Evaluation of the bioavailability of major withanolides of Withania somnifera using an in vitro absorption model system.

Withania somnifera (L.) Dunal, shows several pharmacological properties which are attributed mainly to the withanolides present in the root. The efficacy of medicinally active withanolides constituents depends on the absorption and transportation through the intestinal epithelium. We examined these characteristics by employing the Sino-Veda Madin-Darby canine kidney cells culture system, which under in vitro condition shows the absorption characteristics similar to the human intestinal epithelium. Thus, the aim of the present investigation was to assess the bioavailability of individual withanolides. Withanolides were diluted in Hank's buffered saline at a concentration of 2 µg/ml were tested for permeability studies carried out for 1 h duration. Permeability was measured in terms of efflux pump (P eff) in cm/s. P eff values of withanolide A (WN A), withanone (WNN), 1,2-deoxywithastramonolide (1,2 DWM), withanolide B (WN B), withanoside IV-V (WS IV-V), and withaferin A were 4.05 × 10(-5), 2.06 × 10(-5), 1.97 × 10(-5), 1.80 × 10(-5), 3.19 × 10(-6), 3.03 × 10(-6) and 3.30 × 10(-7) respectively. In conclusion, the nonpolar and low molecular weight compounds (WN A, WNN, 1,2 DWM, and WN B) were highly permeable. As against this, the glycosylated and polar WS IV and WS V showed low permeability. Surprisingly and paradoxically, the highly biologically active withaferin A was completely impermeable, suggesting that further studies possibly using human epithelial colorectal adenocarcinoma (Caco-2) cells may be needed to delineate the absorption characteristics of withanolides, especially withaferin A.

The aqueous extract of Radix Glycyrrhizae stimulates mitogen-activated protein kinases and nuclear factor-kappaB in Jurkat T-cells and THP-1 monocytic cells.

Radix Glycyrrhizae (RG) is a medicinal herb extensively utilized in numerous Chinese medical formulae for coordinating the actions of various components in the recipes and strengthening the body functions. In this report, we demonstrate that the aqueous extract of Radix Glycyrrhizae is capable of stimulating the c-Jun N-terminal kinase and p38 subgroups of mitogen-activated protein kinases (MAPKs), and the nuclear factor-kappaB (NFkappaB) in Jurkat T-lymphocytes. The activation magnitudes of MAPKs and NFkappaB were dose-dependent (EC(50) approximately 1 mg/ml) and time-dependent (maximal around 15-30 minutes). Stimulations of MAPKs and NFkappaB were not associated with changes in intracellular Ca(2+) mobilization. Similar activation profiles of MAPK and NFkappaB were obtained from THP-1 monocytes treated with the extract. In terms of chemotactic activity, the SDF-induced chemotaxis of Jurkat cells and THP-1 cells were inhibited by RG extract at 1-10 mg/ml, while a lower RG concentration (0.1-0.3 mg/ml) potentiated the SDF-induced chemotaxis for the former, but not the latter cell type. Given the fact that MAPKs and NFkappaB are important signaling intermediates for lymphocyte activities, our results suggest that Radix Glycyrrhizae may contain active constituents capable of modulating immuno-responses through various intracellular signaling pathways.

Carvacrol and eugenol differentially stimulate intracellular Ca2+ mobilization and mitogen-activated protein kinases in Jurkat T-cells and monocytic THP-1 cells.

Essential oils are a major active constituent found in many Chinese medicinal herbs. Here, we demonstrate that two components of essential oils, carvacrol and eugenol, dose-dependently trigger intracellular Ca2+ mobilization in Jurkat T-cells and THP-1 monocytic cells. Both carvacrol and eugenol are also capable of stimulating the active phosphorylation of the p38 subgroup of mitogen-activated protein kinases (MAPKs) in both cell types. However, carvacrol selectively activated the ERK subgroup in Jurkat T-cells, and stimulated the JNK subgroup in THP-1 monocytic cells. Eugenol treatment was not linked to ERK or JNK activation in either cell type. EC50 values for the induction of Ca2+ mobilization and MAPK activation were around 10 - 30 microM for both carvacrol and eugenol. Our results suggest that these essential oil components may act as effective agents to modulate the functions of immuno-responsive cells via different intracellular signaling pathways.

Endotoxin and cytokine released during parenteral nutrition.

BACKGROUND: Apart from its benefits, parenteral nutrition (PN)-related complications have been reported. Studies have shown that PN could alter cytochrome P450 (CYP) activity. One of the possible mechanisms is through cytokine and nitrite release, which is triggered by endotoxin. The purpose of this study is to investigate the potential release of endotoxin, cytokines, and nitrite during PN. METHODS: Rats were randomly assigned into either (a) the PN group, which received continuous PN infusion only; or (b) the control group, which received normal chow with saline infusion. The infusions were administered continuously for 7 days, and then blood was collected and microsomes were prepared from the excised livers. RESULTS: Endotoxin levels in the PN group were significantly higher in portal vein but not in inferior vena cava when compared with those of the controls. TNF-alpha and IL-6 levels were significantly higher in the PN group (p < .05). However, IL-1 beta levels were not significantly different in the 2 groups (p > .05). The nitrite levels, the end product of nitric oxide formation, were found to be almost 2 times higher after PN (p < .05). CONCLUSIONS: It is confirmed that a 7-day infusion treatment of PN in rat may be linked to bacterial translocation, which leads to increased levels of endotoxin. This increase could trigger cytokine release, which could down regulate CYP activities.

Factors responsible for the variability of saquinavir absorption: studies using an instrumented dog model.

PURPOSE: To study the effect of dose and food on the bioavailability of saquinavir in dogs. METHODS: A Youden Square block design was used for six female mongrel dogs (20-24 kg) who received six saquinavir treatments. The six randomized treatments were 1 mg/kg intravenous infusion over 30 min; 200, 400, 600, and 800 mg of saquinavir in the form of 200-mg capsules given orally with food; and 400 mg of saquinavir given orally after an overnight fast. A 200-mg 14C-saquinavir capsule was used to replace one of the 200-mg unlabeled saquinavir capsules in the 200- and 800-mg oral study. RESULTS: Absorption of saquinavir from the gut was variable. (F(A): 49-95%). The 14C-saquinavir study shows that the total radioactivity absorbed from the gut was insignificantly different from that of unlabeled saquinavir, suggesting first-pass gut metabolism was unimportant. The bioavailability of saquinavir under fasting condition was significantly lower (8.41 +/- 4.7% vs. 20.3 +/- 2.6%, p < 0.05). Saquinavir underwent significant first-pass liver metabolism because hepatic clearance values (22 to 30 ml min(-1) kg(-1)) approached that of liver blood flow. CONCLUSIONS: Incomplete gut absorption and extensive first-pass liver metabolism are the causes for low bioavailability of saquinavir in dogs. Absorption was further reduced under fasted conditions.

Direct analysis of nine pharmaceuticals in culture media by use of cartridge separation with electrospray mass spectrometric detection.

A 2-cm cartridge has been used for separation before electrospray mass spectrometric analysis of pharmaceutical compounds in cell culture media, alleviating the need for sample extraction and desalting procedures. Nine representative pharmaceuticals listed in the biopharmaceutical classification system (BCS) were chosen as the candidate compounds and Hank's balanced salt solution with Hepes buffer (HBSS-Hepes buffer) was used as the cell-culture medium in an effort to study permeability of chemicals through cell monolayers. Effects of several conditions, e.g. pH and buffer concentration in the mobile phase, flow rate, and temperature on separation efficiency were examined. The nine pharmaceuticals were separated within 2 min by use of a 2-cm C(8) cartridge. Relative standard deviations (RSD) from repeated analysis within the same day or over five days were 0.03-0.2% for retention times and 0.6-5.3% for peak areas; antipyrine was used as internal standard. Calibration curves based on peak-area measurements were linear over the range 0.1-20 micro mol L(-1). The HBSS-Hepes buffer did not interfere with separation and detection; identical separation and peak intensity were obtained when the samples were separately prepared in distilled water or in the culture medium.

Liquid chromatography-electrospray mass spectrometric studies of ginkgolides and bilobalide using simultaneous monitoring of proton, ammonium and sodium adducts.

A liquid chromatographic-electrospray mass spectrometric method was developed for the determination of ginkgolides and bilobalide and was applied to the analysis of commercial products of Ginkgo biloba leaf extracts. Adducts of these compounds with ammonium, proton and sodium were detected and their relative abundance depended on the electrospray fragmentor voltage. The relative standard deviation (RSD) was improved from > 17% to < 6%, when three adduct ions of (M + H)+, (M + NH4)+ and (M + Na)+ were used for quantification compared with single ion monitoring. The characteristic mass spectra of bilobalide were different from those of ginkgolides; in particular, dimers of (2M + Na)+ were observed for bilobalide only. Analysis of 26 commercial ginkgo products revealed large variations in the composition and concentrations of ginkgolides and bilobalide in herbal products.

Hydroxypropyl-beta-cyclodextrin-flutamide inclusion complex. II. Oral and intravenous pharmacokinetics of flutamide in the rat.

PURPOSE: The objective of this work was to determine the pharmacokinetics of flutamide (FLT) and its active metabolite, 2-hydroxy-flutamide (FLT-2-OH) in rats, following formulation in hydroxypropyl-Beta-cyclodextrin (FLT-HPBetaCyD). METHODS: The pharmacokinetics of FLT-HPBetaCyD, FLT-suspension (FLT-SUSP), and FLT-solution (FLT-COSOLV) were compared after oral (p.o.) and intravenous (i.v.) administration, respectively. In a non-crossover design, male Sprague-Dawley rats received each formulation as a single oral dose [15 mg (54 micro mol) FLT/kg] by oral gavage, or single i.v. dose [1.6 mg (5.8 micro mol) FLT/kg] via an indwelling jugular vein catheter. FLT and its metabolite, FLT-2-OH, were determined in plasma and urine aliquots by an HPLC method. RESULTS: In a preliminary in vitro experiment, using the dialysis bag dissolution method, 80% of a test dose of FLT was released from lyophilized FLT-HPBetaCyD into simulated gastric juice within 2 h, compared to less than 5% release from commercial FLT powder (FLT-SUSP). Following oral FLT-HPBetaCyD, the mean area under the plasma concentration curve (AUC(0- infinity)) for FLT, was 1580 +/- 228 ng x h/mL, with the maximum plasma concentration (Cmax; 1297 +/- 127 ng/mL) at 0.5 h (Tmax) after administration. The AUC(0- infinity) and C(max) were significantly higher than after FLT-SUSP (AUC(0- infinity) 748 +/- 206 ng x h/mL; C(max) 230 +/- 111 ng/mL and T(max) 2.33 +/- 0.29 h, respectively). After i.v. FLT-HPBetaCyD, the FLT AUC(0- infinity) was 1355 +/- 162 ng x h/mL, compared to 1421 +/- 283 ng x h/mL for FLT-COSOLV. FLT C(max) were 714 +/- 144 mL/h and 735 +/- 88 mL/h, respectively. The respective volumes of distribution (V(z)) were 369 +/- 191 mL and 242 +/- 25 mL. The plasma concentration-time profile and pharmacokinetic parameters of FLT after FLT-HPBetaCyD and FLT-COSOLV did not differ significantly. The pharmacokinetic parameters for FLT-2-OH were formulation independent after i.v. dosing, but AUC(0- infinity); C(max) and T(max), values were substantially greater with the FLT-HPBetaCyD in the oral study (40269 +/- 5875 ng x h/mL, 4062 +/- 502 ng/mL, and 3.50 +/- 0.41 h, respectively). CONCLUSIONS: FLT from FLT-HPBetaCyD was released rapidly into solution in vitro and in vivo. FLT-HPBetaCyD improved oral bioavailability relative to FLT-SUSP. Intravenous pharmacokinetic profiles for both FLT and FLT-2-OH were identical following either FLT-HPBetaCyD or FLT-COSOLV, indicating that the FLT-HPBetaCyD formulation behaved as a true solution.