Identification of tanshinone IIA metabolites in rat liver microsomes by liquid chromatography-tandem mass spectrometry

Tanshinone IIA, the major component extracted from Radix salvia miltiorrhiza, has been observed to possess various kinds of pharmacological activities including antioxidant, prevention of angina pectoris and myocardial infarction and anticancer. Tanshinone IIA was incubated with rat liver microsomes and the resulting metabolites were identified by liquid chromatography/tandem mass spectrometry. The results showed the formation of three main hydroxyl metabolites. The three hydroxyl metabolites of tanshinone IIA were proved to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A by comparing the tandem mass spectra and the chromatographic retention time with that of the respective authentic compounds. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A are all the chemical components of total tanshinones. It was reasonable to presume that the three hydroxy metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA and the total tanshinones.     

Electrochemical and spectroelectrochemical studies of four tanshinones from Salvia miltiorrhiza Bunge

The spectra of four tanshinones in the potentiostatic reduction process, including tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone, were investigated using spectroelectrochemical cell and UV spectrophotometer. Their cyclic voltammograms （CVs） were recorded with a glassy carbon electrode （GCE）. The experiment results show that the antioxidant activity of these tanshinones, in the structure, where A, B and C rings connect through a single double bond, is weaker than that where A ring does not have double bond. Moreover, the increasing angle strain in the reduction process could enhance the antioxidant activity. In summary, the rank of antioxidant activities of these tanshinones, from weak to strong, is tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone.     

Characterization of metabolites of tanshinone IIA in rats by liquid chromatography/tandem mass spectrometry

The metabolism of tanshinone IIA was studied in rats after a single-dose intravenous administration. In the present study, 12 metabolites of tanshinone IIA were identified in rat bile, urine and feces with two LC gradients using LC-MS/MS. Seven phase I metabolites and five phase II metabolites of tanshinone IIA were characterized and their molecular structures proposed on the basis of the characteristics of their precursor ions, product ions and chromatographic retention time. The seven phase I metabolites were formed, through two main metabolic routes, which were hydroxylation and dehydrogenation metabolism. M1, M4, M5 and M6 were supposedly tanshinone IIB, hydroxytanshinone IIA, przewaquinone A and dehydrotanshinone IIA, respectively, by comparing their HPLC retention times and mass spectral patterns with those of the standard compounds. The five phase II metabolites identified in this research were all glucuronide conjugates, all of which showed a neutral loss of 176 Da. M9 and M12 were more abundant than other identified metabolites in the bile, which was the main excretion path of tanshinone IIA and the metabolites. M12 was the main metabolite of tanshinone IIA. M9 and M12 were proposed to be the glucuronide conjugates of two different semiquinones and these semiquinones were the hydrogenation products of dehydrotanshinone IIA and tanshinone IIA, respectively. This hydrogenized reaction may be catalyzed by the NAD(P)H: quinone acceptor oxidoreductase (NQO). The biotransformation pathways of tanshinone IIA were proposed on the basis of this research.     

Study of the phase I and phase II metabolism of a mixture containing multiple tanshinones using liquid chromatography/tandem mass spectrometry

Metabolism of a mixture containing four dominant components in lipid solubles of Danshen was studied both in vitro and in vivo. The parent compounds and their metabolites were simultaneously detected by using liquid chromatography coupled with ion trap mass spectrometry. The results indicated that oxidation was the major pathway in phase I metabolism. O-Glucuronidation of the hydroxylated tanshinones was identified in the rat urine samples collected after the oral administration of the tanshinone components. The metabolic rates obtained from the in vitro metabolism study of each individual component were significantly different from those obtained from the incubation study of the four components in a cassette. Metabolite identification showed that tanshinone IIA and tanshinone I were the major metabolites of cryptotanshinone and dihydrotanshinone I, respectively. The obtained results demonstrated the metabolic change between the active components in Danshen and suggested the need to study the multiple components or even the extract from the herbal medicines.     

Metabolism of tanshinone IIA, cryptotanshinone and tanshinone I from Radix Salvia miltiorrhiza in zebrafish

The study aimed to investigate the potential of zebrafish in imitating mammal phase I metabolism of natural compounds. Three diterpenoid quinones from Radix Salvia miltiorrhiza, namely tanshinone IIA (TIIA), cryptotanshinone (Cry) and tanshinone I (TI) were selected as model compounds, and their metabolites mediated by zebrafish were characterized using a high-performance liquid chromatography coupled ion-trap mass spectrometry (HPLC/IT-MSn) method with electrospray ionization in positive mode. The separation was performed with a Zorbax C-18 column using a binary gradient elution of 0.05% formic acid acetonitrile/0.05% formic acid water. According to the MS spectra and after comparison with reference standards and literature reports, hydroxylation, dehydrogenation or D-ring hydrolysis metabolites of TIIA and Cry but not of TI were characterized, which coincided with those reported using regular in vivo or in vitro metabolic analysis methods, thus verifying that zebrafish can successfully imitate mammalian phase I metabolism which instills further confidence in using zebrafish as a novel and prospective metabolism model.     

Simultaneous determination of tanshinone IIA and its three hydroxylated metabolites by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of tanshinone IIA and its three hydroxylated metabolites, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, in a rat liver microsome was developed and fully validated. A single step of liquid-liquid extraction with ethyl acetate was utilized in this method. Chromatographic separation of the sample matrix from the analytes and the internal standard diazepam was performed using a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1-500 ng/mL for all analytes. The intra- and inter-day precisions (RSD %) were within 15% and deviations of the assay accuracies were within 15.0% for all analytes. The analytes proved to be stable during sample storage, preparation and analyses. This validated method was successfully applied to the enzyme kinetic study of tanshinone IIA in liver microsome. The elimination of tanshinone IIA and formation of tanshinone IIB and hydroxytanshinone IIA in the liver microsome all exhibited a sigmoidal kinetics profile. The formation of przewaquinone A shows a typical hyperbolic profile. In addition, this method has now been applied in the analysis of other bio-samples including plasma, urine, bile and feces.     

Microwave-assisted extraction of tanshinones from Salvia miltiorrhiza bunge with analysis by high-performance liquid chromatography

A novel microwave-assisted extraction (MAE) method has been developed for the extraction and determination of tanshinones (tanshinone IIA, cryptotanshinone and tanshinone I) from the root of Salvia miltiorrhiza bunge with analysis by HPLC. Various experimental conditions were investigated to optimize the percentage extraction. Under appropriate MAE conditions, such as ethanol concentrations of 95% (v/v), MAE for 2 min, liquid/solid ratio of 10:1 (ml/g), the percentage extraction can reach high in a short time. The percentage extraction (tanshinone IIA: 0.29%; cryptotanshinone: 0.23%; tanshinone I: 0.11%) by MAE was the same or even higher than conventional extraction methods. MAE only needs 2 min, but extraction at room temperature, heat reflux extraction, ultrasonic extraction and Soxhlet extraction need 24 h, 45 min, 75 min and 90 min, respectively. MAE was also available in pilot plant form for larger scale extraction.     

Molecular imprinting in ionic liquid-modified porous polymer for recognitive separation of three tanshinones from Salvia miltiorrhiza Bunge

Five ionic liquid-modified porous polymers with different imidazolium-based functional groups were obtained. A molecular imprinting technique was introduced to form the ordered functional groups in the porous structure. The adsorption isotherm was applied to investigate the interactions between the polymers and target compounds: cryptotanshinone; tanshinone I; tanshinone IIA. Thorough comparison revealed that the polymer with a carboxyl group possessed the highest reorganization of the three compounds. After that, the obtained polymer was applied as the sorbent in the solid-phase extraction process to separate the target compounds from methanol extract. The loading volume of extract solution on the sorbent was determined by adsorption isotherm equation and practical test. Under optimized washing and elution conditions, 0.35 mg/g of cryptotanshinone, 0.33 mg/g of tanshinone I, and 0.27 mg/g of tanshinone IIA from plant were obtained by quantitative HPLC analysis. Moreover, six commercial functional drinks containing tanshinones were purified and analyzed.     

Efficient separation of tanshinones by polyvinylpyrrolidone‐stabilized graphene‐modified micellar electrokinetic chromatography

In this work, a PVP‐stabilized graphene was used in MEKC for the separation of tanshinones. Seven structurally similar tanshinones were studied, that is, tanshinone IIB, dihydrotanshinone I, tanshinone I, cryptotanshinone, 1,2‐dihydrotanshinone I, miltirone, and tanshinone IIA. To achieve optimal conditions, graphene concentration, sample solvent composition, SDS concentration, 2‐propanolconcentration, and buffer pH were investigated. At a separation voltage of 30 kV and a 41.5 cm effective length fused‐silica capillary, good resolution within 12 min was performed using 10 mM borate buffer (pH 9.3) containing 30 mM SDS, 10% v/v 2‐propanol and 6 μg/mL graphene. The method was validated in terms of linearity (r² > 0.9970), intra‐ and inter‐day precision were less than 3.56 and 4.83%, respectively. The proposed method was then successfully applied to Danshentong capsule, an herbal preparation from Salvia miltiorrhiza. Our results indicated the high separation efficiency of PVP‐stabilized graphene provided new opportunities for the analysis of complex samples.     

Characterization of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen) by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

The qualitative analysis of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen in Chinese) was performed using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (ESI-MS(n)). Tanshinones are the major bioactive constituents of Dan-shen, which is used in China for the treatment of haematological abnormalities and cardiovascular diseases. The ESI-MS(n) fragmentation behavior of tanshinones was investigated. For tanshinones with the tanshinone I nucleus, the fragmentation was triggered by loss of a molecule of CO except bearing a substituent at C17 or C18, followed by sequential eliminations of CO. If C(15-16) was a saturated bond, the fragmentation was triggered by elimination of a molecule of H2O. For tanshinones with the tanshinone IIA nucleus, the fragmentation was triggered by loss of a molecule of H2O, followed by successive eliminations of CO. Ions corresponding to loss of a molecule of propylene (Delta m = 42) were also observed. Moreover, when C(15-16) was a saturated bond, ions corresponding to losses of CH3, H2O and propylene were more abundant. If no D-ring existed, the presence of isopropyl resulted in an elimination of a molecule of H2O with an adjacent CO or OH. In addition, the extension of the pi-conjugation in the A-ring (especially at C(1-2)) induced the fragmentation by loss of a molecule of CO. These fragmentation rules were applied to the identification of tanshinones in a chloroform/methanol (3:7) extract of Dan-shen, which was separated on a C18 column with gradient elution. A total of 27 tanshinones were identified, including five new constituents. The established method could be used for the sensitive and rapid identification of tanshinones in the Dan-shen drug and its pharmaceutical preparations.     

Simultaneous determination of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone in rat plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study of a standardized fraction of Salvia miltiorrhiza, PF2401-SF

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone, the active components of Salvia miltiorrhiza in rat plasma, was developed. After liquid-liquid extraction with tariquidar as an internal standard, tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone were eluted from an Atlantis dC18 column within 5 min with a mixture of methanol and ammonium formate (10 mm, pH 6.5; 85:15, v/v). The analytes were detected by an electrospray ionization tandem mass spectrometry in the selected reaction monitoring (SRM) mode. The standard curves were linear (r=0.999) over the concentration range of 0.25-80 ng/mL for tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone in rat plasma. The coefficients of variation and the relative errors of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone for intra- and inter-assay at four quality control (QC) concentrations were 1.1-5.1% and -4.0-6.0%, respectively. The lower limit of quantification for tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone was 0.25 ng/mL from 100 microL of plasma. This method was successfully applied to the pharmacokinetic study of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone after oral administration of PF2401-SF, the standardized fraction of Salvia miltiorrhiza enriched with tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone to male Sprague-Dawley rats.     

Simultaneous determination of four tanshinones in Salvia miltiorrhiza by pressurized liquid extraction and capillary electrochromatography

A pressurized liquid extraction (PLE) and CEC were developed for the simultaneous determination of four tanshinones (dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA) in Salvia miltiorrhiza. High extraction efficiency (>98.5%) was achieved under the optimum PLE conditions. A good separation was obtained by using a Hypersil C18 capillary (3 microm, 100 microm/25 cm) with a mixture of 30 mM Tris-HCl (pH 8.5)-ACN (1:3, v/v) as BGE solution running at 20 kV and 20 degrees C within 12 min. All the calibration curves showed good linearity (r2 >0.9958) within test ranges. The developed method showed good repeatability for the quantification of four investigated components in S. miltiorrhiza with intra- and interday variations of less than 4.4 and 6.8%, respectively. The validated method was successfully applied to quantify four tanshinones in S. miltiorrhiza, which is helpful to control the quality of S. miltiorrhiza.     

Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC‐MS/MS

A fast, sensitive and reliable ultra performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantitation and pharmacokinetic study of five tanshinones (tanshinone I, tanshinone IIA, tanshinone IIB, dihydrotanshinone I, cryptotanshinone), the bio‐active ingredients of Huo Luo Xiao Ling Dan (HLXLD) in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim‐pack XR‐ODS column (75 × 3.0 mm, 2.2 µm particles) and eluted with a mobile phase consisting of acetonitrile–0.05% formic acid aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min, and the total run time was 7.0 min. The detection was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.050–0.400 ng/mL for all the analytes. Linearity, precision and accuracy, the mean extraction recoveries and matrix effects all satisfied criteria for acceptance. This validated method was successfully applied to a comparative pharmacokinetic study of five bio‐active components in rat plasma after oral administration of HLXLD or Salvia miltiorrhiza extract in normal and arthritic rats. The results showed that there were different pharmacokinetic characteristics among different groups. Copyright © 2016 John Wiley & Sons, Ltd.     

Separation of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of tanshinones from the roots of Salvia miltiorrhiza Bunge by stepwise elution. A set of three solvent systems and other experimental conditions were determined by analytical HSCCC. Using the optimized conditions, the preparative HSCCC separation was performed on 50 mg of crude light petroleum extract yielding pure tanshinones of tanshinone HA (7 mg), tanshinone I (3 mg) and cryptotanshinone (4 mg) all at purities of over 95% in a single run.     

One single LC-MS/MS analysis for both phenolic components and tanshinones in Radix Salviae Miltiorrihizae and its medicinal products

A LC-MS/MS method was developed for the separation and simultaneous determination of phenolic components including danshensu, protocatechuic acid, protocatechuic aldehyde and caffeic acid as well as tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA in samples of Radix Salviae Miltiorrhizae and Salviae Miltiorrhizae tablet. Triple quadrupole mass spectrometry was optimized in both positive and negative ion multiple reaction monitoring modes for the simultaneous quantitative analysis of the two different types of active components by using a time-segment program. The method gave recoveries of 85.4-106.4% with relative standard deviations of 2.4-8.0% for the spiked herb samples. The limits of detection were 0.30-0.83 μg/g for the analysis of 1.0 g Radix Salviae Miltiorrhizae or tablet samples.     

Preparative isolation and purification of six diterpenoids from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of six diterpenoids. dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude diterpenoids were obtained by extraction with ethanol-n-hexane (1:1, v/v) from S. miltiorrhiza Bunge. Preparative HSCCC with the two-phase solvent systems A composed of n-hexane-ethanol-water (10:5.5:4.5, v/v) and B composed of n-hexane-ethanol-water (10:7:3, v/v) was successfully performed in a stepwise elution yielding six relatively pure diterpenoids from 300 mg of the crude extract in a single run. The purities of dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B were 88.1, 98.8, 97.6, 93.5, 96.8 and 94.3%, respectively.     

Comparative in vivo constituents and pharmacokinetic study in rats after oral administration of ultrafine granular powder and traditional decoction slices of Chinese Salvia

Salvia miltiorrhiza, one of the most well‐known herbal medicines, is commonly used for the treatment of coronary heart diseases in China. Besides traditional decoction slices (TDS), another relatively new product of S. miltiorrhiza, ultrafine granular powder (UGP; D90 < 45 μm), is also increasingly being used. In this paper, a UHPLC‐LTQ‐Orbitrap MS technique was developed for a metabolite profile study after oral administration of UGP and TDS of S. miltiorrhiza. The results showed that the number of in vivo absorbed compounds from UGP was much greater than that from TDS, and different types of products from S. miltiorrhiza will have different metabolic processes in vivo. Furthermore, a UHPLC‐Q‐Trap MS/MS method for simultaneously determining four tanshinones (tanshinone IIA, dihydrotanshinone I, tanshinone I and cryptotanshinone) was established and applied to assess the pharmacokinetics of the two types of products. All of the analytes displayed significant higher area under the concentration–time curve and peak concentration after oral administration of UGP than after TDS, indicating that ultrafine powder product could improve the bioavailability and absorption of cryptotanshinon,tanshinone II A,dihydrotanshinonE I and tanshinone I in vivo. The present study provides scientific information for further exploration of the pharmacology of these two types of S. miltiorrhiza and offers a reference for clinical administration of S. miltiorrhiza.     

Determination of Three Tanshinones from Radix Salvia Miltiorrhiza by Molecularly Imprinted Solid-phase Extraction

A selective molecularly imprinted solid‐phase extraction procedure was developed for the selective separation of tanshinone I, tanshinone IIA, and cryptotanshinone from Radix Salvia Miltiorrhiza samples. Tanshinone IIA imprinted polymers (MIP) synthesized in ethanol‐dodecanol system show high affinity to tanshinone IIA and its structure analogs in aqueous environment and the affinity can be controlled by adjusting the intensity of the eluents. By using 60% water‐40% methanol (volume ratio) and 99.5% methanol‐0.5% trifluoroacetic acid (volume ratio) as washing and eluting solvents, most interferences originating from the salvia matrix were eliminated. The extracts were sufficiently clean enough to be directly injected into HPLC for further chromatographic analysis. Good linearity was obtained from 0.4 to 500.0 mg·L^?1 (r²=0.999) with the relative standard deviations less than 4.2%. The mean recoveries of tanshinone IIA in Radix Salvia Miltiorrhiza were more than 85.6% at three different concentrations and the limits of detection were 0.06–0.09 mg·L^?1. This method is a viable alternative tool to the existing HPLC methods for analyzing the content of the three tanshinones in Radix Salvia Miltiorrhiza samples.     

Diterpenoid tanshinones and phenolic acids from cultured hairy roots of Salvia miltiorrhiza Bunge and their antimicrobial activities

Four diterpenoid tanshinones and three phenolic acids were isolated from the crude ethanol extract of the cultured hairy roots of Salvia miltiorrhiza Bunge by bioassay-guided fractionation. By means of physicochemical and spectrometric analysis, they were identified as tanshinone ΙΙA (1), tanshinone Ι (2), cryptotanshinone (3), dihydrotanshinone Ι (4), rosmarinic acid (5), caffeic acid (6), and danshensu (7). These compounds were evaluated to show a broad antimicrobial spectrum of activity on test microorganisms including eight bacterial and one fungal species. Among the four tanshinones, cryptotanshinone (3) and dihydrotanshinone Ι (4) exhibited stronger antimicrobial activity than tanshinone ΙΙA (1) and tanshinone Ι (2). The results indicated that the major portion of the antimicrobial activity was due to the presence of tanshinones and phenolic acids in S. miltiorrhiza hairy roots, which could be used as the materials for producing antimicrobial agents for use in agricultural practice in the future.     

Cryptotanshinone but not tanshinone IIA inhibits angiogenesisin vitro

In the course of screening of angiogenesis inhibitor from natural products, cryptotanshinone from Salvia miltiorrhiza was isolated as a potent small molecule inhibitor of angiogenesis. Cryptotanshinone inhibits bFGF-induced angiogenesis of BAECs at ten micromolar ranges in vitro without cytotoxicity. Tanshinone IIA, another tanshinone isolated from S. miltiorrhiza, which is structurally very similar to cryptotanshinone except C-15 position of dihydrofuran ring does not inhibit angiogenesis induced by bFGF. These results demonstrate that cryptotanshinone is a new anti-angiogenic agent and double bond at C-15 position of the dihydrofuran ring plays a crucial role in the activity.