Comparative pharmacokinetics study of leonurine and stachydrine in normal rats and rats with cold-stagnation and blood-stasis primary dysmenorrhoea after the administration of Leonurus japonicus houtt electuary.

Leonurus japonicus houtt, a well-known herb of traditional Chinese medicine, is widely used to treat gynaecological diseases. In this study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method for simultaneously quantifying leonurine and stachydrine, the two main bioactive components in Leonurus japonicus houtt, was developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile and separation by a Hewlett Packard XDB-C8 column (150 × 4.6 mm, id, 5 µm) equipped with a gradient elution system containing methanol-water and 0.1% formic acid at a flow-rate of 0.4 mL/min. Components were then detected by a mass spectrometer in positive electrospray ionization mode. This method showed good linearity, precision, accuracy, recovery, stability, and negligible matrix effects, which were within acceptable ranges. The method was successfully applied to compare the pharmacokinetics in normal rats and rats with cold-stagnation and blood-stasis primary dysmenorrhoea treated with Leonurus japonicus houtt electuary. The result showed significant differences (p < 0.05) in the pharmacokinetic parameters between the primary dysmenorrhoea and normal groups. This result implied that Leonurus japonicus houtt electuary remained longer and was absorbed slower in rats with primary dysmenorrhoea and exhibited higher bioavailability and peak concentration.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. METHODS: Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. RESULTS: Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. CONCLUSIONS: Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

Flow Cytometry Method Analysis of Apoptosis: No Significant Difference Between EDTA and EDTA-free Trypsin Treatment Procedure.

Flow cytometry method (FCM) is a generally accepted tool to analyze apoptosis. Although apoptosis assay kit was applied by many companies, the manufacturers were not consistent with whether using Trypsin with EDTA to collect the adherent cells. In another words, the influence of EDTA on apoptotic ratio is not clear. In this work, we compared the proportion of apoptotic cells with EDTA or EDTA-free Trypsin treatment by FCM. We concluded that Trypsin with or without EDTA has little influence on the proportion of apoptotic cells. In addition, we found that the ratio of necrosis and apoptosis was different in cells collected by scraping. WAVE2 protein was analyzed as a typical example for movement related protein. WAVE2 expression is elevated in the EDTA Trypsin treated group, compared with EDTA-free Trypsin treatment and scrapping group.

Herb-drug interaction prediction based on the high specific inhibition of andrographolide derivatives towards UDP-glucuronosyltransferase (UGT) 2B7.

Herb-drug interaction strongly limits the clinical application of herbs and drugs, and the inhibition of herbal components towards important drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reasons. The present study aims to investigate the inhibition potential of andrographolide derivatives towards one of the most important phase II DMEs UDP-glucuronosyltransferases (UGTs). Recombinant UGT isoforms (except UGT1A4)-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation were employed to firstly screen the andrographolide derivatives' inhibition potential. High specific inhibition of andrographolide derivatives towards UGT2B7 was observed. The inhibition type and parameters (Ki) were determined for the compounds exhibiting strong inhibition capability towards UGT2B7, and human liver microsome (HLMs)-catalyzed zidovudine (AZT) glucuronidation probe reaction was used to furtherly confirm the inhibition behavior. In combination of inhibition parameters (Ki) and in vivo concentration of andrographolide and dehydroandrographolide, the potential in vivo inhibition magnitude was predicted. Additionally, both the in vitro inhibition data and computational modeling results provide important information for the modification of andrographolide derivatives as selective inhibitors of UGT2B7. Taken together, data obtained from the present study indicated the potential herb-drug interaction between Andrographis paniculata and the drugs mainly undergoing UGT2B7-catalyzed metabolic elimination, and the andrographolide derivatives as potential candidates for the selective inhibitors of UGT2B7.

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.

Three new triterpenoids from Panax ginseng exhibit cytotoxicity against human A549 and Hep-3B cell lines.

Three new triterpenoid derivatives, 3-O-ß-D-glucopyranosyl-20(S)-protopanaxtriol (1), 3-formyloxy-20-O-ß-D-glucopyranosyl-20(S)-protopanaxtriol (2) and 26-hydroxyl-24(E)-20(S)-protopanaxtriol (3), along with six known ginsenosides, were isolated from leaves of Panax ginseng. Their structures were established on the basis of spectral analysis (IR, 1D and 2D NMR, HRESI-MS). Compounds 1-3 exhibited various degree of cytotoxicity towards human A549 pulmonary carcinoma cells and Hep3B hepatoma cells.

[Protective effects of tianji capsule on vascular endothelial cells from oxidative injury induced by hydrogen peroxide in vitro].

OBJECTIVE: To study the protective effects of Tianji capsule (TJ) on vascular endothelial cells from oxidative injury induced by hydrogen peroxide and its possible mechanism of anti-oxidation. METHODS: The effect of TJ on the proliferation of normal human umbilical vascular endothelial cells (HUVECs) as well as its cytotoxicity was evaluated with methylthiazolyl tetrazolium (MTT) assay. After the establishment of oxidative injury model of HUVECs, control, oxidative injury model, TJ and CoQ10 treatment groups were set up. HUVECs were incubated with 37.5, 75, 150 and 300 microg/mL TJ or 100 microg/mL CoQ10 for 24 h, and 0.1 mmol/L H2O2 (final concentration) was added to HUVECs in each groups for 30 min. Then collected the cells for proliferation detection with MTT assay, and the levels of MDA and NO, the activities of SOD, GSH-Px and NOS, as well as the releasing rate of LDH in HUVECs were also determined. RESULTS: No cytotoxicity was observed in HUVECs with less than 400 microg/ mL TJ incubated for 48 h, but increased proliferation rates were noticed. Pretreated with TJ (37.5, 75, 150 and 300 microg/mL), increased proliferation rate, the activities of SOD, GSH-Px, NOS were observed, but the decreased level of MDA and releasing rate of LDH were also found. CONCLUSION: TJ could protect HUVECs against oxidative injury induced by H2O2.

Constituents with α-glucosidase and advanced glycation end-product formation inhibitory activities from Salvia miltiorrhiza Bge.

The 75% ethanol extract from roots of Salvia miltiorrhiza Bge. (Dan shen) afforded two new compounds, 3-hydroxy-2-(2'-formyloxy-1'-methylethyl)-8-methyl-1,4-phenanthrenedione (1), (8'R)-isosalvianolic acid C methyl ester (2), and 14 known compounds. Their structures were established on the basis of spectral analysis. The ability of the compounds to inhibit α-glucosidase activity and formation of advanced glycation end-products (AGEs) was evaluated. All compounds displayed various degrees of inhibitory effects against α-glucosidase; moreover, compounds 2, 6, 11, 14, and 16 exhibited much more potent inhibition against AGEs than the positive control (aminoguanidine, AG, IC(50) 0.11 µM). This is the first time that compounds from this plant have been reported to have inhibitory activity against α-glucosidase.

[Study on protective effect of Tianji soft capsule on blood lipids, internal antioxidant system and vascular endothelial system in hyperlipidemia rats].

OBJECTIVE: To study the protective effects of Tianji soft capsule (TJSC) on blood lipids, internal antioxidant system and vascular endothelial system in hyperlipidemia rats. METHODS: Seventy two healthy male rats were divided into six groups. The rats in control group were administered with ordinary diet. The rats in model group were fed with high cholesterol/lipid diet to induce hyperlipidemia. The rats in TJSC and CoQ10 groups were fed with high cholesterol/lipid diet, and treated with TJSC at different doses of 83 (low-dose group, L), 250 (middle-dose group, M), 750 (high-dose group, H) mg/kg, and CoQ10 at the dose of 83 mg/kg, respectively. All animals were put to death after four weeks, effects on lipid level; antioxidant system and endothelial system were evaluated through detection of total cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL), atherogenic index (AI), malondidehyde (MDA) and plasma endothelin (ET), HDL/TC ratio, activities of superoxide dismutase (SOD) and nitric oxide (NO). RESULTS: Compared with model group, serum TC, TG, VLDL, AI, MDA and ET reduced and the HDL/TC ratio increased, meanwhile activities of SOD and NO were enhanced. CONCLUSION: TJSC can regulate the lipid metabolism, enhance antioxidant system and protect the vascular endothelia system in hyperlipiemic rats.

[Comparative study on anti-hypercholesterolemia activity of diosgenin and total saponin of Dioscorea panthaica].

OBJECTIVE: To compare the anti-hypercholesterolemic and cholesterol absorption inhibitory activities between total saponin of Dioscorea panthaica (TSDP) and diosgenin (Dio). METHOD: TSDP and Dio were given ig or i.p. to mice or rats treated with cholesterol feed to evaluate their preventive and therapeutic effect on hypercholesterolemia. TSDP or Dio and cholesterol were mixed with pig bile to form the micelle, then the freeing cholesterol was detected to evaluate inhibitory effect of the both compounds on cholesterol absorption. RESULT: Dio (80 and 160 mg.kg-1) showed significantly therapeutic and preventive effect on hypercholesterolemia in mice, while TSDP showed a certain preventive activity only at a big dose (400 mg.kg-1). The intraperitoneal injection of Dio (20 and 40 mg.kg-1) to mice suffered from hypercholesterolemia was effective, but TSDP showed no effective. The serum total cholesterol level was decreased when rats were pre-treated with TSDP (200 and 400 mg.kg-1, ig) and Dio (200 and 100 mg.kg-1, ig). However, the hypercholesterolemia-preventing activity of Dio was stronger than that of TSDP. In addition, inhibitory effect of Dio on cholesterol micelle formation was still stronger than that of TSDP. CONCLUSION: The preventive and therapeutic activity of Dio against hypercholesterolemia indused by cholesterol in mice or rats is stronger than that of TSDP. The anti-hypercholesterolemia mechanism of Dio is probably related with its cholesterol absorption inhibitory activity.

[Intestinal bacteria metabolism of TSDP and characterization of metabolites in rats].

OBJECTIVE: To study the rat intestinal bacteria metabolism of total saponins of Dioscorea pathaica (TSDP) in vitro, and characterize the metabolites in serum and urine of rats after oral administration of TSDP 900 mg.kg-1. METHOD: TSDP metabolites were detected with thin-layer chromatography (TLC) and combination of electrospray ionization mass spectrometry (ESI-MS) and sequential tandem mass spectrometry (MSn). RESULT: In vitro, TSDP was decomposed easily by rat intestinal bacteria, and metabolites DP-1, DP-2, DP-4, DP-5 and diosgenin (Dio) were observed with prolongation of incubation time by ESI-MS2. In vivo, in the full-scan positive mass spectrum of the rat urine sample, the ion peak at m/z 415 (M-H) and its characteristic fragmentations at m/z 397 and m/z 271 in the MS/MS spectrum were identified with that of metabolite Dio, therefore metabolite Dio was deduced to exist in the rat urine, and metablite Dio was allso detected in the rat serum sample. CONCLUSION: TSDP is decomposed easily by rat intestinal bacteria and metabolite diosgenin is absorbed into blood after oral administration of TSDP.