Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Pharmacokinetic Evaluation of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranon in Rats.

Recently, potent neuroprotective and anti-diabetic effects of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), a sesquiterpenoid isolated from Tussilago farfara Linnaeus, have been elucidated. To facilitate further pre-clinical evaluation in rats, an analytical method for the determination of ECN in rat plasma was developed and optimized by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples were pretreated by the protein precipitation method with an acetonitrile solution of losartan (LST) as the internal standard. Chromatographic separation was performed using a an Octadecyl-silica (ODS) column (2.6 µm, 100 x 4.6 mm) in the isocratic mode. The mobile phase, comprising 10 mM ammonium formate in water pH 5.75) and acetonitrile (11:89, v/v), was eluted at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed in the multiple reaction monitoring mode with positive electrospray ionization, and the mass transitions of ECN and LST were m/z 431.3 to 97.3 and m/z 423.1 to 207.2, respectively. The calibration curves of spiked plasma samples were linear in the 10.0-10,000 ng/mL range (r2 > 0.996). The lower limit of quantification (LLOQ) was determined as 10.0 ng/mL. Validation was conducted in the LLOQ, and three quality control (QC) sample levels (10.0, 25.0, 3750, and 7500 ng/mL) were studied. Among them, the relative standard deviation for the within- and between-run precisions was under 9.90%, and the relative error of the accuracies was within the -8.13% to 0.42% range. The validated method was successfully employed to investigate the pharmacokinetic properties of ECN in rats, which revealed the linear pharmacokinetic behavior of ECN for the first time.

A modified data filtering strategy for targeted characterization of polymers in complex matrixes using drift tube ion mobility-mass spectrometry: Application to analysis of procyanidins in the grape seed extracts.

BACKGROUNDS: Polymers, widely existing in food or dietary materials, have been attracting researchers, facing challenges, and needing effective strategies on targeted characterization in complex matrixes. METHODS: A modified data filtering strategy (including locating with drift time and m/z ranges, multiple mass defect filtering, validating MS information, and evaluating MS/MS spectra) was developed and applied for procyanidins in the grape seed extracts (GSE) using drift tube ion mobility-mass spectrometry. The procyanidin ions' trendlines were predicted by multi-model regression. Their collision cross-sections (CCSs) were calculated using single-field methods. RESULTS AND DISCUSSION: Totally, 769 CCSs belonging to 686 procyanidins with polymer degrees at 1-15 were characterized. The exponent regression was the most reasonable model (r2 ≥ 0.9379) to reveal the trendlines. The change tendency of CCSs with their polymer degrees, charge states, and linkage types were investigated. CONCLUSION: This study provided an innovative strategy for targeted characterization of polymers in complex matrixes.

Rapid screening and characterization of natural antioxidants in Polygonum viviparum by an on-line system integrating the pressurised liquid micro-extraction, HPLC-DAD-QTOF-MS/MS analysis and antioxidant assay.

Rapid discovery of active ingredients from complex matrices is one of great challenges for modern drug development. Traditional methods often require many sample treatment steps, including an extraction step with exclusively dedicated solvents followed by repeated separation and activities assessment. This present work described an integrated analytical setup for natural antioxidants discovery in which the online extraction (OLE) of a solid sample is directly coupled to its analysis by high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) antioxidant assay (OLE-HPLC-DAD-QTOF-MS/MS-ABTS). This developed approach makes sample extraction, chromatographic separation and chemical detection, and antioxidant assay integrated into a single HPLC injection and was successfully applied for the rapid discovery of natural antioxidant bioactives from Polygonum viviparum. A total of 21 secondary metabolites were characterized according to their retention times, ultraviolet (UV) spectra, exact mass and fragmentation ions in MS/MS spectra, and 18 of them displayed antioxidant activity (response as negative peaks in antioxidant assay). This work describes a simple, green and efficient approach to minimize the sample consumption (only 0.4 mg was required) and eliminate complex sample treatment procedures. The developed OLE-HPLC-DAD-QTOF-MS/MS-ABTS system offers new perspectives for rapid chemical profiling of natural products and their antioxidants discovery.

Combination of an AccQ•Tag-Ultra-Performance Liquid Chromatographic Method with Tandem Mass Spectrometry for the Analysis of Amino Acids.

Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ•Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra-performance liquid chromatography (UPLC) and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors and thoroughly tested for a variety of samples such as P. falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ•Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in Arabidopsis thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.

Direct Analysis of Underivatized Amino Acids in Plant Extracts by LC-MS/MS (Improved Method).

In this chapter we describe a method for quantification of 20 proteinogenic amino acids by liquid chromatography-mass spectrometry which affords neither derivatization nor the use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5 µ SCX 100 Å) with 5% acetic acid in water (A) and 75 mM ammonium acetate in water (B). Quantification is accomplished by use of d2-phenylalanine as internal standard achieving limits of detection of 5-50 nM. The method was successfully applied for the determination of proteinogenic amino acids in plant extracts.

Fully automated method based on on-line molecularly imprinted polymer solid-phase extraction for determination of lovastatin in dietary supplements containing red yeast rice.

A fully automated method for the determination of lovastatin in dietary supplements containing red yeast rice has been developed. It uses a sequential injection analysis system combined with solid-phase extraction applying highly selective molecularly imprinted polymer sorbent. A miniaturized column for on-line extraction was prepared by packing 4.5 mg of the sorbent in a 5.0 × 2.5-mm-i.d. cartridge, which was used in the flow manifold. Sequential injection analysis manifold enabled all steps of lovastatin extraction and continuous spectrophotometric detection at 240 nm. A limit of detection of 60 µg g-1, a limit of quantitation of 200 µg g-1, and a linear calibration range of 200-2000 µg g-1 were achieved. Intra-day and inter-day precision values (RSD) were ≤ 6.7% and ≤ 4.9%, respectively, and method recovery values of spiked red yeast rice extracts at 200, 1000, and 2000 µg g-1 concentration levels were 82.9, 95.2, and 87.7%. Our method was used for determination of lovastatin lactone in four dietary supplements containing red yeast rice as a natural source of lovastatin, also known as monacolin K. The extracted samples were subsequently analyzed by the reference UHPLC-MS/MS method. Statistical comparison of results (F test, t test, α = 0.05) obtained by both methods did not reveal significant difference. A substantial advantage of the new automated approach is high sample throughput thanks to the analysis time of 7.5 min, miniaturization via down-scaling the extraction column, and smaller sample and solvent consumption, as well as reduced generation of waste. Graphical abstract ᅟ.

Development of a validated LC-MS/MS method for the in vitro and in vivo quantitation of sunitinib in glioblastoma cells and cancer patients.

Sunitinib is a multi-targeted tyrosine kinase inhibitor approved for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumor and is currently being investigated against other forms of malignant tumors. Recently great interest has emerged for the application of sunitinib to glioblastoma treatment. In order to have a method with broad applicability it will be of importance to have access to a method that could be applied both in human plasma and cell uptake studies. No method has been reported thus far for the estimation of sunitinib uptake in glioma cells. We therefore set out to develop a method that could be applied for quantifying sunitinib in human plasma and in cell uptake studies. The method was validated and accredited according to ISO 17025:2005 guideline in human plasma and successfully applied to cancer patient plasma. Also, the method was effectively recruited to establish a protocol for the evaluation of sunitinib accumulation into M095K glioma cells. This method could significantly contribute to developmental phases in repurposing this drug in different cancer types.

Separation and analysis of flavonoid chemical constituents in flowers of Juglans regia L. by ultra-high-performance liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry.

The flowers of Juglans regia L. have demonstrated their medicinal value as a part of edible plants. In this study, an analytical methodology for identification of flavonoid chemical constituents in flowers of Juglans regia L. was established using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Thirty-six flavonoid compounds were identified based on highly accurate mass measurements, and the mass spectrometric fragmentation pathways of eleven representative compounds were proposed, which was helpful for the identification of different types of flavonoids. The study has laid the foundation for the research and development of effective drugs from flowers of Juglans regia L.

A novel strategy for rapid screening of the complex triterpene saponin mixture present in the methanolic extract of blackberry leaves (Rubus cv. Loch Ness) by UHPLC/QTOF-MS.

Triterpene saponins are important chemical constituents in blackberry leaves and have significant antimicrobial activity, among other healthful properties. In this study, a new UHPLC-MS method was optimized with outstanding efficiency for a non-targeted metabolic approach and comprehensive analysis of bioactive compounds in blackberry leaves (Rubus cv. Loch Ness). With minimum sample treatment, phenols and triterpene saponins, among others, were separated according to their elution times. Once separated, the major triterpene saponins were classified by their aglycone moieties and their structures tentatively identified based on their accurate mass spectra in positive and negative ESI mode. By the use of UHPLC coupled to a TOF, a high-resolution and accuracy mass analyzer, reliable molecular formulas of the detected compounds were predicted, and along with the generated MS spectra in full scan mode, allowed the tentative identification and classification of the most abundant triterpene saponins presented the samples analyzed. A rapid and comprehensive strategy to study the complex saponin profile is presented. Unlike other LC-MS/MS methods, the proposed method requires just one mass analyzer, and to our knowledge, this is the first systematic study on triterpene saponins in blackberry leaves.

Application of pressurized liquid extraction (PLE) to obtain bioactive fatty acids and phenols from Laminaria ochroleuca collected in Galicia (NW Spain).

The increase of pathologies like cardiovascular diseases, obesity or diabetes due to the nature of diet is a matter of concern in our society. Because of this, there is a high interest in healthy natural products that could prevent the appearance of such diseases. This paper aims to study the content of fatty acids (FAs) and phenolic compounds of brown alga Laminaria ochroleuca (L. ochroleuca) and to determine the nutritional quality of the lipids extracted using pressurized liquids extraction (PLE) technique. PLE was applied to the algae using four solvents of different polarity (hexane, ethyl acetate, ethanol and ethanol:water 1:1). Results showed that the higher yield (52%), is obtained with ethanol: water solvent, however, both ethyl acetate and ethanol enrich unsaturated fatty acid (USFA) (palmitoleic, linolenic, linoleic, oleic, araquidonic and eicosapenataenoic) in the lipid fraction of L. ochroleuca, providing extracts up to 55% of their total fatty acid content compared to other solvents. The nutritional quality of the lipids in all PLE extracts was assessed by considering the ω-6/ ω-3 fatty acid ratio and two dietary indexes involved in the risks of coronary heart disease, atherogenic (AI) and thrombogenic (TI). The lower (best) index values are for ethanol extract, 4.4 (ω-6/ ω-3), 0.74 (AI) and 1.05 (TI), followed of ethyl acetate, 4.4 (ω-6/ ω-3), 0.87 (AI) and 1.24 (TI). Finally, the antioxidant capacity of PLE alga extracts in terms of total phenol content (TPC) was analyzed by the Folin-Ciocalteu method. The ethanol: water extracts showed the highest TPC with a concentration up to 173.65 mg eq. gallic acid / g PLE extract.

A novel strategy to screen inhibitors of multiple aminoglycoside-modifying enzymes with ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry.

Resistance to aminoglycoside antibiotics occurs primarily as a result of aminoglycoside-modification enzymes (AMEs) that modify the antibiotics. In this work, a novel strategy to combat the effects of antibiotic resistance was developed by screening multiple AMEs inhibitors with ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF MS). The method screened inhibitors of three AMEs (AAC(6')-APH(2"), AAC(6') and APH(2")) simultaneously through measuring the acetyltransferase activity and phosphotransferase activity of AAC(6')-APH(2") enzyme in a single assay. Screening inhibitors of multiple targets could greatly improve the screening efficiency at early-stages of drug discovery. In this study, enzyme reaction conditions including cosubstrate, enzyme concentration and cosubstrate concentration were optimized. The inhibition constants (Ki) for two known inhibitors, paromomycin and quercetin, were determined to be 1.23 and 20.27 µM, respectively. The assay was further validated through the determination of a high Z' factor value of 0.73. The developed assay was applied to screen a chemical library against bifunctional AAC(6')-APH(2'') enzyme. Using this assay, two pyrimidinyl indole derivatives were found to be potent, and effective AAC(6')-APH(2'') inhibitors. The assay of exploring the selective inhibitory effect on two AAC(6')-APH(2'') active sites was further performed. Two pyrimidinyl indole derivatives were found to exhibit striking inhibitory activities on AAC(6').

Determining the Mode of Action of Antimalarial Drugs Using Time-Resolved LC-MS-Based Metabolite Profiling.

Methods for assessing the mode of action of new antimalarial compounds identified in high throughput phenotypic screens are needed to triage and facilitate lead compound development and to anticipate potential resistance mechanisms that might emerge. Here we describe a mass spectrometry-based approach for detecting metabolic changes in asexual erythrocytic stages of Plasmodium falciparum induced by antimalarial compounds. Time-resolved or concentration-resolved measurements are used to discriminate between putative targets of the compound and nonspecific and/or downstream secondary metabolic effects. These protocols can also be coupled with 13C-stable-isotope tracing experiments under nonequilibrative (or nonstationary) conditions to measure metabolic dynamics following drug exposure. Time-resolved 13C-labeling studies greatly increase confidence in target assignment and provide a more comprehensive understanding of the metabolic perturbations induced by small molecule inhibitors. The protocol provides details on the experimental design, Plasmodium falciparum culture, sample preparation, analytical approaches, and data analysis used in either targeted (pathway focused) or untargeted (all detected metabolites) analysis of drug-induced metabolic perturbations.

Qualitative and quantitative analysis of Yifei Tongluo granules to identify main bioactive components using LC-DAD/MS and pharmacokinetic studies.

A standard fingerprint containing twelve common peaks was constructed from ten batches of Yifei Tongluo granules to evaluate batch-to-batch consistency by using HPLC-DAD. Additionally, the corresponding medicinal material attributes of these chemical constituents were analyzed according to the data acquired from the HPLC method and the identification was further carried out using the LC-MS/MS method. Comparing the retention time or accurate mass with previous studies or standards, the common components were tentatively identified in 50 min for ten batches of samples. At the same time, a reliable LC-MS/MS method was established to quantify marker substances simultaneously in 25 min, and the linear relationship of the standard curves was good in the experimental range. The validations of the method were successfully applied to the quality control and pharmacokinetic study. The results obtained from this study suggest that militarine was most abundant and the components in the granules caused pharmacokinetic herb-drug interactions in rats. This study provides a meaningful basis for evaluating the viability of Yifei Tongluo granules for clinical applications.

A multidimensional analytical approach based on time-decoupled online comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of ginsenosides from white and red ginsengs.

Here, time-decoupled comprehensive two-dimensional ultra-high liquid chromatography (UHPLC) coupled with an ion mobility (IM)-high resolution mass spectrometer (HRMS) was established and used to analyze ginsenosides from the main roots of white ginseng (WG) and red ginseng (RG), which enabled the separation of complex samples in four dimensions (2D-LC, ion mobility, and mass spectrometry). The incompatibility of mobile phases, dilution effect, and long analysis time, which are the main shortcomings of traditional comprehensive 2D-LC methods, were largely avoided in this newly established 2D-UHPLC method. The orthogonality of this system was 55%, and the peak capacity was 4392. Under the optimized 2D-UHPLC-IM-MS method, 201 ginsenosides were detected from white and red ginseng samples. Among them, 10 pairs of co-eluting isobaric ginseng saponins that were not resolved by 2D-UHPLC-HRMS were further resolved using 2D-UHPLC-IM-MS. In addition, 24 ginsenoside references were analyzed by UHPLC-IM-MS to obtain their collision cross section (CCS) values and ion mobility characteristics. Finally, the established new method combined with multivariate statistical analysis was successfully applied to differentiate WG and RG, and 9 ginsenosides were found to be the potential biomarkers by S-Plot and the values of max fold change, which could be used for classifying WG and RG samples. Overall, the obtained results demonstrate the applicability and potential of the established time-decoupled online comprehensive 2D-UHPLC-IM-MS system, and it will be extended to the analysis of other targeted or untargeted compounds, especially co-eluting isomers in more herbal extracts.

Simultaneous determination of seven flavonoids, two phenolic acids and two cholesterines in Tanreqing injection by UHPLC-MS/MS.

A new ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry was developed to evaluate the quality of Tanreqing injection. Seven flavonoids (Rutin, Baicalin, Scutellarin, Chrysin-7-O-Beta-d-glucoronide, Oroxylin A-7-O-ß-d-glucoronide, Wogonin, Luteolin-7-O-glucoside), two phenolic acids (Chlorogenic acid, Caffeic acid) and two cholesterines (Ursodeoxycholic acid, Chenodeoxycholic acid) in Tanreqing injection could be measured simultaneously. For the determination of the eleven compounds, the conditions were set as follows: The mobile phase was a gradient of 0.1% aqueous formic acid solution (A) and acetonitrile (B); the flow rate was 0.2 mL min-1, the column was Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm); and the multiple-reaction monitoring (MRM) with a negative electro spray ionization interface (ESI-) was selected. Within the test ranges, all the standard regression curves showed excellent linear regression (r > 0.99). In terms of (relative standard deviation) RSDs, the precision, repeatability and stability of the eleven compounds were all lower than 3%. The recovery rates of Tanreqing injection and the RSD were 97.8-103.7% and 0.4%-2.0%, respectively. The RSD value was in accordance with the requirements of less than 3.0%. This method has been successfully used in the analysis of Tanreqing injection. In summary, a fast, accurate and reliable UPLC-ESI--MS/MS method was successfully developed for the simultaneous detection of the eleven major active ingredients with different chemical structures in Tanreqing injection, and can be used for the quality control of Tanreqing injection as well.

New insights into phenol and polyphenol composition of Stevia rebaudiana leaves.

The diversity in phenols and polyphenols of stevia leaf has been simplified applying sequential fractionation techniques on its ethanol extract through ultrasound assisted maceration. Two of the fractions obtained by reverse-phase column chromatography resulted differently active in an extensive antioxidant and cytotoxic screening. Both fractions were chemically profiled by ultra-performance liquid chromatography (UHPLC) coupled with electrospray ionization (ESI) quadrupole/time-of-flight (QqTOF) mass spectrometry (MS). One of the fractions was composed mainly of chlorogenic acids and flavonol triglycosides, whereas the other was rich in flavonoids mono- and diglycosides and in their hydroxycinnamoyl derivatives. Among the fifty compounds identified, non-phenol metabolites, such as benzyl primeveroside and roseoside, as well as a lignan polyphenol (5'), are reported for the first time as constituents of the Stevia leaf.

Interactions of pharmacokinetic profiles of Ginkgotoxin and Ginkgolic acids in rat plasma after oral administration.

Ginkgolic acids (GAs) and Ginkgotoxin (4'-O-methylpyridoxine, MPN) are main toxic compounds in Ginkgo biloba seeds which are widely used in the treatment of coughing in China. To evaluate the pharmacokinetics of GAs, MPN and their metabolites in rat plasma, a highly sensitive method followed by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has been developed and validated. The proposed method is selective, precise and accurate enough of MPN and its metabolites (4-pyridoxic Acid, pyridoxal, and pyridoxine) for the pharmacokinetic study. After oral administration of MPN, the plasma concentrations of MPN and its metabolites were increased rapidly. Meanwhile, an investigation was carried out to compare the interactions of the pharmacokinetic profiles of MPN and GAs. Five GAs and main metabolites of GA (15:1) and GA (17:1) were also analyzed by using our previous method. After coadministration GAs with MPN, Tmax of MPN delayed and Cmax decreased. Meanwhile, Tmax of 4-pyridoxic Acid, pyridoxal, and pyridoxine were also showed a certain degree of delay. The concentrations of hydroxylation products of GA (15:1) and GA (17:1) increased at a slower rate and the area under the curves was significantly reduced. However, glucuronidation metabolites of GA (15:1) and GA (17:1) were increased faster than administered of GAs alone. The interactions of the pharmacokinetic profiles of GAs and MPN in rat plasma after oral administration were obviously observed.

Rapid discrimination between red and white ginseng based on unique mass-spectrometric features.

Red ginseng (RG) and white ginseng (WG), two processed products of Panax ginseng C. A. Meyer, are in high demand due to their unique features. In this study, some of these unique features were identified and confirmed as biomarkers of RG by using ultra-high-performance liquid chromatography-mass spectrometry, data mining, support vector machine, and artificial neural network. Principal component analysis showed clear separation between the RG and WG extracts, indicating the presence of potential discriminators. In addition, 20 features that are dominant in RG were found by data mining. Samples of Panax quinquefolium (PQ) and Panax notoginseng (PN), close relatives of Panax ginseng C.A.Meyer, were investigated and it was found that 17 features which were absent in PQ and PN samples, were present in RG and WG. Five of these markers were identified as nitrogen-containing compounds that have not been previously reported. Finally, we found that RG can be identified among different ginseng medicinal herbs including RG, WG, PQ, and PN samples, by loading four feature markers corresponding to nitrogen-containing compounds into a discriminating model, based on a support vector machine or an artificial neural network. Thus, this study provides an efficient tool to identify RG during pharmacological research.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Rapid identification of urokinase plasminogen activator inhibitors from Traditional Chinese Medicines based on ultrafiltration, LC-MS and in silico docking.

The urokinase plasminogen activator (uPA) is regarded as the crucial trigger for plasmin generation, which is involved in several diseases especially for neoplasm metastasis. In this study, an efficient approach integrating ultrafiltration, LC/MS, bioassay and in silico docking, was proposed for rapidly detecting uPA ligands from Traditional Chinese Medicines (TCMs). Forty-two TCMs were initially assessed, and as illustrative case studies, Galla Chinensis and Sanguisorbae Radix, which appeared significant inhibitory activities on uPA, were chosen to develpe and verify the strategy. A total of seven uPA ligands were successfully detected and identified. Two of them, pentagalloylglucose and 28-O-ß-d-glucopyranosyl pomolic acid, were demonstrated to be potential inhibitors, with IC50 at 1.639 µM and 37.82 µM repectively. Furthermore, a combinatorial compound library screening combined with in silico docking assay, was revealed that ursolic acid (IC50 = 2.623 µM) was also speculated to be a potent parent structure for inhibition of uPA. This approach offers a multidimensional perspective to discover uPA-binding leading compounds from TCMs or other complex mixtures, which would provide an efficient route for drug discovery.