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.

Anti-Salmonella mode of action of natural L-phenyl lactic acid purified from Lactobacillus plantarum ZJ316.

Salmonella is a serious foodborne pathogen responsible for more than 90 million cases of gastroenteritis worldwide annually. Due to the gradual increase in antibiotic-resistant Salmonella strains, the identification of natural antibacterial substances is urgently needed. Herein, we purified natural L-phenyl lactic acid (L-PLA) from Lactobacillus plantarum ZJ316 and revealed its antimicrobial mode against Salmonella enterica subsp. enterica ATCC 14028. L-PLA (98.14% pure) was obtained using the macroporous resin XAD-16, solid-phase extraction (SPE), reverse-phase high-performance liquid chromatography (RP-HPLC), and chiral chromatography. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results showed that the cell morphology was severely compromised. Transmembrane electrical potential (ΔΨ), transmembrane pH gradient (ΔpH), intracellular ATP level, extracellular electrical conductivity (EC), and genomic DNA analyses were employed to evaluate the antibacterial mode of action of L-PLA. The proton motive force (PMF) and ATP of Salmonella cells rapidly dissipated, and the EC markedly increased. The gel retardation assay demonstrated that L-PLA could bind to genomic DNA and intercalate into the nucleic acids. The anti-Salmonella mode of action of L-PLA was attributed to the destruction of the cell membrane and genomic DNA binding. This research suggests that L-PLA has potential applications as an antimicrobial agent in food, medicine, and other fields. KEY POINTS: • Natural L-PLA was purified from L. plantarum ZJ316 with a purity of 98.14%. • L-PLA effectively inhibited Salmonella strains by antibacterial activities and MICs. • Membrane destruction and binding with DNA are the anti-Salmonella modes of L-PLA.

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.

The attenuation of HIV-1 Tat-induced neurotoxicity by Salvianic acid A and Danshen granule.

The neurotoxicity of HIV-1 Tat protein contributes significantly to the pathogenesis of HAND, and hence the attractive therapeutic strategies focusing on Tat-induced neurotoxicity are warranted. Salvia miltiorrhiza have been known to antioxidant property and neuroprotective effects. The Danshen granule is the pharmaceutical dosage forms of Salvia miltiorrhiza and Salvianic acid A is an essential chemical constituent of Salvia miltiorrhiza. However, the protective effects of Salvianic acid A and Danshen granule on Tat-induced neurotoxicity remain unknown. Here, we found that Salvianic acid A and Danshen granule remarkable inhibited Tat-induced cell death, blocked LDH release and rescued dendritic spine loss. Furthermore, Salvianic acid A and Danshen granule significantly ameliorates Tat-induced intracellular ROS and MDA production, attenuates cell apoptosis. In addition, Salvianic acid A and Danshen granule pretreatment obviously increases antioxidant enzymatic activity of CAT, SOD and GSH-Px and inhibits apoptotic pathways. In conclusion, this study demonstrated that Salvianic acid A and Danshen granule provides substantial neuroprotection against Tat-induced neurotoxicity, which may be new therapeutic agent in Tat induced HAND or neurodegenerative diseases.

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.

Longterm storage of post-packaged bread by controlling spoilage pathogens using Lactobacillus fermentum C14 isolated from homemade curd.

One potent lactic acid bacterial strain C14 with strong antifungal activity was isolated from homemade curd. Based on morphological as well as biochemical characters and 16S rDNA sequence homology the strain was identified as Lactobacillus fermentum. It displayed a wide antimicrobial spectrum against both Gram-positive and Gram-negative pathogenic bacteria, and also against number of food spoilage, plant and human pathogenic fungi. The cell free supernatant (CFS) of the strain C14 was also effective against the fungi tested. Inhibition of radial growth of Penicillium digitatum, Trichophyton rubrum and Mucor sp. was noticed in the presence of CFS of C14 even at low concentration (1%). More than 94.3 ± 1.6% and 91.5 ± 2.2% inhibition of conidial germination of P. digitatum and Mucor sp. were noticed in the presence of 10-fold-concentrated CFS of C14. Massive deformation of the fungal mycelia was observed by SEM studies, and losses of cellular proteins and DNA are also evident upon its treatment with C14. HPLC analysis revealed the presence of phenyl lactic acid, lactic acid along with some unidentified compounds in the antifungal extract. Challenge experiment showed immense potential of the strain C14 in preventing the spoilage of bread samples caused by Mucor sp. and Bacillus subtilis. The bread samples remained fresh upto 25 days even after inoculation with Mucor sp. (3.7 × 104 spores /ml) and B. subtilis (4.6 × 104 CFU /ml). Along with the antifungal properties, the isolated lactic acid bacterial strain also showed very good antioxidant activities. Unchanged level of liver enzymes serum glutamic pyruvic transaminase and serum glutamic oxaloacetic transaminase in albino mice upon feeding with C14 also suggested non-toxic nature of the bacterial isolate.

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.

Enantioseparation of 3-phenyllactic acid by chiral ligand exchange countercurrent chromatography.

3-Phenyllactic acid is an antimicrobial compound with broad-spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3-phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3-phenyllactic acid with a synthesized chiral ligand. A two-phase solvent system was composed of n-butanol/hexane/water (0.4:0.6:1, v/v/v) to which N-n-dodecyl-l-hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid-liquid extraction. Baseline enantioseparation of racemic 3-phenyllactic acid by analytical high-speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3-phenyllactic acid reached 99.0%, as determined by chiral high-performance liquid chromatography.

Efficient magnesium lactate production with in situ product removal by crystallization.

In this paper, attempts were made to develop an in situ product removal process for magnesium lactate production based on crystallization. The crystallization was conducted at 42°C without seed crystal addition. The product concentration, productivity and yield of fermentation coupled with in situ product removal (ISPR) reached 143 g L(-1), 2.41 g L(-1)h(-1) and 94.3%. In four cycles of crystallization, the average reuse rate of fermentation medium and removal rate of product reached 64.0% and 77.7%. At the same time, ISPR fermentation saved 40% water, 41% inorganic salts and 43% yeast extract (YE) as compared to fed-batch fermentation. The process introduces an effective way to reduce the amount of waste water and the raw material cost in magnesium lactate fermentation.

Separation of salvianic acid A from the fermentation broth of engineered Escherichia coli using macroporous resins.

Salvianic acid A (also known as danshensu) is a plant-derived polyphenolic acid, and has a variety of physiological and pharmacological activities. Our laboratory previously constructed an unprecedented artificial biosynthetic pathway in Escherichia coli and established the fermentation process to produce salvianic acid A. Here, we developed an efficient method for separating salvianic acid A from the fermentation broth of engineered Escherichia coli by macroporous resins. Among ten tested macroporous resins, the static and dynamic adsorption/desorption experiments demonstrated that X5 resin was the best to separate salvianic acid A from fermentation broth. Other parameters during static and dynamic procedures were also investigated. Under the optimum separation conditions, the average adsorption capacity of SAA were 10.66±0.54 mg/g dry resin and the desorption ratio was 85.6±4.1%. The purity and recovery yield of salvianic acid A in the final dry product were 90.2±1.5 and 81.5±2.3%, respectively. The results show that adsorption separation with macroporous resin X5 was an efficient method to prepare salvianic acid A from fermentation broth. This work will benefit the development and application of plant-derived salvianic acid A and its derivatives.

Efficient calcium lactate production by fermentation coupled with crystallization-based in situ product removal.

Lactic acid is a platform chemical with various industrial applications, and its derivative, calcium lactate, is an important food additive. Fermentation coupled with in situ product removal (ISPR) can provide more outputs with high productivity. The method used in this study was based on calcium lactate crystallization. Three cycles of crystallization were performed during the fermentation course using a Bacillus coagulans strain H-1. As compared to fed-batch fermentation, this method showed 1.7 times higher average productivity considering seed culture, with 74.4% more L-lactic acid produced in the fermentation with ISPR. Thus, fermentation coupled with crystallization-based ISPR may be a biotechnological alternative that provides an efficient system for production of calcium lactate or lactic acid.

Bioorganic research of Galactites tomentosa Moench. Honey extracts: enantiomeric purity of chiral marker 3-phenyllactic acid.

Thistle (Galactites tomentosa Moench.) honey organic extracts were obtained by headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) and analyzed by gas chromatography (GC-FID and GC-MS) for the first time. Most abundant headspace compounds were terpenes, particularly linalool derivatives (hotrienol was predominant with a range of 38.6-57.5%). 3-Phenyllactic acid dominated in the solvent extracts (77.4-86.4%) followed by minor percentages of other shikimate pathway derivatives. After determination of an adequate enantioseparation protocol on Chirallica PST-4 column, the honey solvent extracts were analyzed by high-performance liquid chromatography (HPLC). The chiral analysis revealed high enantiomeric excess (>95%) of (-)-3-phenyllactic acid in all samples. Therefore, previous findings of chemical markers of thistle honey were extended, providing new potential for advanced chemical fingerprinting (optical pure chemical marker).

Isolation, characterization, and determination of a new compound in red wine.

Chromatographic separation using fluorescence as a detection mode revealed, besides a series of flavan-3-ols, the recurrent presence of an undefined compound in Bordeaux red wine. Its isolation and structure characterization by complementary means (high-resolution mass spectrometry, nuclear magnetic resonance, and chemical synthesis) has permitted us to identify it as the nitrogen-containing glycoconjugate 3-indolyl-(2R)-O-ß-D-glucosyl-lactic acid. Its quantification was performed for different wines of different vine varieties and terroirs with the aim to assess whether this compound may be used as a terroir, variety, or wine process tag.

Salvianolic acid A protects against vascular endothelial dysfunction in high-fat diet fed and streptozotocin-induced diabetic rats.

Salvianolic acid A (SalA) is one of the main active ingredients of Salvia miltiorrhizae. The objective of this study was to evaluate the effect of SalA on the diabetic vascular endothelial dysfunction (VED). The rats were given a high-fat and high-sucrose diet for 1 month followed by intraperitoneal injection of streptozotocin (30 mg/kg). The diabetic rats were treated with SalA (1 mg/kg, 90% purity) orally for 10 weeks after modeling, and were given a high-fat diet. Contractile and relaxant responses of aorta rings as well as the serum indications were measured. Our results indicated that SalA treatment decreased the level of serum Von Willebrand factor and ameliorated acetylcholine-induced relaxation and KCl-induced contraction in aorta rings of the diabetic rats. SalA treatment also reduced the serum malondialdehyde, the content of aortic advanced glycation end products (AGEs), and the nitric oxide synthase (NOS) activity as well as the expression of endothelial NOS protein in the rat aorta. Exposure of EA.hy926 cells to AGEs decreased the cell viability and changed the cell morphology, whereas SalA had protective effect on AGEs-induced cellular vitality. Our data suggested that SalA could protect against vascular VED in diabetes, which might attribute to its suppressive effect on oxidative stress and AGEs-induced endothelial dysfunction.

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.

Enantioseparation of amino acids and alpha-hydroxy acids on ligand-exchange continuous beds by capillary electrochromatography.

A new chiral stationary phase based on continuous bed (CB) technology using L-prolinamide as a chiral selector was prepared. Its ability for enantioseparation of amino acids and alpha-hydroxy acids by ligand-exchange CEC was compared with that of a CB containing L-4-hydroxyproline as chiral selector. Preparation was done by a one-step in situ copolyermerization procedure using methacrylamide as monomer, vinylsulfonic acid as charge providing agent and piperazine diacrylamide as crosslinker. L-Prolinamide showed marked enantioselective properties for the separation of amino acids and alpha-hydroxy acids. Furthermore, we show the possibility of simultaneous enantioseparation of amino acids and alpha-hydroxy acids using the CB with L-4-hydroxyproline as chiral selector.

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.