OBJECTIVE: To determinate triptolide and wilforlide A in biological samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and to verify the method. METHODS: After 0.4 mL blood, urine or 0.4 g hepatic tissues with internal standard were extracted by ethyl acetate, they were separated on a Allure PFP Propyl (100 mm x 2.1 mm, 5 µm) with a mobile phase of methanol-20 mmol/L ammonium acetate using gradient elution. For mass spectrometric detection, electrospray ionization (ESI⁺) in positive mode was elected and the data was collected using multiple-reaction monitoring (MRM). RESULTS: The linearity was good (r > 0.995 0) and the limit of detection was 2 ng/mL or 2 ng/g for triptolide and wilforlide A. The recovery was 61.08%-102.98%. The intra-day and inter-day precision was less than 12.58% for each biological sample, and the accuracy was 90.61%-105.80%. CONCLUSION This method is simple, convenient and good selective, and could be applied to analysis of triptolide and wilforlide A in different biological samples. And the method may provide technical support for forensic medicine identification, clinical diagnosis and treatment of tripterygium wilfordii Hook. f. poisoning.
Chromatography, High Pressure Liquid ; Diterpenes/urine* ; Epoxy Compounds/urine* ; Humans ; Oleanolic Acid/urine* ; Phenanthrenes/urine* ; Sensitivity and Specificity ; Tandem Mass Spectrometry

An efficient method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap (UHPLC-LTQ-Orbitrap) mass spectrometer was established to elucidate the metabolites of tanshinone Ⅰ and tanshinone ⅡA in rats. Urine and plasma samples were collected after oral gavage. After processing biological sample by solid phase extraction, Waters ACQUITY HPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid (A) - acetonitrile (B) solution as the mobile phase for gradient elution. The plasma, urine and the blank samples were then analyzed by ESI-LTQ-Orbitrap equipped with an ESI ion source under positive ion mode. On the basis of the accurate mass measurements, multiple mass spectra and comparison of data with published literature, a total of 26 metabolites were tentatively identified and characterized in the rat samples. Among them, 7 metabolites were derived from tanshinone Ⅰ through metabolic pathways of glucuronide conjugation, hydroxylation, reduction reaction, demethylation reaction, methylation, sulfate conjugation and their composite reactions. Nineteen metabolites were derived from tanshinone ⅡA through metabolic pathways of hydroxylation, reduction reaction, methylation, sulfate conjugation, glucuronidation, glucosylation and their complicated reactions. The results showed that the metabolism of tanshinone Ⅰ and tanshinone ⅡA in rats could be comprehensively clarified by using UHPLC-LTQ-Orbitrap mass spectrometer, providing material basis for the further research in terms of pharmacodynamics, toxicology, and secondary development of Chinese medicine.
Animals ; Chromatography, High Pressure Liquid ; Diterpenes, Abietane ; blood ; metabolism ; urine ; Rats

OBJECTIVETo observe the change of urinary monocyte chemottractant protein-1 (MCP-1) in patients with diabetic nephropathy (DN), and to explore the therapeutic effect and mechanism of triptolide (TL) in treating DN.

METHODSThirty-five patients in the treated group were treated with TL plus benazepril and thirty two patients in the control group were treated with benazepril alone for six months. The change of urinary MCP-1 was measured before and after treatment.

RESULTSLevel of urinary MCP-1 in DN patients was significantly higher than that in healthy subjects (P < 0.01), but it could be significantly decreased after TL treatment, showing significant difference as compared with that in the control group (P < 0.05).

CONCLUSIONDetermination of urinary MCP-1 level is beneficial to know the degree of kidney inflammation in DN patients. TL can inhibit inflammatory reaction to decrease the level of urinary MCP-1, and thus improve the renal function.

Adult ; Aged ; Chemokine CCL2 ; urine ; Diabetic Nephropathies ; drug therapy ; urine ; Diterpenes ; therapeutic use ; Epoxy Compounds ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Male ; Middle Aged ; Phenanthrenes ; therapeutic use ; Phytotherapy ; Prospective Studies

The metabolic profile of pseudolaric acid B (PB) was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the specific metabolite of PB in plasma, urine, bile and feces using HPLC and HPLC-ESI/MS(n) after both oral and intravenous administration to rats, and almost no prototype was detected in all kinds of samples. The metabolic behaviors of PB orally administered in rats treated with antibiotics to eliminate intestinal microflora were identical with those in untreated rats, demonstrating that the metabolism of PB is independent of intestinal microflora. PB was stable in 48 h respective incubation with artificial gastric juice and artificial intestinal juice, suggesting that neither pepsin nor trypsin is in charge of metabolism of PB, and also demonstrating that PB is stable in both pH environments of gastric tract and intestinal tract. In vitro research on metabolism of PB in rat liver microsomes incubation revealed that little PB was metabolized and that the proposed metabolites were the demethoxy and demethoxydecarboxy products of the prototype. The amount of metabolites was extremely low compared with the prototype, indicating that liver microsomes are not responsible for the metabolism of PB either. PB was gradually metabolized into PC2 during 1 h in whole blood incubation in vitro, and the metabolic process showed dynamically dependent manner with incubation time. Once absorbed into blood, PB was quickly metabolized into PC2, accordingly, little prototype was detected in all kinds of samples. The metabolism was attributed to the rapid hydrolysis of C-19 ester bond by plasma esterase. These results clarified the metabolic pathway of PB for the first time, which was of great significance to identify the in vivo active form and interpret acting mechanism of the active compounds of P. kaempferi.
Administration, Intravenous ; Administration, Oral ; Animals ; Bile ; metabolism ; Diterpenes ; blood ; metabolism ; urine ; Esterases ; metabolism ; Feces ; chemistry ; Hydrolysis ; Male ; Metabolic Networks and Pathways ; Microsomes, Liver ; metabolism ; Pinaceae ; chemistry ; Plant Bark ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley

AIMInvestigations on reducing the toxicity of triptolide through poly(D, L-lactic acid) nanoparticles as a drug carrier by oral administration to Wistar rats.

METHODSTriptolide-loaded poly (D, L-lactic acid) nanoparticles (TP-PLA-NPs) were prepared by modified spontaneous emulsification solvent diffusion (modified-SESD). The shape of nanoparticles was observed by transmission electron microscope (TEM). The size distribution and mean diameter were measured by laser light scattering technique. The entrapment efficiency and contents of drug loading were determined by RP-HPLC. The physical state of drug loaded in nanopartiles were primarily investigated by X-ray powder diffractometry. TP-PLA-NPs release behavior in vitro was carried out. After oral administration of the nanoparticles to Wistar rats in 15d, the toxicity for liver and kidney were studied by determining aspartate transaminase (AST), alanine transaminase (ALT) and blood urea nitrogen in serum and concentration of protein in urine.

RESULTSThe preparation process adapted to the formulation was as follows: the volume ratio of the aqueous and organic phases was 40/15; the surfactant concentration was 1%; the drug concentration was 0.3%; triptolide-PLA was 1:15 (w/w). The mean diameter was 149.7 nm and the polydispersity index was 0. 088 for the nanoparticles prepared by above conditions. The entrapment efficiency and content of drug loading were 74.27% and 1.36%, respectively. The release behavior of drug in vitro showed an initial burst effect, subsequently a slower rate stage. The results indicated that the liver toxicity (P < 0.01) and kidney toxicity (P < 0.05) caused by triptolide could be decreased significantly by nanoparticles carrier.

CONCLUSIONPLA-NPs might be used as a new oral carrier for triptolide.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Blood Urea Nitrogen ; Delayed-Action Preparations ; Diterpenes ; administration & dosage ; isolation & purification ; toxicity ; Drug Carriers ; Drug Delivery Systems ; Epoxy Compounds ; Lactic Acid ; Male ; Nanotechnology ; Particle Size ; Phenanthrenes ; administration & dosage ; isolation & purification ; toxicity ; Polyesters ; Polymers ; Proteinuria ; urine ; Rats ; Rats, Wistar ; Tripterygium ; chemistry