Structural characterization of an inulin neoseries-type fructan from Ophiopogonis Radix and the therapeutic effect on liver fibrosis in vivo.

Ophiopogonis Radix is a well-known Traditional Chinese Medicine and functional food that is rich in polysaccharides and has fructan as a characteristic component. In this study, an inulin neoseries-type fructan designated as OJP-W2 was obtained and characterized from Ophiopogonis Radix, and its potential therapeutic effect on liver fibrosis in vivo were investigated. Structural studies revealed that OJP-W2 had a molecular weight of 5.76 kDa and was composed of glucose and fructose with a molar ratio of 1.00:30.87. Further analysis revealed OJP-W2 has a predominantly lineal (1-2)-linked ß-D-fructosyl units linked to the glucose moiety of the sucrose molecule with (2-6)-linked ß-D-fructosyl side chains. Pharmacological studies revealed that OJP-W2 exerted a marked hepatoprotective effect against liver fibrosis, the mechanism of action was involved in regulating collagen deposition (α-SMA, COL1A1 and liver Hyp contents) and TGF-ß/Smads signaling pathway, alleviating liver inflammation (IL-1ß, IL-6, CCL5 and F4/80) and MAPK signaling pathway, and inhibiting hepatic apoptosis (Bax, Bcl-2, ATF4 and Caspase 3). These data provide evidence for expanding Ophiopogonis Radix-acquired fructan types and advancing our understanding of the specific role of inulin neoseries-type fructan in liver fibrosis therapy.

Protective effects and regulatory mechanisms of Shen-shuai-yi recipe on renal fibrosis in unilateral ureteral obstruction-induced mice.

Renal fibrosis (RF) is a common pathological feature of chronic kidney disease (CKD), which remains a major public health problem. As now, there is still lack of chemical or biological drugs to reverse RF. Shen-shuai-yi Recipe (SSYR) is a classical Chinese herbal formula for the treatment of CKD. However, the effects and mechanisms of SSYR in treating RF are still not clear. In this study, the active constituents SSYR for treating RF were explored by UHPLC-Q-Orbitrap HRMS. Bioinformatics analyses were employed to analyze the key pharmacological targets and the core active constituents of SSYR in the treatment of RF. In experimental validation, vehicle or SSYR at doses of 2.12 g/kg/d and 4.25 g/kg/d were given by orally to unilateral ureteric obstruction (UUO) mice. 13 days after treatment, we detected the severity of renal fibrosis, extracellular collagen deposition and pre-fibrotic signaling pathways. Bioinformatics analysis suggested that signal transducer and activator of transcription 3 (STAT3) was the core target and lenticin, luteolin-7-O-rutinoside, hesperidin, kaempferol-3-O-rutinoside, and 3,5,6,7,8,3',4'-heptamethoxyflavone were the key constituents in SSYR for treating RF. SSYR significantly reduced the expressions of fibronectin (FN), α-smooth muscle actin (α-SMA), collagen-I and alleviated renal interstitial collagen deposition in UUO kidneys. In mechanism, SSYR potently blocked the phosphorylation of STAT3 and Smad3 and suppressed the expression of connective tissue growth factor (CTGF). Collectively, SSYR can ameliorate RF via inhibiting the phosphorylation of STAT3 and its downstream and reducing the collagen deposition, suggesting that SSYR can be developed as a novel medicine for treating RF.

Astragalus saponins and its main constituents ameliorate ductular reaction and liver fibrosis in a mouse model of DDC-induced cholestatic liver disease.

Cholestatic liver disease (CLD) is a chronic liver disease characterized by ductular reaction, inflammation and fibrosis. As there are no effective chemical or biological drugs now, majority of CLD patients eventually require liver transplantation. Astragali radix (AR) is commonly used in the clinical treatment of cholestatic liver disease and its related liver fibrosis in traditional Chinese medicine, however its specific active constituents are not clear. Total astragalus saponins (ASTs) were considered to be the main active components of AR. The aim of this study is to investigate the improvement effects of the total astragalus saponins (ASTs) and its main constituents in cholestatic liver disease. The ASTs from AR was prepared by macroporous resin, the content of saponins was measured at 60.19 ± 1.68%. The ameliorative effects of ASTs (14, 28, 56 mg/kg) were evaluated by 3, 5-Diethoxycarbonyl-1, 4-dihydrocollidine (DDC)-induced CLD mouse model. The contents of hydroxyproline (Hyp), the mRNA and protein expression of cytokeratin 19 (CK19) and α-smooth muscle actin (α-SMA) in liver tissue were dose-dependently improved after treatment for ASTs. 45 astragalus saponins were identified in ASTs by UHPLC-Q-Exactive Orbitrap HRMS, including astragaloside I, astragaloside II, astragaloside III, astragaloside IV, isoastragaloside I, isoastragaloside II, cycloastragenol, etc. And, it was found that ductular reaction in sodium butyrate-induced WB-F344 cell model were obviously inhibited by these main constituents. Finally, the improvement effects of astragaloside I, astragaloside II, astragaloside IV and cycloastragenol (50 mg/kg) were evaluated in DDC-induced CLD mice model. The results showed that astragaloside I and cycloastragenol significantly improved mRNA and protein expression of CK19 and α-SMA in liver tissue. It suggested that astragaloside I and cycloastragenol could alleviate ductular reaction and liver fibrosis. In summary, this study revealed that ASTs could significantly inhibit ductular reaction and liver fibrosis, and astragaloside I and cycloastragenol were the key substances of ASTs for treating cholestatic liver disease.

Pharmacokinetics and anti-liver fibrosis characteristics of amygdalin: Key role of the deglycosylated metabolite prunasin.

BACKGROUND: Amygdalin (Amy) is a cyanoside and is one of the chief active ingredients in Persicae Semen, Armeniacae Semen Amarum, and Pruni Semen. Amy has extensive and remarkable pharmacological activities, including against anti-hepatic fibrosis. However, the pharmacokinetic and anti-liver fibrosis effects of Amy and its enzyme metabolite prunasin (Pru) in vivo have not been studied and compared, and studies on Pru are limited. PURPOSE: To investigate the pharmacokinetic characteristics and anti-liver fibrosis effect of Amy and its metabolite Pru in vivo and in vitro, and elucidate whether the metabolism of Amy in vivo for Pru is activated. METHODS: Pru was prepared from Amy via the enzymatic hydrolysis of ß-glucosidase, and isolated by silica gel column chromatography. An efficient and sensitive ultrahigh-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry was developed and validated to determine simultaneously Amy and Pru in rat plasma after dosing intravenously and orally for pharmacokinetic studies. The affinities of Amy and Pru for ß-glucosidase were compared by enzyme kinetic experiments to explain the possible reasons for the differences in pharmacokinetic behavior. In vitro, the inhibitory effects of Amy and Pru on hepatic stellate cell activation and macrophage inflammation on JS1 and RAW 264.7 cells were determined. In vivo, the ameliorative effects of Amy and Pru on liver fibrosis effects were comprehensively evaluated by CCl4-induced liver fibrosis model in mice. RESULTS: The standard curves of Amy and Pru in rat plasma showed good linearity within the concentration range of 1.31-5000.00 ng/ml, with acceptable selectivity, carry-over, detection limit and quantification limits, intra- and inter-day precision, accuracy, matrix effect, and stability. The Cmax and AUC(0-∞) of Pru (Cmax = 1835.12 ± 268.09 ng/ml, AUC(0-∞) = 103,913.17 ± 14,202.48 ng•min/ml) were nearly 79.51- and 66.22-fold higher than those of Amy (Cmax = 23.08 ± 5.08 ng/ml, AUC(0-∞) = 1569.22 ± 650.62 ng•min/ml) after the oral administration of Amy. The oral bioavailability of Pru (64.91%) was higher than that of Amy (0.19%). The results of enzyme hydrolysis kinetics assay showed that the Vmax and Km of Pru were lower than those of Amy in commercial ß-glucosidase and intestinal bacteria. In vitro cellular assays showed that Amy and Pru were comparable in inhibiting the NO production in the RAW264.7 cell supernatant and the mRNA expression of α-SMA and Col1A1 in JS1 cells. Amy and Pru were also showed comparable activity in ameliorating CCl4-induced liver fibrosis in mice. CONCLUSION: The pharmacokinetic characteristics of Amy and Pru in rat plasma were significantly different. After the separate gavage of Amy and Pru, Amy was absorbed predominantly as it's metabolite Pru, whereas Pru was absorbed predominantly as a prototype. The anti-liver fibrosis effects of Amy and its deglycosylated metabolite Pru were comparable in vivo and in vitro. The deglycosylated activated metabolite Pru of Amy plays an important role in anti-liver fibrosis. These findings will facilitate the further exploitation of Amy and Pru.