Salvianolic acid A attenuates steroid resistant nephrotic syndrome through suPAR/uPAR-αvß3 signaling Inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvianolic acid A (SAA) is extracted from traditional Chinese medicine Salvia miltiorrhiza and is the main water-soluble and the biologically active ingredient. SAA possesses a variety of pharmacological activities and has an excellent protective effect on kidney disease, especially steroid resistant nephrotic syndrome (SRNS), and has advantages in improving the efficacy of glucocorticoids, but its mechanism needs to be further explored. PURPOSE: The study was designed to explore the effect of suPAR and uPAR in SRNS patients and evaluate the potential effect of SAA in improving podocyte steroid resistance and explore its mechanism. METHODS AND MATERIALS: The ELISA kits were used to detect the levels of suPAR in the blood and urine of subjects. The levels of uPAR, GRα, and GRß expression in renal tissues of SRNS patients was detected by immunohistochemistry and analyzed using the Pearson method. In vitro studies, steroid resistance model was induced by the TNF-α and IFN-γ. The protein and mRNA expression of Nephrin, GR, GRα and GRß were analyzed using western blot and qRT-PCR. The activity of GR-DNA binding was detected by using TransAM™ GR kits. Adriamycin further induced steroid resistance podocyte. Flow cytometry was used to detect the effect of SAA on podocyte apoptosis. ELISA assay was used to detect the suPAR expression in the podocyte supernatant. Western blot and qRT-PCR were used to detect the protein and mRNA expression of uPAR and Nephrin in podocytes. RESULTS: The serum and urine levels of suPAR were conspicuously higher in SRNS patients than healthy volunteers and SSNS patients, and the expression of uPAR in renal tissue of SRNS patients is negatively correlated with GRα, but positively correlated with GRß. The combination of TNF-α and IFN-γ could conspicuously increase the GRß expression and reduce GRα/GRß, and induce steroid resistance in podocytes. Moreover, we found that SAA could reduce the apoptosis of podocytes and suppress the expression of suPAR/uPAR, and increase the expression of Nephrin. CONCLUSION: The level of suPAR and uPAR expression may have important value in predicting glucocorticoids resistance in patients with idiopathic nephrotic syndrome (INS). The combination of TNF-α and IFN-γ induce podocytes can establish steroid resistance model in vitro. SAA could improve glucocorticoids resistance of podocyte which can be attributed in part to regulate the suPAR/uPAR-αvß3 signaling pathway.

Salvia clandestina L.: unexploited source of danshensu.

We report the characterisation of Salvia clandestina L. shoots and roots aqueous extract using HPLC-ESI/MS-TOF. Among the 29 compounds detected, we observed the presence of danshensu (3-(3,4-dihydroxy-phenyl) 2-hydroxy-propinic acid), a powerful antioxidant and a cardio-protective agent. The danshensu content found in the shoots of S. clandestina was considerable (4.96 mg g-1 DW) if compared with previous studies on S. miltiorrhiza Bunge, the main source of this compound. We also determined the total phenolic concentration and we evaluated their antioxidant activity by ABTS, FRAP and Superoxide anion scavenging methods. All the three assays confirmed a greater antioxidant activity for the shoots in comparison to roots. S. clandestina shoots may represent a valuable and natural unexploited source of danshensu and other phenolic compounds, so that it may be useful for future applications in functional foods and pharmaceutical industries.

Salvianolic acids from antithrombotic Traditional Chinese Medicine Danshen are antagonists of human P2Y1 and P2Y12 receptors.

Many hemorheologic Traditional Chinese Medicines (TCMs) that are widely-used clinically lack molecular mechanisms of action. We hypothesized that some of the active components of hemorheologic TCMs may function through targeting prothrombotic P2Y1 and/or P2Y12 receptors. The interactions between 253 antithrombotic compounds from TCM and these two G protein-coupled P2Y receptors were evaluated using virtual screening. Eleven highly ranked hits were further tested in radioligand binding and functional assays. Among these compounds, salvianolic acid A and C antagonized the activity of both P2Y1 and P2Y12 receptors in the low µM range, while salvianolic acid B antagonized the P2Y12 receptor. These three salvianolic acids are the major active components of the broadly-used hemorheologic TCM Danshen (Salvia militorrhiza), the antithrombotic molecular mechanisms of which were largely unknown. Thus, the combination of virtual screening and experimental validation identified potential mechanisms of action of multicomponent drugs that are already employed clinically.

New depsides from the roots of Salvia miltiorrhiza and their radical-scavenging capacity and protective effects against H2O2-induced H9c2 cells.

Phytochemical investigation of the roots of Salvia miltiorrhiza led to isolations of two new depsides (1-2), along with thirteen known compounds (3-15). Their structures and relative stereochemistry were elucidated by NMR spectral (1H and 13C NMR, HSQC, HMBC), CD and HR-ESIMS data analyses. The absolute configuration of 1 was determined by comparison of the experimental and calculated ECD spectra. All the depsides (1-10) were found to exhibit stronger free radical scavenging capacity (ranging from 2.62 to 22.05µM) than diterpenoids (11-15, IC50>100µM), among which Salvianolic acid A and Salvianolic acid B were the most potent compounds. Additionally, most of depsides (1, 3, 5, 7, 8, 9, 10) possessed significant protective effects against H2O2-induced H9c2 apoptosis in low concentrations. The negative mode collision-induced dissociations of compound 1 and 2 were featured by the α-cleavage and ß-cleavage to lose danshensu (198Da) and caffeic acid (180Da), respectively, while α,ß-dihydroxybenzenes depsides (8-hydroxy) showed characteristic neutral elimination of H2O.

Study on secondary metabolite content of Helleborus niger L. leaves.

Phytochemical investigation of Helleborus niger L. (Ranunculaceae) leaf methanol extract allowed to isolate a phenolic glucoside derivative and two flavonoid glycosides characterized as phenyllactic acid 2-O-ß-D-glucopyranoside (1), quercetin 3-O-2-(E-caffeoyl)-α-L-arabinopyranosyl-(1 → 2)-ß-D-glucopyranoside-7-O-ß-D-glucopyranoside (2), and kaempferol 3-O-α-L-arabinopyranosyl-(1 → 2)-ß-D-galactopyranoside-7-O-ß-D-glucopyranoside (3), respectively. Compounds 1 and 2 were isolated for the first time and their structural characterization was obtained on the basis of extensive NMR spectral studies.

[Research progress of salvianolic acid A].

Salvianolic acid A is a water-soluble component from Danshen, which is frequently used in traditional Chinese medicine. High performance liquid chromatography was often used to analyze content of salvianolic acid A. The yield of salvianolic acid A increased by the technological improvement of extraction and separation. Salvianolic acid A possessed multiple pharmacological activities, including antioxidants, myocardial ischemic protection, antithrombatic, neuroprotection, anti fibrosis, prevention of diabetes and complications. Recently, preliminary pharmacokinetics characteristics of salvianolic acid A were clarified. Based on the research literature and study work from author's laboratory, this review will focus on recent developments concerning the chemistry, pharmacology and pharmacokinetic of salvianolic acid A, and prospect further research.

Isolation of salvianolic acid A, a minor phenolic carboxylic acid of Salvia miltiorrhiza.

Salvianolic acid A (Sal A), a caffeic acid derivative present in Salvia miltiorrhiza (Danshen), has shown significant biological activities, some of which are more potent than those of salvianolic acid (Sal B), the most abundant and bioactive compound in Danshen. However, its low content in the raw material makes it difficult to be used in the clinic. In this study, a novel procedure was developed to isolate Sal A from Salvia miltiorrhiza. Using this procedure, Sal A could be enriched to 1.7% in the raw material by hydrolyzing Sal B. The solution was further purified by macroporous resin and ODS column chromatography. The yield of Sal A was 0.5% with a purity of 98.0%. The procedure is efficient and convenient and is considered suitable for the separation of Sal A from Danshen.

Biphasic effects of sodium danshensu on vessel function in isolated rat aorta.

AIM: To investigate the effects of sodium danshensu on vessel function in isolated rat aortic ring. METHODS: Thoracic aortae from normal rats were isolated and equilibrated in organ bath with Krebs-Henseleit buffer and ring tension was recorded. Effects of sodium danshensu on basal tonus of the vessel and its effects on vessel contraction and relaxation with or without endothelium were observed. RESULTS: In thoracic arteries under basal tonus, sodium danshensu (0.3-3 g/L) produced a dose-dependent transient contraction. In phenylephrine-precontracted thoracic arteries with or without endothelium, low concentration (0.1-0.3 g/L) of sodium danshensu produced a weak contraction, while high concentrations (1-3 g/L) produced a pronounced vasodilator after a transient vasocontraction. Pre-incubation with sodium danshensu could inhibit vessel contraction induced by phenylephrine and potassium chloride in a concentration-dependent way. Sodium danshensu inhibited phenylephrine- and CaCl(2)-induced vasoconstriction in Ca(2+)-free medium. Pre-incubation with tetraethylammonium, a non-selective K(+) channel blocker, and apamin, a small-conductance calcium-activated K(+) channel blocker partially antagonized the relaxation response induced by sodium danshensu. However, iberiotoxin (big-conductance calcium-sensitive K(+) channel blocker), barium chloride (inward rectifier K(+) channel blocker), and glibencalmide (ATP-sensitive K(+) channel blocker) had no influence on the vasodilation effect of sodium danshensu. CONCLUSION: Sodium danshensu showed a biphasic effects on vessel tension. While low dosage of sodium danshensu produced small contraction possibly through transient enhancement of Ca(2+) influx, high dosage produced significant vasodilation mainly through promoting the opening of non-selective K(+) channels and small-conductance calcium-sensitive K(+) channels in the vascular smooth muscle cells.

[Intestinal absorption properties of three components in salvianolic acid extract and the effect of borneol on their absorption in rats].

This study aimed to investigate the effects of concentration, intestinal section and borneol on the intestinal absorption of salvianolic acids. The experiment not only studied the intestinal absorption properties of three concentrations of rosmarinic acid, salvianolic acid B and salvianolic acid A at duodenum, jejunum and ileum, but also of salvianolic acids compatible with borneol at different concentrations using single-pass intestinal perfusion model in rat with phenol red as the marker. The results showed that salvianolic acids was stable under weak-acid condition and affected by metabolism enzyme; The Peff and Ka significantly different among three concentrations of rosmarinic acid and salvianolic acid B, whose intestinal absorption were saturated in high concentration, suggesting that the transport mechanisms of rosmarinic acid and salvianolic acid B were similar to active transport or facilitated diffusion; However, there was inconspicuousness in the Peff and Ka of salvianolic acid A at different concentrations, whose absorption was not saturated in high concentration, indicating that the transport mechanisms of salvianolic acid A was passive diffusion; The Peff and Ka in the ileum obviously higher than those in the duodenum and jejunum, namely the ileum was the best absorption section; When concentration of borneol increased, the enhancing effect of intestinal absorption of salvianolic acids increased, but significantly decreased when borneol increased to some degree. The enhancing effect of medium borneol concentration was the optimum. This implied that borneol can enhance the intestinal absorption of salvianolic acids, and the capacity of enhancing effect was influenced by the concentration of borneol.

Effect of Guanxin No.2 decoction on gene expression in different areas of the myocardial infarcted heart of rats using microarray technology.

OBJECTIVES: We have used microarray technology to detect the effect of Guanxin No.2 decoction on gene expression in different areas of the myocardial infarcted heart of rats. METHODS: Male Sprague-Dawley rats (180-200 g) were randomly divided into three groups: sham-operated; coronary artery ligation; and coronary artery ligation plus administration of Guanxin No.2 decoction (10.0 g raw materials/kg per day by gavage). The experiment was carried out on day seven after ligation. KEY FINDINGS: We found that the gene expression using microarray technology showed many differences in the border infarcted left ventricular area compared with the remote noninfarcted left ventricular area after administration of Guanxin No.2 decoction. CONCLUSIONS: Guanxin No.2 decoction has a long history in treating ischaemic cardiomyopathy in China, but the molecular mechanism has been unclear. In this study we found that some important genes may have contributed to the cardioprotective effect of Guanxin No.2 decoction.

[Absorption and isolation of macroporous resin for five salvianolic acids from Salviae mitiorrhizae].

OBJECTIVE: To study the adsorption of the macroporous resin for the five salvianolic acids (danshen su, rosmarinic acid, protocate chualdehyde, salvianolic acid B, salvianolic acid A, extracted from Salviae mitiorrhizae. METHOD: The five salvianolic acids were employed as an index, and the change of them in the static and dynamic absorbent was detected by HPLC, respectively. RESULT: HP20 resin was a suitable marcoporous resin to purify salvianolic acids. The dynamic adsorption capacity of rosmarinic acid, salvianolic acid B and salvianolic acid A was 30.506 mg x g(-1) (dry resin), 36.996 mg x g(-1), (dry resin), 43.85 mg x g(-1) (dry resin) respectively. CONCLUSION: It is not suitable that danshensu and protocate chualdehyde are the evaluation indexes for using 8 macroporous resins to purify salvianolic acids.

Interaction of salvianolic acids and notoginsengnosides in inhibition of ADP-induced platelet aggregation.

Salvia miltiorrhiza and Panax notoginseng were both considered to be beneficial to cardiovascular diseases in traditional Chinese medicine and often used in combination. To examine the possible interaction between them, the effects of the active fractions of these two herbs, salvianolic acids (SA) and notoginsengnosides (NG), on platelet aggregation were checked respectively or in combination in vitro and in vivo. Both the platelet aggregation of platelet rich plasma (PRP) and washed platelet after ADP induction were checked. In vitro study showed that both SA and NG had an inhibitory effect on platelet aggregation. However, there is no synergistic effect of the combination of SA and NG in vitro. In vivo study showed that i.g. 550 mg/kg/day SA or NG for 5 days could significantly inhibit ADP-induced platelet aggregation of PRP. Moreover, combination of SA and NG at a ratio of 5:1 had a synergistic effect on platelet aggregation of PRP. The mechanism for the synergism of SA and NG in vivo was not clear. High performance liquid chromatography analysis of the plasma of rats received SA, NG or combination of SA and NG showed that co-administration of NG caused change in the plasma distribution profile of SA. The influence of combination on the absorption and/or metabolism of SA may be one of the reasons for the synergism of SA and NG in vivo.

Three new derivatives of anti-HIV-1 polyphenols isolated from Salvia yunnanensis.

During the study of anti-HIV-1 active components of the aqueous extracts of the roots of Salvia yunnanensis, three new derivatives of polyphenols, namely: methyl salvianolate A (2), ethyl salvianolate A (3) and cis-lithospermic acid (5) were isolated along with two known polyphenols, salvianolic acid A (1) and lithospermic acid (4) their structures were elucidated on the basis of NMR and MS spectral analyses. The anti-HIV-1 activities of the 5 polyphenols were tested for the inhibition of P24 antigen in HIV-1 infected MT-4 cell cultures and HIV-1 replicative enzymes in vitro.

One-step purification of 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde from Salvia miltiorrhiza Bunge by isocratic stepwise hydrogen bond adsorption chromatography on cross-linked 12% agarose.

Three major active components of the traditional Chinese medicinal herb Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde, are separated and purified from a crude water extract in one step by isocratic hydrogen bond adsorption chromatography on cross-linked 12% agarose (Superose 12 HR 10/30). Separation is achieved by stepwise elution with mobile phases composed of mixtures of ethanol and acetic acid: 0-50 mL, 5% ethanol, 5% acetic acid; 50-100 mL, 20% ethanol, 20% acetic acid; and 100-200 mL, 30% ethanol, 30% acetic acid. The 3,4-dihydroxyphenyllactic acid is obtained with a purity of 97.3% and with a recovery of 88.1%. The corresponding figures for protocatechualdehyde are a purity of 99.4% with a recovery of 90.7%, and for salvianolic acid B a purity of 90.4% with a recovery of 50.3%, respectively. At a sample load of 40 mg crude extract dissolved in 0.5 mL mobile phase (corresponding to a load of 1.6 mg/mL gel), a 3,4-dihydroxyphenyllactic acid purity of approximately 94% with a recovery of 80.2% is obtained.

[Studies on polyphenolic chemical constitutents from root of Salvia yunnansis].

OBJECTIVE: To study the chemical constituents in the root of Salvia yunnansis. METHOD: Compounds were isolated and purified by Diaion HP20, Sephadex LH - 20, ODS chromatography. Their structures were determined by spectral analysis and chemical evidence. RESULT: Twelve compounds were isolated and identified from the root of S. yunnansis protocatechaldehyde (1), caffeic acid (2), ferulic acid (3), rosmarinic acid (4), salvianolic acid A (5), salvianolic acid C (6), lithospermicacid (7), lithospermicacid B (8), 9'-methyl lithospermate B (9), 9"'-methyl lithospermate B (10), 9',9'''-dimethyl lithospermate B (11), 9'-ethyl lithospermate B (12). CONCLUSION: The compounds 1, 2, 3, 5, 6, 9, 10, 11 and 12 were first isolated from S. yunnanensis.

Chromatographic fingerprinting analysis of Danshen root (Salvia miltiorrhiza Radix et Rhizoma) and its preparations using high performance liquid chromatography with diode array detection and electrospray mass spectrometry (HPLC-DAD-ESI/MS).

Danshen root (Salvia miltiorrhiza Radix et Rhizoma) is a representative Chinese herb containing multiple components contributing to its polyvalent bioactivities. Advanced analysis approaches are needed to obtain a comprehensive picture of the targeting constituents in complete matrix. In this study, a chromatographic fingerprinting method to monitor simultaneously the hydrophilic and lipophilic constituents was developed for the quality evaluation of Danshen root and its preparations. Ten hydrophilic and nine lipophilic components were identified using HPLC-diode array detection-electrospray-MS (HPLC-DAD-ESI/MS) by comparing with the available references and reported data. Using the established method, 13 Danshen root samples collected from different sources, and 21 batches of Danshen preparations including tablets, injections, capsules, and dropping pills produced by different manufacturers were analyzed and their chromatographic fingerprints (CFP) were constructed. The results showed that the products of Danshen roots such as the tablets and capsules contained both the hydrophilic and lipophilic components, while the injections and dropping pills contained mainly the hydrophilic components. Principal components analysis (PCA) was applied for the statistical analysis of the fingerprinting data of crude herb and its preparations. The established CFPs demonstrate the representative chemical profiling of the existing components and can be applied to the authentication and quality assessment of Danshen roots and other Danshen containing formulated preparations.

[Studies on chemical constituents of Prunella vulgaris].

OBJECTIVE: To study the chemical constituents of Prunella vulgaris. METHOD: To separate the constituents of P. vulgaris by using various kinds of chromatography and identify their structures on the basis of spectral analysis. RESULT: Seven compounds were isolated from the spikes of P. vulgaris. Their structures were established as autantiamide acetate (1), rhein (2), tanshinone I (3), danshensu (4), stigmast-7, 22-dien-3-one (5), 3, 4, alpha-trihydroxy-methyl phenylpropionate (6), butyl rosmarinate (7). CONCLUSION: Compounds 1-4 were isolated from this genus for the first time.

Protective effect of salvianic acid a on acute liver injury induced by carbon tetrachloride in rats.

Previous research has shown that salvianic acid A [2-(3,4-dihydroxyphenyl)-2-hydroxy-propanoic acid, SA] extracted from Salvia miltiorrhiza BGE (Danshen) markedly inhibits lipid peroxidation of mitochondrial membrane of hepatic cells in vitro. The present study was conducted to examine protective effect of SA on liver injury induced by carbon tetrachloride (CCl4) and its possible mechanism in vivo. Male Sprague-Dawley rats weighing 180-200 g were used in the experiments. Five mmol/kg CCl4 in olive oil was given to rats i.p. Spectrophotometrical method was used to measure activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum, activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) as well as malondialdehyde (MDA) level in hepatic tissue and the rate of superoxide anion (O2*-) generation in hepatic submitochondrial particles. Hepatic histological structure was observed under light microscopy. CCl4 caused significant changes of activities of the enzymes, MDA level, and the rate of O2*- generation and histopathological changes of acute hepatic injury were noted. SA reversed the significant changes induced by CCl4. These results demonstrate that SA produces protective action on acute hepatic injury induced by CCl4 via an antioxidative mechanism.

Relaxant effects of danshen aqueous extract and its constituent danshensu on rat coronary artery are mediated by inhibition of calcium channels.

In this study, we have investigated the actions of danshensu, an active, water-extractable component of the medicinal herb danshen (Salvia miltiorrhiza), on rat isolated coronary artery rings precontracted with 1 microM 5-hydroxytryptamine (5-HT) and its action compared to the water-extractable fraction of the herb. Extraction of the water-soluble fraction from danshen (S. miltiorrhiza) provided yield of 17.5% (35 g/200 g). The amount of danshensu determined in the crude danshen herb and in its aqueous fraction was 0.45 mg/g (0.045%) and 3.28 mg/g (0.33%). The danshen aqueous extract was 13 times less potent than danshensu in relaxing 5-HT-precontracted coronary artery rings; IC50 values were 930.3+/-133.5 microg/ml and 71.5+/-11.0 microg/ml. Removal of the endothelium did not significantly affect their vasodilator potencies; IC50 values were 842.1+/-123.8 microg/ml and 84.8+/-8.8 microg/ml. On the other hand, a potassium channel inhibitor tetraethylammonium (TEA, 10 mM) shifted their concentration-response curves by 1.7 and 2.2 folds. The possible involvement of Ca2+ channels was investigated in artery rings incubated with Ca2+-free buffer and primed with 1 microM 5-HT or 60 mM KCl for 5 min prior to addition of CaCl2 to elicit contraction. In 5-HT-primed preparations, the CaCl2-induced vasoconstriction was abolished by 2 mg/ml danshen aqueous extract and 200 microg/ml danshensu, whereas, in KCl-primed preparations, 10 mg/ml danshen aqueous extract and 600 microg/ml danshensu were required to abrogate the vasoconstriction. These findings suggest the vasorelaxant actions of danshen aqueous extract and danshensu were produced by inhibition of Ca2+ influx in the vascular smooth muscle cells. The opening of K+ channels had a minor contribution to the response, but endothelium-dependent mechanisms were not involved.

[The qualitative analysis and quantitative analysis of purification of salvianolic acids by macroreticular resin].

OBJECTIVE: To purify salvianolic acids by macroreticular resin,then mensurate the contents of salvianolic acids and analyse the chromatogram with HPLC. METHOD: Make salvianolic acids with macroreticular resin; mensurate the content of Salvianolic acids with UV spestrophotometry: the control compound is protocaechuic aldehyde, and the wavelength is 281 nm. Analysis the chromatogram with HPLC, and compare the chromatogram in different technics: zorbax ODS column (4.6 mm x 250 mm, 5 microm), mobilephase: 1% aceticacid-water and methanol in different proportions, the wavelength is 281 nm. RESULT: The contents of salvianolic acids is 53.8%; HPLC chromatogram indicate that the method is reasonable to make salvianolic acids. CONCLUSION: Determination of contents and HPLC chromatogram can control the quality of Salvianolic acids more accurately.