Identification of active ingredients mediating anti-platelet aggregation effects of BuyangHuanwu decoction using a platelet binding assay, solid phase extraction, and HPLC-MS/MS.

BuyangHuanwu decoction (BHD) is widely used as a traditional herbal medicine because of its antithrombotic effect, which is attributed to the inhibition of platelet aggregation; however, its active compounds remain unknown. In this study, we developed a method involving platelet binding, solid-phase extraction, and HPLC-MS/MS for screening BHD compounds with potential anti-platelet aggregation properties. Five compounds showing platelet binding affinity were identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-ß-d-glucoside, galloylpaeoniflorin, and formononetin-7-O-ß-d-glucoside. The results of anti-platelet aggregation experiments in vitro confirmed that these compounds inhibited adenosine diphosphate-induced platelet aggregation. Our results suggest that a platelet binding assay combined with solid-phase extraction and HPLC-MS/MS is an effective method for screening anti-platelet aggregation agents in traditional Chinese medicines.

Biospecific isolation and characterization of angiogenesis-promoting ingredients in Buyang Huanwu decoction using affinity chromatography on rat brain microvascular endothelial cells combined with solid-phase extraction, and HPLC-MS/MS.

Buyang Huanwu decoction (BHD) was reported to exert angiogenesis-promoting effects, but its active ingredients remain unknown. In this study, we developed a method to screen potential angiogenesis-promoting compounds in BHD, which involved biospecific isolation using live rat brain microvascular endothelial cells (rBMECs) and characterization using solid-phase extraction (SPE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Six compounds showed binding affinity to rBMECs and were further identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-ß-D-glucoside, galloylpaeoniflorin, formononetin-7-O-ß-D-glucoside, and (3R)-7,2'-hydroxy-3',4'-dimethoxy-isoflavan. The results indicated that five of them except 6-hydroxykaempferol-di-O-glucoside showed a protective effect against oxygen glucose deprivation/reperfusion injury in rBMECs and upregulated the secretion of vascular endothelial growth factor and basic fibroblast growth factor, suggesting a mechanism underlying their angiogenic activity. Our findings suggest that biospecific live cell-based isolation combined with SPE and HPLC-MS/MS is an effective method for screening potential bioactive components in traditional Chinese medicines.

[Three methods for controlling presacral massive bleeding during pelvic operations].

OBJECTIVE: To evaluate three different methods for controlling presacral massive bleeding during pelvic operations. METHODS: Clinical data of 11 patients with presacral massive bleeding during pelvic operation at The Sixth Affiliated Hospital of Sun Yat-sen University and 157 Branch Hospital of Guangzhou General Hospital of Guangzhou Military Command from January 2001 to January 2016 were analyzed retrospectively. Hemostasis methods for presacral massive bleeding during operation included gauze packing (whole pressure), drawing pin (local pressure) and absorbable gauze (absorbable gauze was adhered to bleeding position with medical glue after local pressure). Efficacy of these 3 methods for controlling bleeding was evaluated and compared. RESULTS: Ten patients were male and 1 was female with average age of 65.2 (40 to 79) years old. Eight cases were rectal cancer, 2 were presacral malignancies and 1 was rectal benign lesion. Bleeding volume during operation was 300 to 2 500 (median 800) ml. From 2001 to 2012, 4 cases received gauze packing, of whom, 3 cases were scheduled Dixon resection before operation and then had to be referred to Hartman resection; 3 cases died of systemic failure due to postoperative chronic errhysis and infection, and 1 underwent re-operation. At the same time from 2001 to 2012, 5 cases received drawing pin, of whom, bleeding of 3 cases was successfully controlled and Dixon resection was completed. In other 2 cases with hemostasis failure, 1 case underwent re-operation following the use of gauze packing, and another 1 case received absorbable gauze hemostasis. All the 5 patients were healing. From 2013 to 2016, 2 cases completed scheduled anterior resection of rectum after successful hemostasis with absorbable gauze and were healing and discharged. CONCLUSIONS: Gauze packing hemostasis is a basic method for controlling presacral massive bleeding. Drawing pin and absorbable gauze hemostasis are more precise and may avoid the change of surgical procedure. But drawing pin has the possibility of hemostasis failure. Absorbable gauze hemostasis with medical adhesive is effective, simple and fast.

Effect of stimulating the acupoints Feishu (BL 13) and Dazhui (GV 14) on transdermal uptake of sinapine thiocyanate in asthma gel.

OBJECTIVE: To investigate the effect of stimulating the acupoints Feishu (BL 13) and Dazhui (GV 14) on the transdermal uptake of sinapine thiocyanate contained in a gel used for the management of asthma. METHODS: Thirty Sprague-Dawley rats were randomly divided into three equal groups using a random number table: the Feishu (BL 13) acupoint group, the Dazhui (GV 14) acupoint group, and the nonacupoint group or control group. Using microdialysis technology, preprocessed skin probes were implanted into the rats at Feishu (BL 13), Dazhui (GV 14), and a nonacupoint site. Asthma gel was then placed on the skin at Feishu (BL 13), Dazhui (GV 14) acupoints, and the nonacupoint for all groups. Dialysate was collected every 30 min for 12 h. The normalized concentration of sinapine thiocyanate in the skin was determined by high-performance liquid chromatography. RESULTS: The rat in vivo transdermal experiment demonstrated that the quantity-time equation showed a good linear correlation with zero-order kinetics (r > 0.99). The transdermal behavior was in accordance with the first-order rate open model in which the transdermal penetration rates and the accumulative amounts of sinapine thiocyanate in the skin at the acupoint sites were greater than those through the skin of the nonacupoint site. The systemic maximum concentration and the area under the curve of sinapine thiocyanate in the acupoint groups were significantly greater than in the nonacupoint group. A lag time was observed in both acupoint groups, but not in the nonacupoint group. CONCLUSION: Stimulating the acupoints promotes the percutaneous absorption of sinapine thiocyanate and also controls its release, reducing concentration fluctuations in the blood.

Effect of ferulic acid on the brain pharmacokinetics of tetramethylpyrazine in conscious rats.

1. In traditional Chinese medicine, Angelica sinensis is often coprescribed with Ligusticum chuanxiong Hort for the treatment of ischemic cerebrovascular diseases. Tetramethylpyrazine (TMP) is one of the most important active ingredients isolated from Ligusticum chuanxiong Hort; ferulic acid (FA) is the main water-soluble component of Angelica sinensis. 2. The purpose of this study is to investigate the possible effect of FA on the brain pharmacokinetics of TMP in conscious Sprague-Dawley rats. The pharmacokinetic parameters of TMP were investigated in brain microdialysates after oral and intravenous administration of TMP (4 mg/kg) to rats in the absence and presence of FA (5 mg/kg). Samples were collected at timed intervals for the measurement of TMP by a rapid and sensitive UPLC-MS/MS method. 3. The pharmacokinetic parameters were calculated by noncompartmental analysis for brain microdialysates. The brain pharmacokinetic data for TMP showed significant increases in Cmax, t1/2, AUC0-inf and MRT0-inf after combination with FA. After intragastric administration with FA, there were significant decreases in the Tmax (from 38.33 ± 5.77 to 21 ± 5.48 min; p < 0.01) of TMP. This study indicated that potential drug-drug interaction between TMP and FA should be taken into consideration and the combined administration is beneficial in improving the bioavailability of TMP in the brain.

Microdialysis combined with UPLC-MS/MS method for determination of tetramethylpyrazine and ferulic acid in striatum of awake and anesthetic rats subjected to cerebral ischemia.

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous determination and pharmacokinetic investigation of Tetramethylpyrazine (TMP) and Ferulic acid (FA) in rat striatum. The method was validated over the concentration range of 1.15-505ng/mL for TMP and 3.23-101ng/mL for FA, with a lower limit of quantitation (LLOQ) of 1.15ng/mL and 3.23ng/mL, respectively. This method can be successfully applied in pharmacokinetic studies of TMP and FA in striatum of awake and anesthetic rats. The cerebral blood flow velocity (CBF) during middle cerebral artery occlusion (MCAO) was monitored by Laser speckle contrast imaging, to observe whether the compatibility of TMP and FA could improve CBF against cerebral ischemia/reperfusion (I/R) injury. Infarct volume was examined to evaluate severity of ischemic brain injury. The pharmacokinetic study indicated that T1/2, Cmax, MRT and AUC0-inf were changed after combined administration of TMP and FA, when compared with either drug alone both in awake and anesthetic groups. The pharmacodynamics results showed that co-administration of drugs could enhance the CBF during middle cerebral artery occlusion and reduced the infarct volume. Taken together, the compatibility treatment of TMP and FA might be a promising therapeutic strategy for ischemic stroke. Further study is required to optimize the compatibility proportion.

Nogo-p4 Suppresses TrkA Signaling Induced by Low Concentrations of Nerve Growth Factor Through NgR1 in Differentiated PC12 Cells.

BACKGROUND: Regeneration of injured axons in adult mammalian central nervous system (CNS) is not spontaneous. Nogo is a major inhibitory molecule contributing to axon regeneration failure. The molecular mechanisms of Nogo inhibition of axon regeneration are not completely understood. To further investigate the underlying mechanisms, we studied the effects of Nogo-p4, a 25-amino acid core inhibitory fragment of Nogo, on nerve growth factor (NGF)-induced TrkA signaling. METHODS: NGF-differentiated PC12 cells were used as cell models. The effects of Nogo-p4 on two key components of TrkA signaling, phosphorylated Erk1/2 and Akt, were analyzed by western blot. Co-immunoprecipitation experiments were performed to detect the formation of NgR1/p75 complexes. Neurite growth was quantified by measuring the neurite length. RESULTS: Nogo-p4 did not significantly affect TrkA signaling induced by 100 ng/ml NGF, but signaling was suppressed when an NGF concentration of 5 ng/ml was used. Further investigation demonstrated that Nogo-p4 affected TrkA signaling in an NGF concentration-dependent manner. Nogo-p4 suppression of TrkA signaling was strong at low (1 and 5 ng/ml), moderate at intermediate (25 ng/ml), but absent at high (50 and 100 ng/ml) NGF concentrations. NEP1-40 attenuated, and NgR1 overexpression enhanced, Nogo-p4 suppression of TrkA signaling induced by low concentrations of NGF. High but not low concentrations of NGF reduced the formation of NgR1/p75 complexes triggered by Nogo-p4. Nogo-p4 strongly inhibited neurite growth induced by low rather than high concentrations of NGF. CONCLUSION: Nogo-p4 binding with NgR1 suppresses TrkA signaling induced by low concentrations of NGF in differentiated PC12 cells. Suppression of NGF-induced TrkA signaling may be another mechanism by which Nogo inhibits neurite growth.

Optimization and evaluation of pluronic lecithin organogels as a transdermal delivery vehicle for sinomenine.

The purpose of the present study was to prepare and optimize sinomenine (SIN) pluronic lecithin organogels system (PLO), and to evaluate the permeability of the optimized PLO in vitro and in vivo. Box-Behnken design was used to optimize the PLO and the optimized formulation was pluronic F127 of 19.61%, lecithin of 3.60% and SIN of 1.27%. The formulation was evaluated its skin permeation and drug deposition both in vitro and in vivo compared with gel. Permeation and deposition studies of PLO were carried out with Franz diffusion cells in vitro and with microdialysis in vivo. In vitro studies, permeation rate (Jss) of SIN from PLO was 146.55 ± 2.93 µg/cm(2)/h, significantly higher than that of gel (120.39 µg/cm(2)/h) and the amount of SIN deposited in skin from the PLO was 10.08 ± 0.86 µg/cm(2), significantly larger than that from gel (6.01 ± 0.04 µg/cm(2)). In vivo skin microdialysis studies showed that the maximum concentration (Cmax) of SIN from PLO in "permeation study" and "drug-deposition study" were 150.27 ± 20.85 µg/ml and 67.95 µg/ml, respectively, both significantly higher than that of SIN from gel (29.66 and 6.73 µg/ml). The results recommend that PLO can be used as an advantageous transdermal delivery vehicle to enhance the permeation and skin deposition of SIN.

[Study on recovery and its influencing factors of ferulic acid and tetramethylpyrazine in cerebral microdialysis probe].

To establish a method for detecting microdialysis recovery of tetramethylpyrazine (TMP) and ferulic acid (FA) and investigating the influencing factors, providing the basis for further in vivo microdialysis experiments. The concentration of FA and TMP in dialysates were determined by high pressure liquid chromatography ( HPLC) and probe recovery were calculated respectively. The influence of the flow rates, medium concentration, temperature and in vivo probe stability on the recovery of FA and TMP were investigated by using concentration difference method (incremental method and decrement method). The recovery obtained by incremental method were similar to by decrement method. The in vitro recovery rate of FA and TMP decreased with the increase of 1-2.5 µL min(-1), and increased obviously with the temperature of 25-42 degrees C under the same conditions. The concentration of FA and TMP had no obvious effect on the probe recovery under the same flow rate. In addition, the recovery of TMP and FA remained stable and showed similar trends under the condition of four concentration cycles, indicating that the intra day reproducibility of the concentration difference method was good. The recovery of brain microdialysis probes in vivo 8 h maintained a relatively stable, but certain differences existed between different brain microdialysis probes, demonstrating that each probe was required for recovery correction in vivo experiment. Microdialysis sampling can be used for the local brain pharmacokinetic study of FA and TMP, and retrodialysis method can be used in probe recovery of FA and TMP in vivo.

The influences of pH and ionic strength on the sorption of tylosin on goethite.

As one of the widely used antibiotics in the world, the environmental risks of tylosin (TYL) received more and more attention. In order to assess its environmental fate and ecological effects accurately, it is necessary to understand the sorption properties of TYL on the soils/sediments. The sorption of TYL on goethite at different pH and ionic strength conditions were measured through a series of batch experiments and the sorption data of TYL were fitted by Freundlich and dual-mode sorption models. It was obvious that sorption was strongly dependent on pH and ionic strength. Sorption capacity of TYL increased as the pH increased and ionic strength decreased. The pH and ionic strength-dependent trends might be related with complexation between cationic/neutral TYL species and goethite. The sorption affinity of TYL on goethite decreased as ionic strength increased, which only occurred at higher TYL concentrations, suggested that inner complex might have dominated process at low concentrations and outer complex might occur at higher concentrations of TYL. Spectroscopic evidence indicated that tricarbonylamide and hydroxyl functional groups of TYL might be accounted for the sorption on mineral surfaces. The experimental data of TYL sorption could be fitted by surface complexation model (FITEQL), indicating that ≡FeOH with TYL interaction could be reasonably represented as a complex formation of a monoacid with discrete sites on goethite. The sorption mechanism of TYL might be related with surface complexation, electrostatic repulsion, and H-bounding on goethite. It should be noticed that the heterogeneous of sorption affinity of TYL on goethite at various environment to assess its environment risk.

[Effect of peritoneal lavage with povine-iodine on prevention of postoperative sepsis after exposure of peritoneal cavity to sea water in rat].

OBJECTIVE: To observe the effects of peritoneal lavage with povine-iodine on prevention of sepsis after exposure of peritoneal cavity to sea water in rat. METHODS: Eighty-four SD rats were randomly divided into two groups, and the peritoneal cavity was exposed to sea water. Rats in group A were not treated (group A, n=42), and the peritoneal cavity was lavage with povine-iodine in group B (n=42). Plasma levels of endotoxin and tumor necrosis factor (TNF) were measured preimmersion, and 0, 12, 24 hours after seawater immersion (n=6), and positive incidence of blood bacterial culture was performed (n=18 in each group) in groups A and B. RESULTS: 1. Plasma levels of endotoxin and TNF in group A and B were increased significantly after exposure of peritoneal cavity to sea water (compared with baseline values, all P<0.05). Plasma levels of endotoxin and TNF in group B became lower than those in group A from 12 hours after seawater immersion (P<0.05 or P<0.01). 2. Positive incidence of bacterial culture in group B was 16.7 % (3/18) and it was lower than that in group A (77.8 % (14/18), P<0.01). CONCLUSION: Povine-iodine lavage in the peritoneal cavity can reduce levels of plasma endotoxin and TNF, and lower positive incidence of bacterial culture in rats after exposure of peritoneal cavity to sea water, thereby preventing the development of postoperative sepremia.

[Effect of hypotonic liquid containing colloid on hemorrhagic dogs with celiac seawater immersion wound].

OBJECTIVE: To determine hypotonic liquid containing colloid on hemorrhagic dogs with celiac seawater immersion wound. METHODS: Thirty-five dogs were randomly divided into five groups: control group (group A), 0.9% NaCl therapy group (group B) and 0.45% NaCl therapy group (group C), 5% glucose therapy group(group D) and hypotonic liquid containing colloid therapy group (group E). Changes of mean artery pressure (MAP), cardiac output (CO), urine volume, plasma osmotic pressure and pathologic changes of lung and brain were observed. RESULTS: hypotonic liquid containing colloid could effectively ameliorate MAP and CO, improve urine volume, decrease plasma osmotic pressure and prevent occurrence of lung and brain edema. CONCLUSION: Hypotonic liquid containing colloid can exert satisfactory therapeutic effects on hemorrhagic dogs with celiac seawater immersion wound.

Consequence alimentary reconstruction in nutritional status after total gastrectomy for gastric cancer.

AIM:To investigate the effect of gastroenteric reconstruction on the nutritional status of patients with gastric cancer after total gastrectomy.METHODS: From 1989-1994, nutritional status was studied in 24 patients, including 12 patients with the gastric reservoir and pyloric sphincter reconstruction (GRPS), 7 with Braun's esophago-jejunostomy (EJ) and 5 with Lawrance's Roux en Y reconstruction (RY).The ability of these patients to ingest and absorb the amount of nutrients was examined and compared, and metabolic balance test was performed to compare the efficiency of those patients to accumulate and use the absorbed nutrients.RESULTS:In the controlled hospital situation, the amount of food ingested by all the patients was greater than that required for maintenance of ideal body weight. In direct contrast, food intake in most patients with EJ or RY reconstruction significantly decreased when the patients returned home and that in EJ patients it was the lowest. The overgrowth of anaerobic bacteria was found in the jejunum in the patients with EJ and RY, due mainly to food stasis in the duodenum or in the Roux limb,caused by the operative procedure itself. In patients with GRPS,because of restoring of the alimentary continuity according to the normal digestive physiologic characters, all the nutritional parameters could fall in the normal range.CONCLUSION:The most common mechanism responsible for postoperative malnutrition was inadequate food intake. Having solved the problem of alkaline reflux esophagitis, it is imperative to preserve the duodenal food passage to reduce malabsorption and other complications after total gastrectomy.