Development and validation of a dried blood spots assay for metabolic profiling of ginsenosides using ultra-high performance liquid chromatography-mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C.A. Meyer., a famous and valuable traditional Chinese medicine with thousand years of history for its healthcare and therapeutic effects. It is necessary and meaningful to study the pharmacokinetic behavior of ginsenosides in vivo as they are the most active components. Dried blood spots (DBS) are a mature and advanced blood collection method with meet the needs for the measurement of numerous analytes. AIM OF THE STUDY: This study aimed to explore the feasibility on DBS in the metabolic profile analysis of complex herbal products. MATERIALS AND METHODS: An ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of ginsenosides. The preparation of DBS samples was conducted by spiking the whole blood with analytes to obtain 20 µL of blood spots on Whatman 903 collection card. A punched dish of 10 mm in diameter was extracted with 70 % methanol aqueous solution, digoxin was used as an internal standard. Target compounds were separated on a Waters T3 column (2.1 × 100 mm, 1.8 µm) with acetonitrile and water (0.1 % formic acid) at a flow rate of 0.4 mL/min. RESULTS: The various ginsenosides showed good linearity in the range of 1-2000 ng/mL. The extraction recoveries and matrix effects of the target analytes were above 82.2%. The intra- and inter-batch accuracy and precision were within the limits of ≤15% for all tested concentrations. Moreover, the collected dried blood spot samples could be stably stored at room temperature for 14 days and 4 °C for 1 month without being affected. And it is delightful that the DBS-based analysis is compatible or even superior to the conventional protein precipitation in terms of sensitivity, linearity, and stability. In particular, the target analytes are stable in the DBS sampling under normal storing condition and the sensitivity for some trace metabolites of ginsenosides, such as 20(S)-Rg3, 20(R)-Rg3, F1, Rk1, Rg5, etc. increases 3-4 folds as evaluated by LLOQ. CONCLUSIONS: The established method was successfully applied to pharmacokinetic studies of ginseng extract in mice, this suggests a more feasible strategy for pharmacokinetic study of traditional and natural medicines both in animal tests and clinical trials.

Study on the mechanism of Shuganzhi Tablet against nonalcoholic fatty liver disease and lipid regulation effects of its main substances in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuganzhi Tablet (SGZT) originates from a famous traditional Chinese herbal formula Chaihu Decoction which can be applied to treat liver diseases, however, the pharmacodynamic mechanism of SGZT needs to be evaluated. AIM OF THIS STUDY: To study the mechanism of SGZT in the treatment of non-alcoholic fatty liver disease (NAFLD), and screen out its effective ingredients. MATERIALS AND METHODS: In this study, firstly, the main components of SGZT were analyzed qualitatively. And a rat model of NAFLD was established by feeding high-fat diet. Serum biochemical indexes and liver pathological analysis were used to evaluate the pharmacodynamic effect of SGZT in the treatment of NAFLD. In order to explore the pharmacodynamic mechanism, proteomics and metabolomics analysis were used. Western blotting was used to verify the expression of important differential proteins. And L02 cells were treated with free fatty acids (FFA) and the main substances of SGZT to establish the cell model of NAFLD in vitro and to reveal the pharmacodynamic substance of SGZT. RESULTS: Twelve components were detected in SGZT, and according to the results of serum biochemical indexes and liver pathological analysis, SGZT could effectively treat NAFLD. Combined with the results of bioinformatics analysis, we found that 133 differentially expressed proteins were reversed in liver samples of rats treated with SGZT. The important proteins in PPAR signaling pathway, steroid biosynthesis, cholesterol metabolism and fatty acid metabolism were mainly regulated to maintain cholesterol homeostasis and improve lipid metabolism. SGZT also affected various metabolites in rat liver, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and taurine. In addition, the main components contained in SGZT (hesperidin, polydatin, naringin, emodin, specnuezhenide, saikosaponin A) and a metabolite (resveratrol) could significantly reduce FFA-induced intracellular lipid accumulation. CONCLUSION: SGZT effectively treated NAFLD, and PPAR-γ, Acsl4, Plin2 and Fads1 may be the main targets of SGZT. And Fads1-EPA/DHA-PPAR-γ may be the potential pharmacodynamic pathway. Cell experiments in vitro revealed that the main components of SGZT and their metabolites, such as hesperidin, polydatin, naringin, emodin, specnuezhenide, saikosaponin A and resveratrol may be the main components of its efficacy. Further research is needed to reveal and validate the pharmacodynamic mechanism.

Swertiamarin, an active iridoid glycoside from Swertia pseudochinensis H. Hara, protects against alpha-naphthylisothiocyanate-induced cholestasis by activating the farnesoid X receptor and bile acid excretion pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Swertiamarin (SW), which belongs to iridoid glycosides, is one of the main components of Swertia plants in Gentianaceae family, including Swertia pseudochinensis H. Hara and Swertia mileensis T. N. Ho et W. L. Shi. There are mainly used in traditional Chinese medicine for the treatment of hepatic and biliary disease such as jaundice. AIM OF THIS STUDY: This experiment aimed to explore the protective mechanism of SW on cholestasis induced by alpha-naphthylisothiocyanate in rats. MATERIALS AND METHODS: Healthy rats were randomly divided into the control, model (ANIT, 50 mg/kg), ursodeoxycholic acid (UDCA, 80 mg/kg), and low-dose (SW, 80 mg/kg), medium-dose (SW, 100 mg/kg), and high-dose (SW, 150 mg/kg) groups. The hepatic protective effect of SW was preliminarily evaluated by measurement of serum biochemical indicators and liver morphological evaluation. Moreover, metabolomics and proteomics analysis were used to explore the protective mechanism of SW on cholestasis. The expression of related proteins was determined by Western blot and polymerase chain reaction, and the important proteins were verified by cell experiments in vitro. RESULTS: SW (100 mg/kg) can reduce the serum levels of the model group. The hepatocyte of the medium-dose treatment group was arranged neatly without evident inflammation. SW can partially reverse the changes in cholestasis metabolites, such as taurocholic acid, SM (d18:1/16:0), all-trans-retinoic acid and other products of rats. The main metabolic pathways affected were primary bile acid synthesis, glycerophospholipid metabolism, sphingolipid metabolism and retinol metabolism. SW medium-dose treatment group showed effective reversal of 25 related proteins and it can remarkably reduce the contents of NTCP and CYP27A1 in rat liver and increase the protein expressions of CYP7A1, CYP8B1, bile salt export pump, multidrug resistance-associated protein and FXR. CONCLUSIONS: SW can alleviate ANIT-induced cholestasis, which by activating the farnesoid X receptor and bile acid excretion pathway.

Metabolite profiling of Shuganzhi tablets in rats and pharmacokinetics study of four bioactive compounds with liquid chromatography combined with electrospray ionization tandem mass spectrometry.

Shuganzhi Tablets (SGZT) is developed on the basis of a clinical empirical formula as a hospital preparation for the treatment of fatty liver. In this study, a rapid and highly sensitive LC-MS/MS method was established and validated for simultaneous determination of ginsenoside Re, ginsenoside Rg1, notoginsenoside R1, naringin, specnuezhenide, emodin, polydatin, hesperidin and saikosaponin A in rat plasma. Multiple reaction monitoring mode played an important role in simultaneous quantitative analysis of multiple components. The analytes were separated by the action of an ACQUITY UPLC® BEH C18 column (2.1 × 50 mm, 1.7 µm) in five minutes. The validated LC-MS/MS method was successfully applied to the pharmacokinetic analysis of hesperidin, emodin, polydatin and naringin of SGZT in rat plasma after administration. A UHPLC system couple with a quadrupole combined with time of flight mass spectrometer was used for qualitatively analyzing of the composition of SGZT and its metabolites in serum, urine, bile and feces of rats. The results showed that a total of 65 components were detected in rat biological samples, including 10 prototype components and 55 metabolites. It was speculated that the ingredients of SGZT experienced mainly the following reactions in rats: phase I reaction such as hydrolysis, oxidation, hydroxylation, carboxylation and dehydroxylation and phase Ⅱ reaction such as glucuronidation and sulfation. These results provide useful information for the further study of its active ingredients.

Exploration of the potential mechanism of Banxia Xiexin Decoction for the effects on TNBS-induced ulcerative colitis rats with the assistance of network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia Xiexin Decoction (BXD), an ancient TCM prescription originating from Treatise on Febrile Diseases (Shang Han Lun) of the Han Dynasty, has been widely used in modern clinical practice, especially for gastrointestinal diseases, including ulcerative colitis (UC). However, the modern decoction method of BXD differs from that of the original method. Thus, an exploration of the influence of the different decoction methods on the pharmacological effects is interesting and significant. AIM OF THE STUDY: This study aimed to systematically compare the pharmacological effects of extracts of BXD on TNBS induce UC rats that were prepared by different methods, the ancient method and the modern method. The findings may provide important information for the further mechanical exploration of the classical prescription, contributing to the rational application and enhancing the understanding of BXD in modern applications or scientific research. METHODS: Fifty-four SD rats were randomly divided into the following nine groups at n = 6/group: control group; model group; salicylazosulfapyridine group; BXD ancient extraction method's low-dose group (BXD-AED-L, 3.6 g BXD-AED/kg), medium-dose group (BXD-AED-M, 7.2 g BXD-AED/kg), and high-dose group (BXD-AED-H, 14.4 g BXD-AED/kg); and BXD modern extraction method's low-dose group (BXD-MED-L, 1 g BXD-MED/kg), medium-dose group (BXD-MED-M, 2 g BXD-MED/kg), and high-dose group (BXD-MED-H, 4 g BXD-MED/kg). All the groups, except the control group, were rectally injected with 70 mg/kg ethanol solution containing TNBS (2,4,6-trinitrobenzenesulfonic acid) to establish the UC models. The pharmacological evaluations including disease activity index, colon weight index, macroscopic and histological evaluation of colon damage, and inflammatory cytokine levels (IL-4, IL-10, IL-1ß, TNF-α, and IL-6)were measured. In the network pharmacology analysis, the "herbs-components-targets-disease" network was constructed and visually analyzed with which the targets with a strong correlation with UC were screened out. RESULTS: The results showed that both BXD-AED and BXD-MED might alleviate the severity of UC with different degrees according to the majority of indices that were evaluated. At similar doses, the BXD-AED groups performed better compared with the BXD-MED groups. With the assistance of the network pharmacology analysis, some key active components (quercetin, baicalein, wogonin, and baicalin) related to the anti-UC/inflammation were screened out. The contents of the components in BXD-AED were higher than those in BXD-MED. The joint results of the study indicated that BXD, an ancient TCM compound prescription, is an effective drug candidate for the modern treatment of UC.

Valence, Size, and Shape Control of Gold Nanoparticles Synthesized by Electron-Assisted Reduction.

An electron-assisted strategy was developed to prepare gold nanoparticles (AuNPs) at room temperature. Glow discharge plasma as electron source was successfully used to control the valence state, size, and shape of AuNPs. Stable Au(I) was obtained in 3 min by plasma, and Au(I) was reduced to zero valence with the increase in treatment time. An increase in the amount of Au did not induce an increase in particle size. A narrow size distribution was also achieved. The narrowest size distribution was observed at 9 min at 600 V. AuNPs grew slowly under glow discharge plasma, which slightly changed the mean size of AuNPs. Moreover, the average size of AuNPs was smaller under alkaline conditions. The initial pH of the solution can affect the nucleation and growth of AuNPs and further affect their particle size. Spherical AuNPs, hexagonal AuNPs, rectangular AuNPs, flower-shaped AuNPs, and Au nanorods were easily obtained within 30 min by adding different additives. The hexagonal AuNPs exhibited the largest current response toward caffeine and showed a good linear range (0.1-1000 µM) with a low detection limit (0.064 µM), because their high-energy planes can increase the electron transfer rate and improve electrocatalytic activity.

Protective effect of picroside I against hepatic fibrosis in mice via sphingolipid metabolism, bile acid biosynthesis, and PPAR signaling pathway.

Picroside I, a hepatoprotectant isolated from Picrorhiza kurroa Royle ex Benth and P. scrophulariiflora Pennell, can reduce liver injury in humans and animals. However, its anti-fibrosis effect remains elusive. This work aimed to explore the mechanism underlying the hepatoprotective effect of picroside I against hepatic fibrosis. Male mice (12 mice per group) were randomly divided into six groups: the control group; the model group, which received thioacetamide (TAA); the positive group, which received TAA + S-(5'-adenosyl)-l-methionine (SAMe, 10 mg/kg); the low-dose group, which received TAA + picroside I (25 mg/kg); the middle-dose group, which received TAA + picroside I (50 mg/kg); and the high-dose group, which received TAA + picroside I (75 mg/kg). Serum biochemical indicators were detected, and histological evaluation was performed. Metabolomics and proteomic analyses were conducted via liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS). Data showed that picroside I could decrease the serum levels of alanine transaminase (ALT), aspartate transaminase (AST), collagen type IV (CIV), N-terminal peptide of type III procollagen (PIIINP), laminin (LN), and hyaluronic acid (HA) and reduced fibrosis area. Picroside I altered metabolomic profiles, including energy, lipid, and glutathione (GSH) metabolism, in ice with fibrosis. Additionally, 25 differentially expressed proteins in the picroside I high-dose-treated group were reversed relative to in the model group. These proteins were involved in the sphingolipid signaling pathway, primary bile acid biosynthesis, and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Moreover, this study revealed how picroside I could protect against TAA-induced liver fibrosis in mice. Results indicated that picroside I can serve as a candidate drug for hepatic fibrosis.

Pharmacokinetics and metabolic profiles of swertiamarin in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

Swertiamarin, a typical compound of secoiridiod glycosides with various pharmacological effects which is the major iridoid glicoside of Swertia. In this study, we have established a fast and sensitive LC-MS/MS method. The aim was to conduct pharmacokinetic studies of swertiamarin in vivo of rats. Gentiopicroside was used as internal standard and a C18 column was employed for the separation of analytes. The selected reaction monitoring transitions were m/z 375→177, 357.1→195 for swertiamarin and the internal standard, respectively, in a positive ion mode. The results showed that swertiamarin had a good linearity in the range of 2-8000 ng/mL (r ＞ 0.997) and its limit of detection (LLOD) was 0.5 ng/mL. The developed method subsequently successfully used in the pharmacokinetic study of swertiamarin in rats after oral administration (50, 100, and 150 mg/kg). We obtained a series of pharmacokinetic parameters, and the half-time of swertiamarin was 1 h, while the oral bioavailability was between 5.6-7.6%. Six metabolites of swertiamarin were identified based on accurate mass measurements of protonated molecules and their MS/MS spectrum by ultra-high-performance chromatography/tandem quadrupole time-of-flight mass spectrometry. Furthermore, metabolites were classified into three groups and the metabolic pathway of swertiamarin was proposed. The finding may help for the understanding of effectiveness and safety of swertiamarin.

Characterization of curcumin metabolites in rats by ultra-high-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: Curcumin is a major constituent of Curcuma longa L. and is a naturally bio-active diketone. Structural changes in curcumin have been shown to result in different biological effects. The present study aims to investigate curcumin metabolites in rat plasma, bile, urine, and feces after administration of a single oral dose of curcumin (170 mg/kg). METHODS: After oral administration of curcumin, the plasma, bile, feces, and urine of the rats were collected for a certain period of time, and then subjected to a series of pretreatments. The metabolic pathway of curcumin in vivo was investigated using ultra-high-performance liquid chromatography (UHPLC) combined with electrospray ionization quadruple time-of-flight tandem mass spectrometry (ESI-QTOF-MS). RESULTS: Twelve metabolites were identified and divided into two groups: curcumin metabolites of phase Ι metabolism (M01-M08), curcumin metabolites of phase ΙΙ metabolism (M09-M12), and metabolites M02, M03 and M04 were reported for the first time. CONCLUSIONS: Results showed that curcumin metabolism can help explain the mechanism of its pharmacological effects, and that UHPLC/Q-TOF-MS can serve as an important analytical platform to gather the metabolic profile of curcumin.

Metalloporphyrin-indomethacin conjugates as new photosensitizers for photodynamic therapy.

Photodynamic therapy (PDT) is a promising cancer treatment approach with the advantages of low toxicity and noninvasive characteristics. In this study, a series of metalloporphyrin-indomethacin conjugates tethered with poly(ethylene glycol) (PEG) chains were prepared and characterized. The singlet oxygen production of the conjugates was evaluated through 2', 7'-dichlorofluorescin (DCFH) method. Because of the heavy atom effect, the metal porphyrin complexes exhibited the higher singlet oxygen (1O2) quantum yield than that of free base porphyrin. The order of 1O2 yield of the synthesized porphyrins was PtPor > PdPor > ZnPor > Por. The MTT assay using HeLa cells verified the low cytotoxicity of porphyrin-indomethacin conjugates in the dark. Upon irradiation, the platinated porphyrin (PtPor) showed the highest therapeutic activity among these conjugates, probably due to its high efficiency of 1O2 generation. The cellular uptake and subcellular localization of the conjugates were further evaluated through a confocal laser scanning microscope. The results showed that the conjugates were primarily localized in the lysosomes of HeLa cells.