Peroxynitrite scavenging activity of lithospermate B from Salvia miltiorrhiza

Peroxynitrite (ONOO-) is produced by the reaction of superoxide (O2-) with nitric oxide. ONOO- damages proteins through nitration or oxidation. For protection from ONOO- induced protein modifications, ONOO- scavengers should be supplemented. Evidence was obtained that lithospermate B extracted from Salvia miltiorrhiza showed the strongest scavenging activity among its constituents. Its ONOO- scavenging activity is via an electron donation mechanism. A dihydroxyl group and a double bond seem to be essential structure requirements. The data from the experiments further confirmed a protective effect of lithospermate B on bovine serum albumin and low-density lipoprotein against ONOO-. This study demonstrated that lithospermate B with hydroxyl groups and double bonds exerts an anti-nitration effect by scavenging ONOO-.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

中药丹参有效成分——丹参酮ⅡA的新的全合成方法

A novel synthesis of Tanshinone IIA via o-quinone(4) in eight steps is described. The reaction sequence is outlined in the text.     

Salvianolic acid B, an aqueous component of danshen (Salvia miltiorrhiza), relaxes rat coronary artery by inhibition of calcium channels

In this study, we have investigated the mechanism of action through which salvianolic acid, a constituent of the medicinal herb danshen (Salvia miltiorrhiza), causes relaxation of isolated coronary artery rings precontracted with 1 microM 5-hydroxytryptamine (5-HT). The vasorelaxant effects of salvianolic acid B were also compared with the aqueous extract of danshen. Extraction of the water-soluble fraction from danshen provided a yield of 17.5%. The amount of salvianolic acid B determined in this aqueous extract was 3.9%, and the extract was 6.3 times less potent than salvianolic acid B in relaxing 5-HT-precontracted coronary artery rings; IC(50) values were 930.3+/-133.5 microg/ml and 147.9+/-17.4 microg/ml, respectively. Removal of the endothelium did not significantly affect their vasodilator potencies; IC(50) values were 842.1+/-123.8 mictog/ml and 160.3+/-25.9 microg/ml. On the other hand, a potassium channel inhibitor tetraethylammonium (TEA, 10 mM) shifted their concentration-response curves by 1.7 and 2.9 folds. The possible involvement of Ca(2+) channels was investigated in artery rings incubated with Ca(2+)-free buffer and primed with 1 microM 5-HT or 60 mM KCl for 5 min prior to adding CaCl(2) to elicit contraction. In 5-HT-primed preparations, 2 mg/ml danshen aqueous extract and 0.72 mg/ml salvianolic acid B abolished the CaCl(2)-induced vasoconstriction, whereas, in KCl-primed preparations, 10 mg/ml danshen aqueous extract and 1.44 mg/ml salvianolic acid B produced 90% inhibition. These findings suggest the vasorelaxant effects of danshen aqueous extract and salvianolic acid B were produced by inhibition of Ca(2+) influx in the vascular smooth muscle cells. The opening of K(+) channels had a minor contribution to their effects, but endothelium-dependent mechanisms were not involved. The high vasodilator potency and the quantity of salvianolic acid B contained in danshen aqueous extract suggest it is an important vasodilator constituent in this medicinal herb.     

Beneficial effects of danshensu, an active component of Salvia miltiorrhiza, on homocysteine metabolism via the trans-sulphuration pathway in rats

Background and purpose:

Elevated plasma total homocysteine (tHcy) level has been established as an independent risk factor for cardiovascular diseases. Danshensu, an active ingredient of Salvia miltiorrhiza, shows wide cardiovascular benefit. However, in terms of its own methylation, danshensu could elevate tHcy level, which would act against its cardiovascular benefit, thus posing a ‘therapeutic paradox’. As this paradox has not been fully assessed, we have evaluated the effects of danshensu on tHcy levels to uncover the underlying mechanisms.

Experiment approach:

We evaluated the influence of danshensu on homocysteine (Hcy) metabolism in rats with normal tHcy levels and in rat models of elevated tHcy (single intravenous methionine loading model and a hyperhomocysteinemic model after 3 weeks methionine dosing, with and without 3 weeks of danshensu treatment). We also quantified some metabolic intermediates (S-adenosyl methionine, S-adenosyl-l-homocysteine, cysteine and glutathione) relevant to Hcy metabolism in rat liver and kidney.

Key results:

Acute treatment with a single dose of danshensu in rats with normal tHcy did not change plasma tHcy. In contrast, danshensu significantly lowered tHcy in rats with elevated tHcy. The relatively higher cysteine and glutathione levels after treatment with danshensu indicated that its tHcy-lowering effect was via increased activity of the trans-sulphuration pathway.

Conclusions and implications:

Our results suggested that danshensu may act both acutely to increase trans-sulphuration and after chronic exposure to up-regulate the activity of the trans-sulphuration enzymes. The tHcy-lowering effect of danshensu is another cardiovascular benefit provided by S. miltiorrhiza and suggests a potential tHcy-lowering therapy.     

Antidepressant-like effects of magnesium lithospermate B in a rat model of chronic unpredictable stress

Abstract

Context: Magnesium lithospermate B (MLB), an active polyphenol acid of Danshen [Radix Salviae miltiorrhizae (Labiatae)], shows neuroprotective and anti-inflammatory effects in vivo and in vitro.

Objective: We hypothesized that MLB might exert antidepressant-like effects by targeting the neuroinflammatory signals.

Materials and methods: Sprague-Dawley rats were subjected to the chronic unpredictable stress (CUS) protocol. Rats in the control group received no CUS during the whole experiment. In the model group, rats were exposed to CUS for 7 weeks. From the beginning of the 5th week, model group rats were randomly grouped and subjected to different treatments. In the experiment, control and model group rats were intraperitoneally (i.p.) injected with saline. MLB was dissolved in saline to give a final concentration, and the rats were injected (i.p.) with 15, 30, or 60?mg/kg MLB once a day for 3 weeks.

Results: MLB administration significantly reduced: (1) the immobility time in the forced swimming test (19?s, p?<?0.05); (2) the immobility time in the tail suspension test (76.3?s, p?<?0.05); (3) the corticosterone (CORT) concentrations in the serum (21.7?nmol/L, p?>?0.05); (4) the pro-inflammatory cytokine levels in the serum – TNF-α (92.1?pg/ml, p?<?0.05), IL-1β (86.9?pg/ml, p?<?0.05), and IL-6 (93.8?pg/ml, p?<?0.05); (5) pro-inflammatory cytokine levels in tissue – TNF-α (3.2?pg/mg protein, p?<?0.05), IL-1β (1.5?pg/mg protein, p?>?0.05), and IL-6 (6.3?pg/mg protein, p?<?0.05); and (6) phospho-NF-κB (1.6, p?<?0.05) and phospho-IκB-α (0.4, p?<?0.05) expression in tissue.

Discussion and conclusion: The results suggested that MLB might exert therapeutic actions on depression-like behavior and the HPA axis hyperactivity in CUS rats, and the mechanisms underlying the antidepressant-like effects of MLB might be mediated by regulation of the expression of NF-κB and IκB-α in rats.     

Pharmacokinetics of lithospermic acid B isolated from Salvia miltiorrhiza in rats

The absorption and pharmacokinetics of an active component of Salvia miltiorrhiza, lithospermic acid B (LSB), was investigated after intravenous and oral administration of doses of 10 or 50 mg LSB/kg to rats. Concentrations of LSB were determined by a validated liquid chromatography/mass spectrometry (LC/MS/MS) assay method. After intravenous administration of 50 mg/kg, dose-normalized (10 mg/kg) area under the curve (AUC) (993 microg.min/ml) was significantly greater than that at 10 mg/kg (702 microg.min/ml). The slower clearance Cl-at 50 mg/kg could be due to saturable metabolism of LSB in rats, and this could be supported by significantly slower Cl(NR) and significantly greater 24-h urinary excretion of LSB at 50 mg/kg than at 10 mg/kg. Following oral administration of LSB, the extent of LSB recovered from the entire gastrointestinal tract at 24 h ranged from 41.2% to 23.3%. Although LSB was not detected (limit of quantitation 10 ng/ml) in plasma after oral dose of 10 mg/kg, the absolute oral bioavailability at 50 mg/kg was 5%. Since LSB was shown to have low permeability through the Caco-2 cell monolayers, the low bioavailability of LSB could be due to poor absorption and metabolism.     

Structure-activity relationship of miltirone, an active central benzodiazepine receptor ligand isolated from Salvia miltiorrhiza Bunge (Danshen).

Twenty one o-quinonoid-type compounds and one coumarin-type compound related to miltirone (1) have been synthesized with the aim to identify the key structural elements involved in miltirone's interaction with the central benzodiazepine receptor. On the basis of their inhibition of [3H]flunitrazepam binding to bovine cerebral cortex membranes, it is apparent that ring A of miltirone is essential for affinity. Although increasing the size of ring A from six-membered to seven- and eight-membered is well-tolerated, the introduction of polar hydroxyl groups greatly reduces binding affinity. The presence of 1,1-dimethyl groups on ring A is, however, not essential. On the other hand, the isopropyl group on ring C appears to be critical for binding as its removal decreases affinity by more than 30-fold. It can, however, be replaced with a methyl group with minimal reduction in affinity. Finally, linking ring A and B with a -CH2CH2- bridge results in analogue 89, which is 6 times more potent than miltirone at the central benzodiazepine receptor (IC50 = 0.05 microM).     

Pharmacokinetics of magnesium lithospermate B after intravenous administration in beagle dogs

AIM: To develop a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the phar-macokinetic study of magnesium lithospermate B (MLB), and study the pharmacokinetics of MLB after iv administration in beagle dogs. METHODS: Each beagle dog was iv administered MLB 3, 6, and 12 mg/kg random. The serum drug concentration was determined by specific liquid chromatography-tandem mass spectrometry (LC-MS/ MS) assays. The pharmacokinetic parameters were calculated by Drug and Statistics version 1.0 program. RESULTS: The calibration curve for MLB was linear over a range of 16-4096μg/L with coefficients of correlation >0.999. The intra- and inter-day precisions (CV) of analysis were <10 %, and accuracy ranged from 90 % to 113 %. After iv administration of MLB at the doses of 3, 6, and 12 mg/kg, the C0 values for MLB were estimated to be of 24, 47, and 107 mg/L, respectively. The AUC increased with the increasing doses for iv administration, and the mean AUC0-t values were 109.3, 247.9, and 5     

Hydrolytic kinetics of lithospermic acid B extracted from roots of Salvia miltiorrhiza

The hydrolytic kinetics of lithospermic acid B (LAB) extracted from the roots of Salvia miltiorrhiza (Chinese herb: danshen) was investigated by using reversed-phase high-performance liquid chromatography (HPLC) with UV-vis detection. The influences of initial drug concentration, pH and temperature on hydrolysis of LAB were studied in aqueous solutions. The results showed that initial concentration of LAB has no effect on the degradation rate at pH 2.0. The hydrolysis followed pseudo-first-order kinetics at 90 degrees C. The log k(obs)-pH profile indicated that the optimal stability range was at pH 2.0-5.0. The rate constant of overall hydrolysis as a function of temperature under the given conditions obeyed the Arrhenius equation. Analysis of the acid-induced degraded solution of LAB by liquid chromatography-mass spectrometry (LC-MS) revealed at least four degradation products [M-H](-) ion at m/z 197, 137, 537 and 537, respectively. Three of these degradation products, i.e. danshensu (DSU), protocatechuic aldehyde (PRO), and lithospermic acid, were further identified by comparing the retention times with standard samples. According to the structure of LAB and its hydrolysis behavior in solution, the other product was proposed to be the isomer of lithospermic acid.     

Isolation and quantitative analysis of cryptotanshinone,an active quinoid diterpene formed in callus of Salvia miltiorrhiza BUNGE

Effect of N(6)-benzyladenine (BA) on tanshinone formation in callus cultures of Salvia miltiorrhiza was examined in an attempt to increase the productivity of the medicinal compound, cryptotanshinone. Primary callus was induced by culturing leaf explants on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg l(-1) of 2,4-dichlorophenoxyacetic acid (2,4-D) in darkness. The callus proliferated further on MS basal medium containing 1.0 mg l(-1) 2,4-D and 0.5 mg l(-1) BA and was analyzed for cryptotanshinone by high performance liquid chromatography (HPLC). The HPLC results indicated that it contained small amounts of cryptotanshinone (0.26+/-0.05 mg/g dry wt). Omission of 2,4-D from the medium resulted in a marked increase in the content of cryptotanshinone in callus. The HPLC analysis revealed that the content of cryptotanshinone in the callus cultured on the MS basal medium supplemented with 0.1, 0.2, 0.5, 1.0, and 2.0 mg l(-1) of BA was significantly higher than the marketed crude drug (processed underground parts of S. miltiorrhiza). Maximum yield of cryptotanshinone (4.59+/-0.09 mg/g dry wt) was observed in the callus cultured on MS basal medium supplemented with 0.2 mg l(-1) BA for 60 d. Cryptotanshinone was isolated from callus through silica gel column chromatography followed by preparative TLC and characterized based on NMR and mass spectral data.     

PTP1B inhibitory effect of abietane diterpenes isolated from Salvia miltiorrhiza

Protein tyrosine phosphatase 1B (PTP1B) acts as a negative regulator of insulin signaling, and selective inhibition of PTP1B has served as a potential drug target for the treatment of type 2 diabetes. In the course of screening for PTP1B inhibitory natural products, the MeOH extract of the dried root of Salvia miltiorrhiza BUNGE (Labiatae) was found to exhibit significant inhibitory effect. Bioassay-guided fractionation and purification afforded three related abietane-type diterpene metabolites 1-3. Compounds 1-3 were identified as isotanshinone IIA (1), dihydroisotanshinone I (2), and isocryptotanshinone (3) mainly by analysis of NMR and MS data. Compounds 1-3 non-competitively inhibited PTP1B activity with 50% inhibitory concentration values of 11.4+/-0.6 microM, 22.4+/-0.6 microM and 56.1+/-6.3 microM, respectively.     

白花丹参与紫花丹参不同部位水溶性成分含量比较

目的:分析比较白花丹参与紫花丹参不同部位的水溶性成分含量。方法:采用毛细管电泳-场强放大样品堆积法对2种丹参的5种水溶成分进行分析比较。结果:2种丹参不同部位的成分含量存在一定差异。结论:2种丹参都具有重要的药用价值。     

山东不同产地种植丹参有效成分含量分析

目的通过对山东不同产地的种植丹参进行有效成分的含量测定,并进行了分析汇集总结,希望对丹参的产销和应用提供一些有益的参考。方法采用高效液相色谱法测定丹参中丹参酮ⅡA、丹酚酸B的含量并进行比较。结果与结论测定结果显示,山东种植丹参有效成分含量均能达到或高于药典规定标准,且省内不同产地种植丹参有效成分含量差异较大。     

丹参在炮制大生产过程中丹酚酸B的损失情况研究

目的考察丹参药材炮制大生产过程中指标性成分丹酚酸B的含量变化及损耗情况。方法进行10批次丹参饮片炮制生产,应用HPLC法,在生产过程各主要工艺点进行跟踪取样,测定丹酚酸B的含量并计算损耗;同时在其中一个批次药材炮制大生产过程中,检测每个工艺环节对丹酚酸B的含量的影响。结果原药材、水洗药材、润透药材和切片4个取样点丹酚酸B的平均含量分别为6.83%,4.72%,4.46%和3.55%,相对原药材的损失情况分别为30.84%,34.59%和47.99%。水洗工艺直接导致丹酚酸B损失22.11%,最终损失高达47.48%。结论严格控制(优化)水洗工艺,可有效保留丹酚酸B的含量,提高丹参的炮制效率。     

丹酚酸B镁盐对自由基的清除作用和对脂质过氧化的抑制作用(英文)

AIM: To study the effect of magnesium lithospermate B (MLB) on the lipid peroxidation and on its free radical scavenging activity. METHODS: MLB was incubated in rat tissue homogenate or in a free radical generating system. MLB induced inhibition of lipid peroxidation and its scavenging activity on superoxide anions and hydroxyl radicals was studied using colorimetric estimation. RESULTS: MLB inhibited the lipid peroxidation induced by either an auto-oxidant or Fe2+/VitC in vitro, in the liver homogenate, the inhibitory rate of MLB (10 mg/L) being 69.2% and 57.7%, respectively. MLB (25 and 50 mg/kg) decreased the amount of thiobarbituric acid reactive substances (TBARS) in rat serum, liver, kidney, and heart. However, it did not inhibit the lipid peroxidation of brain homogenate ex vivo. MLB scavenged superoxide anions generated from xanthine/xanthine oxidase system and iron-dependent hydroxyl radicals. CONCLUSION: MLB is an inhibitor of lipid peroxidation and scavenge superoxide anions and hydroxyl radicals both in vitro and ex vivo.     

HPLC法测定丹参中丹酚酸B的含量

目的建立丹参中丹酚酸B的HPLC定量分析方法.方法取丹参生药细粉,加水适量,置冰浴超声提取后进行HPLC分析.色谱柱为Discovery RP-Amide C16(250 mm×4.6 mm,5 μm);流动相为甲醇-乙腈-水-甲酸(425521),紫外检测波长为285 nm.结果丹酚酸B在进样量为0.08～0.32μg范围内有良好的线性关系;精密度实验的RSD为1.61%;加样提取回收率为99.28%.结论本方法简便可靠,其各项方法学指标均可满足含量测定的基本要求.     

Therapeutic effect of magnesium lithospermate B on neointimal formation after balloon-induced vascular injury

Vascular smooth muscle cell (VSMC) proliferation and migration in response to platelet-derived growth factor (PDGF) play an important role in the development of atherosclerosis and restenosis. Recent evidence indicates that PDGF increases intracellular levels of reactive oxygen species in VSMCs and that both PDGF-induced VSMC proliferation and migration are reactive oxygen species-dependent. Danshen is a representative oriental medicine used for the treatment of vascular disease. Previously, we reported that magnesium lithospermate B, an active component of Danshen, is a potent antioxidant. Thus we investigated the therapeutic potential of magnesium lithospermate B in neointimal formation after carotid artery injury in rats along with its effects on the PDGF signaling pathway for stimulating VSMC proliferation and migration in vitro. PDGF is dimeric glycoprotein composed of two A or two B chains. In this study, we used PDGF-BB, which is one of the isoforms of PDGF (i.e., PDGF-AA, PDGF-BB, and PDGF-AB). Our results demonstrated that magnesium lithospermate B directly scavenged reactive oxygen species in a xanthine/xanthine oxidase system and reduced PDGF-BB-induced intracellular reactive oxygen species generation in VSMCs. In a rat carotid artery balloon injury model, magnesium lithospermate B treatment (10 mg/kg/day, i.p) showed a significant effect on the prevention of neointimal formation compared with vehicle treatment. In cultured VSMCs, magnesium lithospermate B significantly attenuated PDGF-BB-induced cell proliferation and migration as measured by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay and transwell migration assays, respectively. Further, magnesium lithospermate B inhibited PDGF-BB-induced phosphorylation of phospatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways by scavenging reactive oxygen species. Together, these data indicated that magnesium lithospermate B, a potent reactive oxygen species scavenger, prevented both injury-induced neointimal formation in vivo and PDGF-BB-induced VSMC proliferation and migration in vitro, suggesting that magnesium lithospermate B may be a promising agent to prevent atherosclerosis and restenosis following angioplasty.     

复方丹参黄芪胶囊与复方丹参片治疗冠心病的疗效比较

目的：比较香港保健协会提供的复方丹参黄芪（ＳＭ＋ＲＡ）胶囊与广州白云山制药厂生产的复方丹参片（ＳＭ）治疗冠心病心绞痛的疗效。方法：稳定型心绞痛３０例（男性１５例，女性１５例；年龄６０±ｓ８ａ）采用复方ＳＭ＋ＲＡ胶囊２粒（含４０ｍｇ／粒）ｐｏ，ｔｉｄ，共４ｗｋ。另１１例稳定型心绞痛病人（男性８例，女性３例；年龄６３±８ａ）采用复方ＳＭ片，３片，ｐｏ，ｔｉｄ，共４ｗｋ。２组伴有高血压病人（前者１７例，后者４例）继续采用复方降压片，复方罗布麻叶片或卡托普利。结果：心绞痛症状疗效与静息心电图疗效：前者依次为９７％与６４％；后者依次为９１％与３６％，但组间比较，Ｐ＞０．０５。结论：复方ＳＭ＋ＲＡ胶囊治疗冠心病心绞痛疗效好，不良反应少。