Two new chemical constituents from Lonicera japonica.

Two new chemical constituents, japopenoid D (1), and japopenoid E (2), were isolated and identified from the flower buds of Lonicera japonica Thunb. The structures of these compounds were elucidated based on spectroscopic analysis (HR-ESI-MS, NMR), and the absolute configurations of 1 and 2 were determined by comparison of their electronic circular dichroism (ECD) spectra with literature and theoretical calculation. The anti-inflammatory activities of the isolates were evaluated by measuring their inhibitory effects on PGE2 and IL-6 production in LPS stimulated RAW 264.7 macrophages. As a result, compound 1 could reduce PGE2 and IL-6 levels in LPS-activated RAW 264.7 macrophages with IC50 values of 6.78 and 42.07 µM, respectively.[Formula: see text].

Pharmacokinetics of the prototype and hydrolyzed carboxylic forms of ginkgolides A, B, and K administered as a ginkgo diterpene lactones meglumine injection in beagle dogs.

Ginkgo diterpene lactones meglumine injection (GDLI) is a commercially available product used for neuroprotection. However, the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI, i.e., ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), have never been fully evaluated in beagle dogs. In this work, a simple, sensitive, and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was developed, and the prototypes and total amounts of GA, GB, and GK were determined in beagle dog plasma. The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations. For the first time, the pharmacokinetics of GA, GB, and GK were fully assessed in three forms, i.e., the prototypes, the hydrolyzed carboxylic forms, and the total amounts, after intravenous administration of GDLI in beagle dogs. It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma, and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio. All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages. GA, GB, and GK showed a constant half-life approximately 2.7, 3.4, and 1.2 h, respectively, which were consistent for the forms at three dose levels (0.3, 1.0, and 3.0 mg·kg-1) and after a consecutive injection of GDLI for 7 days (1.0 mg·kg-1).

Pharmacokinetic and metabolomic analyses of the neuroprotective effects of salvianolic acid A in a rat ischemic stroke model.

Salvianolic acid A (SAA), a water-soluble phenolic acid isolated from the root of Dan Shen, displays distinct antioxidant activity and effectiveness in protection against cerebral ischemia/reperfusion (I/R) damage. However, whether SAA can enter the central nervous system and exert its protective effects by directly targeting brain tissue remains unclear. In this study, we evaluated the cerebral protection of SAA in rats subjected to transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. The rats were treated with SAA (5, 10 mg/kg, iv) when the reperfusion was performed. SAA administration significantly decreased cerebral infarct area and the brain water content, attenuated the neurological deficit and pathology, and enhanced the anti-inflammatory and antioxidant capacity in tMCAO rats. The concentration of SAA in the plasma and brain was detected using LC-MS/MS. A pharmacokinetic study revealed that the circulatory system exposure to SAA was equivalent in the sham controls and I/R rats, but the brain exposure to SAA was significantly higher in the I/R rats than in the sham controls (fold change of 9.17), suggesting that the enhanced exposure to SAA contributed to its cerebral protective effect. Using a GC/MS-based metabolomic platform, metabolites in the serum and brain tissue were extracted and profiled. According to the metabolomic pattern of the tissue data, SAA administration significantly modulated the I/R-caused perturbation of metabolism in the brain to a greater extent than that in the serum, demonstrating that SAA worked at the brain tissue level rather than the whole circulation system. In conclusion, a larger amount of SAA enters the central nervous system in ischemia/reperfusion rats to facilitate its protective and regulatory effects on the perturbed metabolism.

Exploring the neuroprotective effects of ginkgolides injection in a rodent model of cerebral ischemia-reperfusion injury by GC-MS based metabolomic profiling.

Cerebral ischemia-reperfusion (I/R) injury usually contributes to mortality and disability after ischemic stroke. Ginkgolides injection (GIn), a standard preparation composed of ginkgo diterpene lactones extract, is clinically used for neuroprotective treatment on reconvalescents of cerebral infarction. However, the understanding about its therapeutic mechanism is still lacking. In this study, a gas chromatography-mass spectrometry (GC-MS) based metabolomic approach coupled with multivariate data analysis (MVDA) was applied to explore the neuroprotective effects of GIn in a rodent model of focal ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO). Metabolomic profiling revealed a series of metabolic perturbations that underlie the cerebral I/R pathological events. GIn can reverse the I/R induced brain metabolic deviations by modulating multiple metabolic pathways, such as glycolysis, Krebs cycle, pentose phosphate pathway (PPP), γ-aminobutyrate (GABA) shunt and lipid metabolism. Moreover, the main bioactive components of GIn were distributed to brain tissue much more easily in tMCAO rats than in normal rats after an intravenous administration, suggesting that the increased cerebral exposure to ginkgolides in I/R pathological condition potentially facilitated the neuroprotective effects of GIn by directly targeting at brain. The present study provided valuable information for our understanding about metabolic changes of cerebral I/R injury and clinical application of GIn.

In vitro assessment of the glucose-lowering effects of berberrubine-9-O-ß-D-glucuronide, an active metabolite of berberrubine.

Berberrubine (BRB) is the primary metabolite of berberine (BBR) that has shown a stronger glucose-lowering effect than BBR in vivo. On the other hand, BRB is quickly and extensively metabolized into berberrubine-9-O-ß-D-glucuronide (BRBG) in rats after oral administration. In this study we compared the pharmacokinetic properties of BRB and BRBG in rats, and explored the mechanisms underlying their glucose-lowering activities. C57BL/6 mice with HFD-induced hyperglycemia were administered BRB (50 mg·kg-1·d-1, ig) for 6 weeks, which caused greater reduction in the plasma glucose levels than those caused by BBR (120 mg·kg-1·d-1) or BRB (25 mg·kg-1·d-1). In addition, BRB dose-dependently decreased the activity of α-glucosidase in gut of the mice. After oral administration of BRB in rats, the exposures of BRBG in plasma at 3 different dosages (10, 40, 80 mg/kg) and in urine at different time intervals (0-4, 4-10, 10-24 h) were dramatically greater than those of BRB. In order to determine the effectiveness of BRBG in reducing glucose levels, we prepared BRBG from the urine pool of rats, and identified and confirmed it through LC-MS-IT-TOF and NMR spectra. In human normal liver cell line L-O2 in vitro, treatment with BRB or BRBG (5, 20, 50 µmol/L) increased glucose consumption, enhanced glycogenesis, stimulated the uptake of the glucose analog 2-NBDG, and modulated the mRNA levels of glucose-6-phosphatase and hexokinase. However, both BBR and BRB improved 2-NBDG uptake in insulin-resistant L-O2 cells, while BRBG has no effect. In conclusion, BRB exerts a stronger glucose-lowering effect than BBR in HFD-induced hyperglycemia mice. Although BRB significantly stimulated the insulin sensitivity and glycolysis in vitro, BRBG may have a greater contribution to the glucose-lowering effect because it has much greater system exposure than BRB after oral administration of BRB. The results suggest that BRBG is a potential agent for reducing glucose levels.

Study on chemical profiles and metabolites of Allii Macrostemonis Bulbus as well as its representative steroidal saponins in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Allii Macrostemonis Bulbus (AMB) is increasingly becoming popular as an edible vegetable or traditional folk medicine in East Asia due to its great health and medicinal properties. However, due to a lack of available research, the effective material of AMB still remains unknown. In this study, we applied a strategy utilising ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) to investigate chemical profiles of AMB. In addition, metabolite profiles of five representative single steroidal saponins as well as AMB were investigated. Moreover, the metabolic features of saponins in AMB were summarised. After oral administration, the saponins underwent massive phase I and phase II metabolism. Sequential deglycosylation metabolism in rat intestine was the main metabolic pathway of the steroidal saponins, while oxidation, dehydrogenation, glucuronic acid reactions in liver also take part in further modification. These results expand our knowledge about the metabolism of AMB.

Metabolites profile of Xian-Ling-Gu-Bao capsule, a traditional Chinese medicine prescription, in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis.

Xian-Ling-Gu-Bao capsule (XLGB), a well-known traditional Chinese medicine prescription (TCMP), is widely used for the treatment of osteoporosis. However, due to lack of metabolism research, the effective material of XLGB is still unknown. It entails a huge obstacle for the clinical-safe medication administration and quality control of XLGB. To explore the metabolic fate of multiple components of XLGB, herein, we proposed a "representative structure based homologous xenobiotics identification" (RSBHXI) strategy based on ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) and mass defect filter (MDF) technique. A total of 147 XLGB-related xenobiotics were identified or tentatively characterized in rat biofluids after oral administration of XLGB, including 134 (57 prototype components and 77 metabolites) in plasma, 93 (37 prototype components and 56 metabolites) in urine and 118 (46 prototype components and 72 metabolites) in bile. Our results indicated that prenylated flavonol glycosides from Herba epimedii, prenylated flavonoids from Fructus psoraleae, saponins from Radix dipsaci and Rhizoma anemarrahenae, as well as tanshinones from Radix Salviae Miltiorrhizae were major absorbed chemical components of XLGB. Hydrolysis, glucuronidation and sulfation were major metabolic reactions of XLGB. As more xenobiotics were detected in bile than those in urine, it demonstrated that multiple components of XLGB underwent comprehensive hepatobiliary excretion. The present study expands our knowledge about the metabolism of XLGB which will be conducive to revealing its in vivo pharmacological material basis. In addition, the application of RSBHXI strategy provides a new approach for metabolite identification of TCMPs and other complex mixture.