Bioactive solanidane steroidal alkaloids from Solanum lyratum.

Six previously unreported solanidane steroidal alkaloids, namely lyrasolanosides A-F, were isolated from Solanum lyratum. In addition, five known steroidal alkaloids were also identified. The structures of these compounds were determined through the use of NMR, HRESIMS,UV, IR and ECD analysis. To assess their bioactivities, the cytotoxic effects of the six previously unreported compounds were evaluated on A549 cells. The results revealed that lyrasolanoside B (2) exhibited the highest potency among them. Lyrasolanoside B (2) exhibited significant inhibition of cell migration, invasion, and adhesion dramatically. Mechanistically, it was found to suppress the activity of JAK2/STAT3 signaling pathway by downregulating the expression of phosphorylated JAK2/STAT3 in an exosome-dependent manner. In addition, lyrasolanoside B (2) was found to significantly upregulate the expression of E-cadherin and downregulate the expression of N-cadherin and vimentin. These findings indicate that lyrasolanoside B (2) inhibits the metastasis of A549 cells by suppressing exosome-mediated EMT. These findings suggest that lyrasolanoside B (2) may inhibit the metastasis of lung cancer by regulating A549-derived exosomes.

New steroidal glycosides from the fibrous roots of Ophiopogon japonicus.

Two new steroidal glycosides (named fibrophiopogonins A, B), along with one known glycoside, were isolated from the fibrous roots of Ophiopogon japonicus (Liliaceae). Comprehensive spectroscopic analysis, including 2D NMR spectroscopy, and the results of acid hydrolysis allowed the chemical structure of the compounds to be assigned as 26-[(O-ß-D-glucopyranosyl-(1 → 6)-D-glucopyranosyl)]-barogenin- 3-O-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside and (25R)-26-[(O- ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl)]- 3ß,22α,26- trihydroxyfurost- 5-ene-3-O-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside. This is the first isolation of a cholestane glycoside with disaccharide moiety from a Ophiopogon species. The cytotoxic activities of 1~3 against A375 and MCF-7 cells are described.

Neuroprotective effect of α-mangostin on mitochondrial dysfunction and α-synuclein aggregation in rotenone-induced model of Parkinson's disease in differentiated SH-SY5Y cells.

The study was designed to evaluate the protective effect of α-mangostin and explore its mechanism in an in vitro model of Parkinson's disease (PD) induced by rotenone. SH-SY5Y cells were treated with rotenone and α-mangostin for 24 h. α-Mangostin significantly and concentration-dependently inhibited rotenone-induced cytotoxicity. The rotenone-induced aggregation of α-synuclein and loss of TH were alleviated by α-mangostin. α-Mangostin treatment also reversed the rotenone-induced overproduction of reactive oxygen species, activation of caspases (-8 and -3) and mitochondrial dysfunction, reflected by decrease in mitochondrial membrane potential and cellular ATP levels. These findings suggest that α-mangostin has neuroprotective effects against PD-related neuronal injury.

Salvianolic acid A promotes the acceleration of neovascularization in the ischemic rat myocardium and the functions of endothelial progenitor cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely used Chinese herbal medicines in clinical, containing phenolic compounds and potent antioxidant properties. Salvianolic acid A (SAA) is the most potent component of SM. A modern experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by the use of "angiogens", mediators that induce the formation of blood vessels, or angiogenesis. Studies demonstrated that coronary collateral vessels protect ischemic myocardium after coronary obstruction; therefore, we sought to examine whether SAA could stimulate myocardial angiogenesis. MATERIALS AND METHODS: Male Sprague-Dawley rats myocardial infarct (MI) induced by ligation of left anterior descending coronary artery (LAD) were randomly divided into five groups: sham-operated group; LAD occlusion + administration of physiological saline (vehicle treated group); LAD occlusion + administration of different concentrations of SAA (10, 5.0 and 2.5mg/kg/d). Infarct size and capillary density in the infarct region were measured with a previous experimental method. Immunohistological analysis was performed to measure vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) expressions. The secretion of matrix metalloproteinase type X (MMP-9) was evaluated in serum of post-ischemic rats. We also performed the experiments of SAA on rat endothelial progenitor cells (EPCs) numbers and the capacity of migration and vasculargenesis. RESULTS: SAA potentiated the ischemia-induced neovascularization after 1week post-operation when compared to vehicle treated group. This effect could be attributed to an increased formation of VEGF, VEGFR-2, and MMP-9 as well as the promotion of numbers and functions of EPCs. CONCLUSION: These findings show that SAA has potent proangiogenic properties by promoting the expression of proangiogenic factors, and the functions of EPCs, indicating that SAA might contribute to the protective effect against coronary disease. Chemical compound studied in this paper is salvianolic acid A (PubChem CID: 5281793).

Two new furostanol saponins from the fibrous root of Ophiopogon japonicus.

Two new furostanol saponins ophiopogonins J (1) and K (2) were isolated from the fibrous roots of Ophiopogon japonicus. The structures of 1 and 2 were established as (25R)-26-O-[(ß-D-glucopyranosyl-(1 --> 2)-ß-D-glucopyranosyl)]-14-hydroxy-furost-5,20(22)-diene 3-O-[α-L-rhamnopyranosyl-(1 --> 2)]-ß-D-glucopyranoside (1), and (25R)-26-O-[(ß-D-glucopyranosyl-(1 --> 2)-ß-D-glucopyranosyl)]-furost-5,20(22)-diene 3-O-α-L-rhamnopyranosyl-(1 --> 2)[(ß-D-xylopyranosyl-(1 --> 4)-ß-D-glucopyranoside)] (2) on the basis of spectroscopic means including HRESIMS, 1D, and 2D NMR experiments.

Two new furostanol glycosides from the fibrous root of Ophiopogon japonicus (Thunb.) Ker-Gawl.

Two new furostanol glycosides, ophiopogonins H (1) and I (2), were isolated from the fibrous root of Ophiopogon japonicus. The structures of 1 and 2 were established as (25R)-26-[(O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl)]-22α-hydroxyfurost-5-ene-3-O-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside and (25R)-26-[(O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl)]-20α-hydroxyfurost-5,22-diene-3-O-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside on the basis of spectroscopic means including HR-ESI-MS, 1D and 2D NMR experiments.

Pro-angiogenic actions of Salvianolic acids on in vitro cultured endothelial progenitor cells and chick embryo chorioallantoic membrane model.

AIM OF THE STUDY: Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely-used Chinese herbal medicines in clinical practice. In this study we aimed to demonstrate the pro-angiogenic effects of Salvianolic acids (SAs) to treat illnesses such as ischemic cardiovascular diseases, the main active components of aqueous extract of SM. MATERIALS AND METHODS: To do this, new-born rat spleen mononuclear cells were isolated and endothelial progenitor cells (EPCs) were expanded (not more than 24h) SD. Then the pro-angiogenic activities of SAs were evaluated on in vitro cultured EPCs and chick embryo chorioallantoic membrane (CAM) model. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs identification was also tested by ultrastructural analyses. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. RESULTS: EPCs phenotype was confirmed by the presence of double positive cells for DiI-acLDL uptake and lectin binding and identification of Weibel-Palade body in cytoplasm by ultrastructural analyses. Incubation of EPCs with SAs increased the number of EPCs and promoted EPCs migratory, adhesive and in vitro vasculogenesis capacity. SAs also promoted angiogenesis as evidenced by CAM model. CONCLUSIONS: The results of the present study suggest that SAs may have utility for therapeutic postnatal vasculogenesis of ischemic tissue, contributing to the clinical benefit of SM therapy in patients with coronary artery disease.

[Prescription compatibility effect on the major components absorption in danshen extract and their identification].

An improved everted gut sac method was applied to the study of prescription compatibility effect on the major components in Danshen extracts. With the separation and detection by HPLC-ECD, 5 major peaks could be detected in intestinal absorbed solution after prescription administration. Following the identification by HPLC-MS/MS, peak 2, 3, 4, and 5 were rosmaric acid, lithospermic acid, salvianolic acid B, and salvianolic acid A, respectively, which also confirmed with reference standards of those components. Through paralleling substance identification, peak 2, 3, 4, and 5 could be found as the major components in Danshen extracts, except Salvianolic acid E which is undetectable in intestinal solution. The contents of peak 2, 3, and 4 did not show difference before and after compatible prescription administrated, where the peak 5 had a significant increase in the same process. Those results revealed that peak 5, salvianolic acid A, might lead to an increasing pharmacological effect after prescription compatibility.

[Evaluation of different combinations of components of Chinese formulation shuangshentongguan by using AUC values].

A sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was applied for the quantification of each component: tetrahydropalmatine (THP), dehydrocorydaline (DHC), salvianolic acid B (SAB), ginsenoside Rg1, Re, Rb1 and Rd in the Chinese herbal component SSTG (Shuangshentongguan) with different combinations. The pharmacokinetic data were analyzed with WinNonlin 5.2 software. The results showed that combination can increase the THP AUC value while the AUC values of SAB, ginsenoside Rg1, Re, Rb1 and Rd were reduced. These results showed significant differences. The AUC value of ginsenoside Rb1 was increased when combined with Danshen or Yanhusuo, but reduced when combined with Danshen and Yanhusuo. The DHC concentration in serum was too low to be determined.

Two new homoisoflavonoids from the fibrous roots of Ophiopogon japonicus (Thunb.) Ker-Gawl.

Two new homoisoflavonoids ophiopogonone D (1) and ophiopogonanone G (2) were isolated from the fibrous roots of Ophiopogon japonicus. The structures of these two compounds were determined on the basis of spectroscopic means including HR-ESI-MS, 1D, and 2D NMR experiments. The cytotoxic activities of 1 and 2 against Hela and Hep2 cells are described.

[Pharmacokinetic studies of tetrahydropalmatine and dehydrocorydaline in rat after oral administration of yanhusuo extraction by LC-MS/MS method].

A sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of tetrahydropalmatine (THP) and dehydrocorydaline (DHC) in rat plasma. The compounds were simply pretreated by protein precipitation using acetone. Chromatographic separation was achieved on a reversed-phase SB-C18 column with the mobile phase of acetonitrile-ammonium acetate (0. 1% acetic acid) and step gradient elution resulted at a flow rate of 0.80 mL x min(-1). A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 356. 2 --> m/z 191.9 and m/z 366. 2 --> m/z 350.2 for THP and DHC respectively. The method showed excellent linearity over the concentration range 1-1 000 ng x mL(-1) of two components (r = 0.994 for THP and r = 0.992 for DHC). The low limits of quantification were both 1 ng x mL(-1). The extract recoveries of analytes were from 71.71% to 91.59% for THP and from 83.27% to 103.15% for DHC. The precisions, the accuracy and the stability of the analytes meet the requirements. The method was applied to a pharmacokinetic study of THP and DHC after oral administration of the total alkaloid extraction of Rhizoma Corydalis (Yanhusuo). The AUC were (1.90 +/- 0.04), (2.58 +/- 0.08) and (4.34 +/- 0.19) mg x L(-1) h for low, medium and high doses of THP, respectively. While the DHC concentrations in plasma of low dose and medium dose were too lower to be detected, the AUC of high dose was (0.0896 +/- 0.0002) mg x L(-1) h.

Magnesium lithospermate B is excreted rapidly into rat bile mostly as methylated metabolites, which are potent antioxidants.

To elucidate the in vivo pharmacological activities of magnesium lithospermate B (MLB), an active constituent of Radix Salviae Miltiorrhizae, in the rat, its metabolic fate both in vivo and in vitro was investigated. High-performance liquid chromatography revealed that four major metabolites with lower polarity were excreted into bile after intravenous and oral administration of MLB. The metabolites present in combined samples of bile from rats after intravenous injection were isolated and purified by column chromatography and identified as four meta-O-methylated products, namely 3-monomethyl- (M1), 3,3"'-dimethyl- (M2), 3,3"-dimethyl-, and 3,3",3"'-trimethyl-lithospermic acid B according to their spectroscopic characteristics ((1)H, (13)C NMR, (1)H-(1)H correlation spectroscopy, (1)H-detected multiple quantum coherence, and heteronuclear multiple bond coherence combined with positive ion fast atom bombardment-mass spectroscopy). After administration of MLB at an intravenous dose of 4 mg/kg or an oral dose of 100 mg/kg, the total biliary recovery of the four metabolites after 30 h reached 95.5 +/- 2.4% (with approximately 90% recovered within 2 h) or 5.5 +/- 0.7%, respectively. The metabolic pathway was proposed to involve sequential formation of the four methylated metabolites. Incubation of MLB, M1, M2, or M4 in rat hepatic cytosol in the presence of S-adenosyl-l-methionine demonstrated the formation of all four metabolites, which indicated that the enzyme responsible for the biotransformation is catechol O-methyltransferase. MLB and its main metabolites M1 and M2 showed potent 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activities, the activity of M1 being stronger than those of caffeic acid (the monomer form of MLB) and alpha-tocopherol (a representative antioxidant) but weaker than that of MLB. The rapid and high biliary excretion levels of these metabolites suggested that they could undergo enterohepatic circulation in rats and that they might thereby be largely responsible for the pharmacological effects of MLB.

Extremely low bioavailability of magnesium lithospermate B, an active component from Salvia miltiorrhiza, in rat.

We assessed the bioavailability of magnesium lithospermate B (MLB), a main polyphenolic component of Salvia miltiorrhiza and a potent antioxidant having various pharmacological activities, to evaluate its action in vivo. The plasma concentrations of lithospermic acid B (LSB) showed a biexponential decrease after intravenous administration of MLB to rats at doses of 4 and 20 mg/kg. The values of area under the concentration-time curve (AUC; 87.8 +/- 10.9 and 1130 +/- 329 microg.min/mL), total body clearance (CL (tot); 55.52 +/- 7.07 and 23.51 +/- 5.98 mL/min/kg), and distribution volume at steady state (V (ss); 7.60 +/- 1.03 and 3.61 +/- 1.16 L/kg) suggested non-linear pharmacokinetics between the two doses. After oral administration of MLB at a high dose of 100 mg/kg, the mean AUC was barely 1.26 +/- 0.36 microg.min/mL. Absolute bioavailability of MLB was calculated to be 0.0002 from the AUC values after both intravenous dosing at 20 mg/kg and oral dosing at 100 mg/kg. The extremely low bioavailability was caused mainly by poor absorption from the rat gastrointestinal tract; about 65 % of the dose was retained in the tract even 4 h after oral administration, and most of the dose was retained even 20 min after infusion in an in situ jejunal loop experiment. Urinary and biliary excretion of LSB were only 0.70 % +/- 0.26 % and 5.10 % +/- 2.36 %, respectively, over a 30 h time period after intravenous injection despite the large CL (tot) and V (ss) values, and were much less (0.010 % +/- 0.001 % and 0.12 % +/- 0.04 %) after oral dosing. These findings suggest that extensive metabolism, including a first-pass effect, and wide distribution of LSB besides the poor absorption contributed significantly to the extremely low systemic bioavailability.