AIM: To develop sensetive and specific LC-MS/MS-based methods for measurement of plasma tanshinol (TSN), protocatechualdehyde (PCA), and salvianolic acid A, B, and D (SAA, SAB, and SAD) in supporting the pharmacokinetic evaluation of Cardiotonic(superscript ®) pills, a standardized Salvia miltiorrhiza Bge.-containing cardiovascular phytomedicine.
METHODS: Electrospray ionization (ESI) patterns and efficiency of the analytes, along with their fragmentation, were investigated to achieve sensitive ESI-MS/MS detection. Sample preparation was also studied to effectively recover the analytes from plasma. Meanwhile, chromatographic conditions were optimized to avoid matrix component interference on analyte ESI-detection. The newly developed analytical methods were evaluated with respect to accuracy, precison, selectivity, sensitivity, and stability.
RESULTS: The negative ion ESI gave a high ionization efficiency for the analytes. The precursor-to-production pairs m/z 197?l35, l36?l08, 493?295, 717?519, and 4l7?197 were used for sensitive and specific SRM of TSN, PCA, SAA, SAB, and SAD, respectively. For the best measurement of the analytes, two methods were developed, i. e., one for determination of plasma TSN and the other one for simultanious determination of plasma PCA, SAA, SAB, and SAD. The sample preparation for the two quantification methoids involved acidifying the plasma sample with HCl before EtOAc-based liquid-liquid extraction; the extraction efficiency was 57.7%-61.6% for TSN from plasma or 38.0%-58.9% for PCA? SAA? SAB, and SAD in parallel from plasma. Matrix-matched standard curves of the peak area of a given analytes versus the nominal plasma concentration were linear for concentrations of TSN between 2.7ng/mL and 2 000.0ng/mL, with a correlation coefficient 0.999 and a lower limit of quantification 2.7ng/mL, while linear dynamic ranges for PCA, SAA, SAB, and SAD were 1.37 or 4.1-1000.0ng/mL, with correlation coefficients>0.99 and lower limits of quantification 1.4ng/mL for PCA and 4.1ng/mL for SAA, SAB, and SAD. Both the quantification methods demonstrated a within-run assay accuracy and presion 90%-112% and 2.5%-15%, respectively and a between-run data 91%-110% and 0.7%-9.4%, respectively. Further, the applicability of the newly developed analytical methods was demonstrated in a pilot plasma pharmacokinetic study in a beagle dog receiving an intravenous dose of injectable extract from Cardiotonic(superscript ®) pills.
CONCLUSION: Sensetive and specific LC-MS/MS-based methods for measurement of plasma TSN and for measurement of plasma PCA, SAA, SAB, and SAD in parallel are developed and validated, which are applicable to pharmacokinetic evaluation of Cardiotonic(superscript ®) pills.
Component--Plasma and Urinary Tanshinol from Salvia miltiorrhiza Bge., Can Be Used as Pharmacokinetic Markers for Cardiotonic Pills, a Cardiovascular Herbal Medicine
ABSTRCT:
Cardiotonic pills are a type of cardiovascular herbal medicine. To identify suitable pharmacokinetic marker(s) for indicating systemic exposure to Cardiotonic pills, we examined the in vivo pharmacokinetic properties of putatively active phenolic acids from the component herb Danshen (Radix Salviae Miltiorrhizae). We also performed in vitro and in silico assessments of permeability and solubility. Several phenolic acids were investigated, including: tanshinol, protocatechuic aldehyde, salvianolic acids A, B, and D, rosmarinic acid, and lithospermic acid. Plasma tanshinol exhibited the appropriate pharmacokinetic properties in dogs, including dose-dependent systemic exposure in area under concentration-time curve (AUC) and a 0.5-h elimination half-life. In rats, over 60% of intravenous tanshinol was excreted intact into the urine. In humans, we found a significant correlation between the urinary recovery of tanshinol and its plasma AUC. The absorption rate and bioavailability of tanshinol were not significantly different whether Cardiotonic pills were given orally or sublingually. The gender-specificity in plasma AUC disappeared after body-weight normalization, but the renal excretion of tanshinol was significantly greater in women than in men. Protocatechuic aldehyde was predicted to be highly permeable according to in vitro and in silico studies; however, extensive presystemic hepatic elimination and degradation in the erythrocytes led to extremely low plasma levels and poor dose proportionality. Integrated in vivo, in vitro, and in silico studies on the other phenolic acids showed poor gut permeability and nearly undetectable levels in plasma and urine. In conclusion, plasma and urinary tanshinol are promising pharmacokinetic markers for Cardiotonic pills at the tested dose levels.
1: Zhong Yao Cai. 2007 Dec;30(12):1541-4.
Salvianolic acid B attenuates cyclooxygenase-2 expression in vitro in LPS-treated human aortic smooth muscle cells and in vivo in the apolipoprotein-E-deficient mouse aorta.
Chen YL, Hu CS, Lin FY, Chen YH, Sheu LM, Ku HH, Shiao MS, Chen JW, Lin SJ.
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Inflammation plays an essential role in atherosclerosis and post-angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water-soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol-fed apoE deficient mice were investigated. In unstimulated HASMCs, COX-2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX-1 mRNA and protein. The activation of COX-2 protein synthesis in LPS-stimulated HASMCs was shown to involve the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX-2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E(2) production and NADPH oxidase activity in LPS-treated HASMCs. When apoE-deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010 +/- 0.009%) compared to the apoE-deficient group (0.114 +/- 0.043%) and there was a significant reduction in COX-2 protein expression in the thickened intima. These results demonstrate that Sal B has anti-inflammatory properties and may explain its anti-atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis. (c) 2006 Wiley-Liss, Inc.
VEGF induced hyperpermeability in bovine aortic endothelial cell and inhibitory effect of salvianolic acid B.
Qui Y, Rui YC, Zhang L, Li TJ, Zhang WD.
Department of Pharmacology, Second Medical Military University, Shanghai, China.
AIM: To examine the effect of recombinant human vascular endothelial growth factor (VEGF) on the low density lipoprotein (LDL) permeability of bovine aortic endothelial cells (BAEC) and the inhibitory effect of salvianolic acid B in vitro. METHODS: The confluence BAEC monolayers were cultured with normal medium and with medium containing VEGF or salvianolic acid B at various concentrations and for various time periods. The iodine labeled LDL flux across the monolayers was then performed, and radioactivity was measured by SN-695 automatic liquid scintillation counter. RESULTS: Addition of purified human recombinant VEGF to the BAEC monolayers could significantly increase the permeability of the monolayer to 125I-LDL (P < 0.01). The permeability-increasing activity of VEGF on the BAEC monolayers was both dose and time dependent. Salvianolic acid B could markedly inhibit the VEGF-induced hyperpermeability in BAECs (P < 0.01). CONCLUSION: VEGF plays a role in the formation and development of atherosclerosis, and salvianolic acid B has inhibitory effect on VEGF-induced hyperpermeability in BAEC.
A salvianolic acid B-rich fraction of Salvia miltiorrhiza induces neointimal cell apoptosis in rabbit angioplasty model.
Hung HH, Chen YL, Lin SJ, Yang SP, Shih CC, Shiao MS, Chang CH.
Department of Medicine, National Yang-Ming University, Taipei, Taiwan.
Apoptosis has been suggested to participate in stabilizing cell number in restenosis. Salvia miltiorrhiza (SM) Bunge which is a Chinese herb widely used for the treatment of cardiovascular disorders contains a potent antioxidant, Salvianolic acid B. To determine whether the antioxidant affects vascular apoptosis, the present study examined the frequency of apoptotic cell death in atherosclerotic plaques and in restenotic lesions of cholesterol-fed rabbits. New Zealand White rabbits were treated with a normal diet (normal), a 2% cholesterol diet (HC), a 2% cholesterol diet and endothelial denudation (HC-ED), a 2% cholesterol diet with 5% water-soluble extract of SM (4.8 g/Kg B.W./day) and endothelial denudation (HC-ED-SM), or with a 2% cholesterol diet containing probucol (0.6 g/kg B.W./day) and endothelial denudation (HC-ED-probucol). Apoptosis and associated cell types were examined in serial paraffin sections by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry. The expression of p53, an apoptosis-related protein, was also examined. Apoptosis was mainly detected in the neointima of the three groups with endothelial denudation. The percentage of apoptotic cells in SM-treated group (68.5+/-5.9%) was significantly higher than that of normal (0%), HC (1.9+/-1.2%), HC-ED (46.1+/-5.4%), and probucol-treated (32.8+/-3.9%) groups. The SM treatment markedly reduced the thickness of the neointima which was mainly composed of smooth muscle cells with few macrophages. In accordance with the apoptotic cell counts, positive immunoreactivity for p53 was observed in restenotic lesions from HC-ED, SM-treated and probucol-treated groups but not in the intima of the other two groups. These results suggest that the treatment with salvianolic acid B-rich fraction of SM induces apoptosis in neointima which in turn may help prevent the neointimal thickening.
Increase of vitamin E content in LDL and reduction of atherosclerosis in cholesterol-fed rabbits by a water-soluble antioxidant-rich fraction of Salvia miltiorrhiza.
Wu YJ, Hong CY, Lin SJ, Wu P, Shiao MS.
Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan, ROC.
Antioxidants that prevent LDL from oxidation may reduce atherosclerosis. Salvia miltiorrhiza Bge. is a Chinese herb widely used for the treatment of atherosclerosis-related disorders. Salvianolic acid B (Sal B), a water-soluble polyphenolic antioxidant isolated from the roots of this plant, was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals and inhibit LDL oxidation more effectively than probucol. In order to evaluate the antiatherogenic potential, New Zealand White rabbits were fed for 12 weeks a normal diet, a high cholesterol diet, a high cholesterol diet containing 1% probucol, or a high cholesterol diet containing a 5% water-soluble extract of S miltiorrhiza (SM). Both SM and probucol feeding reduced plasma cholesterol. LDLs from the SM-treated group were more resistant to Cu2+-induced oxidation and contained more vitamin E (21.7+/-2.1 mmol/micromol LDL cholesterol) than did LDLs from the high cholesterol diet group (9.6+/-1.8 nmnol/micromol LDL cholesterol) (P<.005). Endothelial damage, determined at week 6, was reduced by 53% in the SM group (P<.01). SM treatment reduced the atherosclerotic area in the abdominal aorta by 56% (P<.005) and cholesterol deposition in the thoracic aorta by 50% (P<.005). The severity of atherosclerosis in the SM group was significantly reduced after adjustment by using cholesterol exposure as an index of the cholesterol-lowering effect. This study concludes that the reduction of atherosclerosis by SM relies not only on its cholesterol-lowering effect but more heavily on its antioxidant potential to prevent endothelial damage and inhibit LDL oxidative modification in hypercholesterolemic animals.