells were further incubated at 37℃ for 24 h.Mitochondria of human umbilical vein endothelial cell were examined with electron microscopy.Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining,and protein levels of cytochrome C,caspase-9,caspase-3 were analyzed by western blot.Results In the control group (37℃),the structure of mitochondrial was intact in HUVEC under transmission electron microscope.In contrast,mitochondrial swelling was found in the group of 43℃ heat stress.Compared with control group,as increasing in heat stress temperature,the rates of induced apoptosis were 17.8％ at 41℃ and 25.6％ at 43℃,and the levels of cytochrome C,Caspase-9,and caspase-3 were significantly increased (P ＜0.05).There was no obvious change at 39℃ heat stress (P ＞ 0.05).Conclusions Mitochondrial apoptosis pathway is involved in apoptosis of human umbilical vein endothelial cells in the wake of heat stress.The vascular endothelial cells apoptosis may be associated with the occurrence of severe heat stroke.

Objective To establish an ultra-high performance liquid chromatographic fingerprinting analysis method for the hydrophilic component of salvianolic acids and the lipophilic component of tanshinones in Radix Salviae Miltiorrhizae(RSM),and to supply a rapid and scientific method for quality control of RSM.Methods The chromatographic conditions were as follows: Acquity C18 BEH(2.1mm?100 mm,1.7 ?m)chromatographic column at 30℃,gradient elution with acetonitrile(A) and 0.4% formic acid(B)(90%~50% A for 0~10 min,75% A for 15 min),the flow rate being 0.5 mL?min-1 and UV detection wavelength set at 280nm.Results The chromatograms of the hydrophilic component and the lipophilic component were obtained within 15min,peak volume being 85.There were 20 peaks separated completely and identified by using HPLC-diode array detection-electrospray ionization-mass spectrometry.Conclusion The developed method is proved practicable,rapid and reliable for quality control of RSM and its preparations.

Objective To explore the effect of activation of caspase-3 on apoptosis of human umbilical vein endothelial cells induced by heat stress. Methods A model of human umbilical vein endothelial cell heat stress was established. The endothelial cells in the control group were incubated at 37 ℃, while the cells in the heat stress group were incubated at 39 ℃, 41 ℃, or 43 ℃ for 2 h , then all the cells were further incubated at 37 ℃ for 24 h. Apoptosis was detected using staining with Hoechst33258; protein levels of caspase-3 were analyzed by Western blot; and the effect of caspase-3 inhibitor Z-DEVD-FMK on heat stress-induced apoptosis was determined. Results As compared with the control group, with an elevation in heat stress temperature (41℃ to 43 ℃), apoptosis was markedly increased and level of caspase-3 was significantly increased. In addition , caspase-3 inhibitor Z-DEVD-FMK significantly decreased the heat stress-induced apoptosis and levels of caspase-3. Conclusions Caspase-3 mediates apoptosis of human umbilical vein endothelial cells can be induced by heat stress.

OBJECTIVETo establish a high performance liquid chromatographic method for the determination of kirenol in Herba Siegesbeckiae Praeparata.

METHODThe analysis was carried out on a Boston Crest ODS column (4.6 mm x 250 mm, 5 microm) with gradient elution using acetonitrile-water as mobile phases. The flow rate was 1.0 mL x min(-1) and the detection wavelength was at 215 nm.

RESULTThe calibration curve was linear over the range of 0.0202-20.02 microg for kirenol. The correlation coefficient of the calibration curve was 0.99975. The average recovery was 99.62% with relative standard derivation (RSD) of 2.0%.

CONCLUSIONThe results showed that the method is simple, accurate and repeatable and it is suitable for the determination of kirenol in Herba Siegesbeckiae Praeparata.

Acetonitriles ; chemistry ; Adenosine ; analysis ; Asteraceae ; chemistry ; Carcinogens ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Quality Control ; Sweetening Agents ; analysis

Objective To evaluate the effect of propofol (PPF) on stress response and lung injury in rats with traumatic brain injury (TBI).Methods A total of 36 SD rats were randomly (random number)divided into sham group,intralipid group,TBI group,PPF1 h group,PPF 2 h group,PPF 6 h group (n =6 in each group).Fluid percussion brain injury models were used.By intraperitoneal injection,intralipid was administered in intralipid group after sham operations,while propofol 100 mg · kg-1 was given to rats in PPF1 h group,PPF 2 h group and PPF 6 h group 1,2,6 hours following injury,respectively.Nerve motor function were scored at different intervals,serum concentrations of adrenocorticotropic hormone (ACTH),cortisol (COR) and norepinephrine (NE) were measured 12 h after injury.Seventy-two hours later,all rats were sacrificed and brains were harvested for TTC staining,and lungs taken were stained with HE staining for observation under light microscope and electron microscopy.Results Compared with sham and intralipid group,nerve motor function scores were significantly decreased,and serum concentrations of ACTH,COR and NE were increased significantly in rats after injury.Compared with TBI group,the above biomarkers were improved significantly after propofol injection.There were no significant differences in above biomarkersbetweenPPF 1 hand PPF 2 h group (t=-0.816,t=-0.208,t=0.582,P＞0.05).The differences in COR and NE concentrations between PPF 2 h and PPF 6 h group were statistically significant (t =3.018,P =0.013;t =3.662,P =0.004).Light microscopy demonstrated abundant inflammatory cell infiltration and massive thickening of the alveolar walls,Electron microscopy showed Type Ⅱ lung epithelial cell swelling,vacuolar degeneration,osmiophilic lamellar corpuscle emptying in cytoplasm,microvilli protrusions decreases in some cytoplasm in TBI group,and pathological damage was improved significantly in PPF 2 h group.Conclusions Propofol may inhibit stress and protect the lung tissue from damage in TBI rats.

OBJECTIVETo investigate the representation methods of dissolubility property of total alkaloid extract from Peganum hamala in different solvents, and to investigate the evaluation method of the dissolubility property of extracts from traditional Chinese medicine.

METHODThe dissolubility property of the whole extract and markers of harmaline and harmine, as well as the particle diameter distribution of the extract in different solvents were evaluated by precipitation method, solubility test, and the particle diameter test.

RESULTBoth the alkaloid extract and it's index ingredients had good solubility in absolute ethanol, 95% ethanol, and 80% ethanol, while the solubility in 60% ethanol was poor, and worst in water. The sequence of particle diameter of extract in solvents was in the following order water > 95% ethanol > 60% ethanol > 60% ethanol > 80% ethanol.

CONCLUSIONThe extract has good solubility in the ethanol solution whose concentration is over 80%. The results between precipitation method and index components method have certain correlation. The particle diameter method can provide distribution information of the extract in different solvents. Combination of those three methods could reflect the dissolubility property of extracts from traditional Chinese medicine more comprehensively.

Alkaloids ; chemistry ; isolation & purification ; Chemical Fractionation ; methods ; Particle Size ; Peganum ; chemistry ; Plant Extracts ; chemistry ; isolation & purification ; Solubility ; Solvents ; chemistry