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The acetone extract of the roots of Salvia jaminiana, containing the sterols campestanol, stigmasterol and sitosterol, and five known diterpenoids, ferruginol, cryptanol, 6,7-dehydroroyleanon, 6-hydroxysalvinolone and microstegiol, remarkably inhibited the growth of Bacillus subtilis, Staphylococcus aureus ATCC 25923 and Streptococcus alpha-hemolitic.
In continuation of our research on Salvia triloba, acute and chronic antiinflammatory and ulcerogenic activities of chloroform, ethanol, butanol and water extracts were detected at 25 mg/kg p.o. The chloroform extract showed the highest antiinflammatory activity in both acute and chronic models, while the ulcerogenic effects of all tested extracts were found to be less than that of acetyl salicylic acid.
Salvia miltiorrhiza Bunge, a traditional Chinese herbal medicine, is often used for prevention and treatment of cardiovascular disorders such as atherosclerosis. To understand its mechanism of pharmacological action, its effects on endothelial monolayer permeability are studied. The present study demonstrated that extract of S. miltiorrhiza (ESM) and its major ingredients, Danshensu (DSS) and salvianolic acid B (Sal B), inhibited tumor necrosis factor (TNF-alpha) induced endothelial permeability, whereas the other major ingredient, protocatechualdehyde, was ineffective. ESM, DSS and Sal B also repressed expression of vascular endothelial growth factor (VEGF) and extracellular signal-regulated kinase (ERK) activation in TNF-alpha induced HUVEC cells. Furthermore, it was found that ESM attenuated the disorganization of vascular endothelial (VE)-cadherin induced by TNF-alpha. The effect of ESM on TNF-alpha induced endothelial permeability and redistribution of VE-cadherin is likely due to a reduction of VEGF protein expression as a result of modulation of the ERK signaling pathway. Endothelial cell hyperpermeability is implicated in inflammation and subsequent ischemic reperfusion injury and atherosclerosis. Data from this study suggest that one of the mechanisms S. miltiorrhiza exerts its pharmacological effect is through its modulation of endothelial cell permeability.
Lipopolysaccharide (LPS) has been implicated as one of the major cause of Gram-negative bacteria-induced sepsis that are life-threatening syndromes occurring in intensive care unit patients. Many natural products derived from medicinal plants may contain therapeutic values on protecting endotoxemia-induced sepsis by virtue their ability to modulate multiple pro-inflammatory cytokines. In the present study, we show that Salvia miltiorrhiza (SM) BUNGE or Danshen, used in treatment of various systemic and surgical infections in the hospitals of China, was able to block the lethal toxicity of LPS in mice via suppression of TNF-alpha release and protection on liver injury. The ability of SM to suppress LPS-induced TNF-alpha release is further confirmed by in vitro experiments conducted on human peripheral blood leukocytes (PBL) and the RAW 264.7 macrophage cell line. Immunophenotyping by flow cytometry shows improved T-helper cell (CD4) and T-suppressor cells (CD8) ratio in SM-treated PBL and splenocytes of LPS-challenged mice. The drop in plasma glutamate-pyruvate transaminase (GPT) induced by LPS provides evidence that SM can protect hepatic damage. The present study explains some known biological activities of SM, and supports the clinical application of SM in the prevention of inflammatory diseases induced by Gram-negative bacteria.
In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.
A method has been developed to determine the four water-soluble components-Danshensu (I), protocatechuic acid (II), protocatechuic aldehyde (III) and salvianolic acid B (IV) in Chinese medicine plant Salvia miltiorrhiza Bunge using high-performance liquid chromatography with a coulometric electrode array detection (HPLC-CEAD) system. Heat reflux extraction was used to pretreat the sample. This analysis was carried on a column of Hypersil C18 (250 mm x 4.6 mm, 5 microm) with a mobile phase of sodium acetate (pH 2.5, 50 mM) and acetonitrile in gradient mode. An ESA electrochemical detector monitored the four compounds. Potentials of four electrodes in series were set at 100, 150, 200 and 250 mV, respectively. Optimization of the pH of mobile phase and the proportion of acetonitrile were also performed. Calibration curve showed good linearity with correlation coefficients (r) more than 0.9937. Average recoveries of the four compounds were more than 92% and relative standard deviations were less than 6.6%. This method appeared to be stable, sensitive and reproducible for determination of the four water-soluble compounds in Chinese medicine plant S. miltiorrhiza Bunge.
High-performance liquid chromatographic (HPLC) fingerprints were developed for identification of both lipophilic and hydrophilic components of the roots of Salvia miltiorrhiza and four related preparations. These samples were separated with an Agilent Zorbax Extend C(18) reserved-phase column (5 microm, 250 mm x 4.6 mm) by linear gradient elution using water-phosphoric acid (100:0.026, v/v) and acetonitrile as mobile phase. The flow rate was 0.8 ml/min and the detector wavelength was set at 280 nm. Mean chromatograms and correlation coefficients of samples were calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of TCM". The correlation coefficients of Danshen and Fufang Danshen tablets (FDT) samples were in the range of 0.352-0.993 and 0.768-0.987, respectively. The correlation coefficients of Compound Danshen dripping pills (CDDP), Danshen injection (DSI) and Xiangdan injection (XDI) samples were higher than 0.928, 0.850 and 0.960, respectively. It was the first time to identify 34 peaks by comparing with standard compounds and using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS(n)) technique. All results indicated that the developed fingerprint assay could be readily utilized as a quality control method for S. miltiorrhiza and its related preparations.
To standardize and control herbal medicines, a feasible approach and control system is necessary. In this paper, a high-performance liquid chromatography with a coulometric electrode array detector (HPLC-CEAD) system was applied to fingerprint Salvia miltiorrhiza Bunge (S. miltiorrhiza Bunge), a popular herbal medicine, for the first time. pH of mobile phase, working potentials and sample preparation were included in our research. Twenty-five common peaks were obtained from extracts of S. miltiorrhiza Bunge (Shandong province), more than that obtained in previous report. Fingerprints of S. miltiorrhiza Bunge from different locations were also studied. The content of main components varied in different samples. Overlapping ratio of peaks (ORP) in 10 batches of S. miltiorrhiza Bunge (Shandong province) was not less than 72.46%. In method validation, relative standard deviation (RSD) of relative retention times and relative peak areas were of not more than 3%. It was concluded that HPLC-CEAD system can be applied in fingerprinting herbal medicines.
In this study, we evaluate the biosafety and bioactivity (antioxidant potential) of a traditional water infusion (tea) of common sage (Salvia officinalis L.) in vivo in mice and rats by quantification of plasma transaminase activities and liver glutathione-S-transferase (GST) and glutathione reductase (GR) enzyme activities. The replacement of water by sage tea for 14 days in the diet of rodents did not affect the body weight and food consumption and did not induce liver toxicity. On the other hand, a significant increase of liver GST activity was observed in rats (24%) and mice (10%) of sage drinking groups. The antioxidant potential of sage tea drinking was also studied in vitro in a model using rat hepatocytes in primary culture. The replacement of drinking water with sage tea in the rats used as hepatocyte donors resulted in an improvement of the antioxidant status of rat hepatocytes in primary culture, namely a significant increase in GSH content and GST activity after 4 h of culture. When these hepatocyte cultures were exposed to 0.75 or 1 mM of tert-butyl hydroperoxide (t-BHP) for 1 h, some protection against lipid peroxidation and GSH depletion was conferred by sage tea drinking. However, the cell death induced by t-BHP as shown by lactate dehydrogenase (LDH) leakage was not different from that observed in cultures from control animals. This study indicates that the compounds present in this sage preparation contain interesting bioactivities, which improve the liver antioxidant potential.
The leaves of sage (Salvia officinalis L., Lamiaceae) are reported to have a wide range of biological activities, such as anti-bacterial, fungistatic, virustatic, astringent, eupeptic and anti-hydrotic effects. To determine the hypoglycaemic effect of sage leaves, we investigated the effects of essential oil and methanolic effect of the plant on healthy and streptozotocin-induced diabetic rats. The animals were made diabetic using by streptozotocin (70 mg/kg, i.p.). The methanolic extract (100, 250, 400 and 500 mg/kg) and essential oil (0.042, 0.125, 0.2 and 0.4 ml/kg) were injected intraperitoneally. The control groups were administered water and sunflower oil as vehicles of methanolic extract and essential oil, respectively. Blood samples were obtained from retro-orbital sinus before administration and 1, 3 and 5 h after administrations. The serum glucose was measured by the enzymatic method of glucose oxidase. The results showed that the essential oil of sage did not change serum glucose, while the plant extract significantly decreased serum glucose in diabetic rats in 3h without effect on insulin releasing from the pancreas but not in healthy rats. Also, the LD(50) of the methanolic extract is measured (4000 mg/kg, i.p.). The present data indicate that sage extract has hypoglycaemic effect on diabetic animals and the plant should be considered in future therapeutic researches.
The aim of this study is to investigate the mechanisms of the vasorelaxant effect of the crude extract of Salvia miltiorrhiza (family: Labiatae), also known as "Danshen", in rat knee joints. Changes in blood flow of rat knee joints were measured in vivo by a laser Doppler perfusion imager. Topical administration of Danshen onto the exposed knee joint blood vessels produced dose-dependent increases in blood flow. Treatment of the rat knee joint with 2x 1 nmol of atropine, 2x 0.1 nmol of propranolol, or 2x 0.1 nmol of a mixture of pyrilamine plus cimitedine produced no change on the vasodilator response to Danshen. However, significant inhibition of the Danshen-induced vasodilator response was observed in knee joints treated with 2x 100 nmol of N(G)-nitro-L-arginine methyl ester (L-NAME), 2x 100 nmol of flurbiprofen, 2x 10 nmol of the calcitnonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), and also in knee joints but had been denervated by capsaicin treatment or by surgery. Intravenous administration of low doses of Danshen (2.5 and 6 mg/kg) did not affect the systemic blood pressure but significantly increased knee joint blood flow, whereas, high doses of Danshen (167 and 381 mg/kg) produced hypotension with concurrent decreases in knee joint blood flow. These findings indicate that the knee joint blood vessels are more sensitive to the relaxant effect of Danshen compared to blood vessels in the general circulation. The vasorelaxant effect of Danshen was found to be partly mediated by CGRP released from sensory nerves, and nitric oxide and prostaglandins also played a part. However, there is no evidence to support a role for muscarinic receptors, adrenoceptors, or histamine receptors.
A reversed-phase high performance liquid chromatographic method was established for the simultaneous determination of tanshinones in five kinds of traditional Chinese medicinal preparations (TCMPs) containing Radix salvia miltiorrhiza (Chinese herbal name: Danshen). Tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA were successfully separated on a Diamonsil C18 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of methanol, tetrahydrofuran, water and glacial acetic acid (20:35:44:1, v/v/v/v), employing isocratic elution at a flow rate of 1.0 mL/min. Detection was accomplished at 254 nm. The compounds were identified by comparing their retention times and UV spectra in the 200-400 nm range with authentic standards. Regression equations revealed good linear relationship between the peak areas of the constituents and their concentrations (correlation coefficients: 0.9998 for cryptotanshinone, 0.9999 for tanshinone I and 1.0000 for tanshinone IIA). The relative standard deviations (n=6) of retention time and peak area were less than 0.25% and 1.00%, respectively. The recoveries were between 96.2% and 102.5%. The proposed method has been successfully applied to the simultaneous determination of the tanshinones in five kinds of Chinese herbal preparations containing Danshen within 20 min.
A simple, economical and effective non-aqueous micellar electrokinetic chromatography (NAMEKC) method was developed for simultaneous assay of three bioactive components (cryptotanshinone, tanshinone IIA and tanshinone I) in radix Salviae miltiorrhizae and its medicinal preparations for the first time. After optimization of separation conditions, a buffer of 140 mmol l(-1) sodium cholate (SC) in methanol was selected for the separation of the three tanshinones, but baseline separation of tanshinone I and tanshinone IIA in practical samples was not achieved. Therefore, second-order derivative electropherograms were applied for resolving overlapping peaks. Regression equations revealed good linear relationships (correlation coefficients 0.995-0.999) between peak heights in second-order derivative electropherograms and concentrations of the three analytes. The recoveries of three constituents ranged from 91.3 to 105.7%. The results indicated that baseline separation of the analytes was hard to be achieved in practical samples sometimes and second-order derivative electropherograms was applicable for the resolving and analysis of overlapping peaks.
A gradient HPLC-DAD-ESI MS method has been developed for simultaneous determination of multiple bioactive compounds such as abietane-type diterpenes, flavonolignans and phenolic compounds in compound preparations of Silybum marianum and Salvia miltiorrhiza. The separation was completed on an ODS column using 0.5% (v/v) formic acid aqueous solution and methanol as gradient mobiles. Fourteen components can be identified by ESI MS working on ESI(-) and ESI(+) switching mode, respectively. Ten components can be quantified by using external standard method with UV detecting at 254 and 280 nm, respectively. The correlation coefficients of all the calibration curves were found to be higher than 0.992. The recoveries of the standards were about 96-101%. Besides quantification of the components, the chromatograms acquired by this method can be used as the bioactive components fingerprints for the quality control of compound preparations of S. marianum and S. miltiorrhiza.
In this paper, a method using solid-phase extraction (SPE) and HPLC/ESI-MS(n) for the identification of the major saponins in "Danshen Dripping Pill", a Chinese patent medicine consisting of Salvia miltiorrhizae and Panax notoginseng, is described. Through solid-phase extraction process, the saponins in "Danshen Dripping Pill" were separated from the phenolic constituents of S. miltiorrhizae and, meanwhile, efficiently concentrated. Subsequently, these saponins were characterized by HPLC/ESI-MS(n) analysis. Based on the studies of MS and MS2 spectra and the comparison with reference compounds and literature data, a total of 19 saponins were identified.
A high-performance liquid chromatographic method was applied to the determination of danshensu, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid B and salvianolic acid A in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations. The six phenolic acids were simultaneously analyzed with a Zorbax Extend C18 column by gradient elution using 0.026% (v/v) phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1 ml min(-1), and detection wavelength was set at 288 nm. The recovery of the method was in the range of 95.1-104.8%, and all the compounds showed good linearity (r > 0.9997) in a relatively wide concentration range. This assay was successfully applied to the determination of six major phenolic acids in 32 samples. The results indicated that the developed HPLC assay could be readily utilized as a quality control method for S. miltiorrhiza and its related traditional Chinese medicinal preparations.
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.
TTE-50 is a standardized extract of Salvia miltiorrhiza which mainly consisted of tanshinones. A sensitive and specific method using liquid chromatography-diode array detection-electrospray ionization (ESI) ion trap mass spectrometry was established for the study of the constituents and metabolites of TTE-50 in rat bile sample after oral administration. The bile samples were extracted with ethyl acetate (EtOAc) of three-fold volume for three times. The chromatographic separation was carried out on a Zorbax Extend-C18 column with a gradient elution program whereas acetonitrile-water was used as mobile phase. Mass spectra were acquired in positive ionization mode and data-dependant scan was used for the identification of the tanshinones and metabolites in the bile samples. Identification and structural elucidation of the tanshinones and their metabolites in bile samples were performed by comparing their retention-times and full scan MS(n) spectra with those of reference compounds and data in the literatures. Sixteen tanshinones in TTE-50 along with seventeen phase I metabolites were identified simultaneously. The metabolic modification could take place in the C-4 side chain of tanshinone IIA, from methyl to primary alcohol, then to aldehyde group was proposed for the first time. The established method was valuable for the study of the metabolism of complex system such as herbal extracts or traditional Chinese medicine (TCM) formula.