In this study, a new capillary electrophoresis (CE) method was developed for the online determination of platelet aggregation induced by thrombin (THR). The developed CE method was applied to evaluate the anti-platelet aggregation activity of Rhizoma Corydalis extract. Besides, the anti-platelet activity of Rhizoma Corydalis extract was also evaluated by the classic turbidimetry method for comparison. The results showed that platelet aggregation rate could be obtained or calculated based on the peak height or peak area of free platelets in CE analysis, and the anti-platelet aggregation activities of Rhizoma Corydalis extract were both dose-dependent manner determined by the CE and turbidimetry methods. Furthermore, compared with classic turbidimetry for platelet aggregation assay, CE is superior in smaller sample consumption and higher repeatability. Therefore, CE may be a potential method for evaluating anti-platelet aggregation activity of drugs.

As a serine protease, thrombin (THR) plays an important role in the coagulation cascade. In this study, a frontal affinity chromatography (FAC) method was developed for the characterization of interactions between immobilized thrombin and phenolic acids, which have potential thrombin inhibitory activity. First, a simple method to immobilize thrombin was developed, where an immobilized artificial membrane (IAM) chromatographic column was used as the carrier material for entrapping THR. Then the frontal analysis of five compounds was performed both on the THR-IAM column and argatroban-THR-IAM column. Finally, the dissociation constants were determined and the values are 2.46, 12.42, 44.93, 76.91 and 100.69 μmol L−1 for gallic acid, protocatechuic acid, ferulic acid, chlorogenic acid and sinapic acid, respectively. In addition, molecular docking was further applied to study the interaction between the compounds and thrombin, and the rank order of docking energy values is similar to that of the dissociation constants of the compounds determined by FAC. The results of the present study demonstrate that the interaction between the compounds and thrombin can be well characterized by FAC experiments along with molecular docking.

OBJECTIVETo evaluate the anti-platelet aggregation effects of extracts from 31 Traditional Chinese Medicines (TCM) with the property of activating blood and resolving stasis in terms of TCM theory.METHODSThe 31 TCMs extracts were prepared using water, 90% ethanol and ethyl acetate., and the effects on anti-platelet aggregation were tested on a platelet aggregation analyzer in vitro with adenosine 5'-diphosphate, bovine thrombin and arachidonic acid (AA) as aggregation inducers, respectively. Aspirin was the positive control.RESULTSLots of the tested TCMs had inhibitory effects with concentration-dependent manner on platelet aggregations induced by various agonists. Especially, some of the TCMs such as Chuanxiong (Rhizoma Chuanxiong), Yanhusuo (Rhizoma Corydalis Yanhusuo) and Danshen (Radix Salviae Miltiorrhizae) showed good anti-platelet aggregation effect similar or higher than that in positive control group.CONCLUSIONThe study provided scientific references that several TCMs such as Chuanxiong (Rhizoma Chuanxiong), Yanhusuo (Rhizoma Corydalis Yanhusuo) and Danshen (Radix Salviae Miltiorrhizae), possess the property of anti-platelet aggregation.

Hydrophilic interaction liquid chromatography, an alternative liquid chromatography mode, is of particular interest in separating hydrophilic and polar ionic compounds. Compared with traditional liquid chromatography techniques, hydrophilic interaction liquid chromatography offers specific advantages mainly including: (1) relatively green and water-soluble mobile phase composition, which enhances the solubility of hydrophilic and polar ionic compounds; (2) no need for ion-pairing reagents and high content of organic solvent, which benefits mass spectrometry detection; (3) high orthogonality to reverse-phase liquid chromatography, well adapted to two-dimensional liquid chromatography for complicated samples. Therefore, hydrophilic interaction liquid chromatography has been rapidly developed in many areas over the past decades. This review summarizes the recent progress (from 2012 to July 2016) of hydrophilic interaction liquid chromatography in pharmaceutical analysis, with the focus on detecting chemical drugs in various matrices, charactering active compounds of natural products and assessing biotherapeutics through typical structure unit. Moreover, the retention mechanism and behavior of analytes in hydrophilic interaction liquid chromatography as well as some novel hydrophilic interaction liquid chromatography columns used for pharmaceutical analysis are also described.

Isoflavones are the main active components in Radix Puerariae, the dry root of Pueraria lobata or P. thomsonii. We developed a simple capillary zone electrophoresis (CZE) method with an ionic liquid (IL) as an additive for the simultaneous determination of eight isoflavones – ononin, daidzin, genistin, biochanin A, formononetin, puerarin, genistein and daizein – in Radix Puerariae. The experimental conditions, including the concentration of sodium tetraborate, the pH, the type and concentration of the ILs, the applied voltage and the capillary temperature, were investigated. The eight analytes (detection wavelength 260 nm) were well separated within 9 min using a running buffer of 30 mM sodium tetraborate and 50 mM 1-butyl-3-methylimidazolium tetrafluoroborate as an additive at pH 9.5, with an applied voltage of 18 kV and a capillary temperature of 25 °C. The method developed was fully validated (limit of detection, 1.72–4.92 μg mL−1; limit of quantification, 3.64–9.84 μg mL−1; intra-day precision, 1.1–4.7% RSD; inter-day precision, 2.1–6.6% RSD; and recovery, 93.1–107.5% with 4.0–5.9% RSD) and was successfully applied to the determination of the eight analytes in three Radix Puerariae samples. The results indicated that P. lobata contains more isoflavones than P. thomsonii. CZE with an IL as an additive is a promising method for the analysis of natural products.

Isoflavones are a very important group of natural products. This study investigated the separation of eight isoflavones, namely ononin, daidzin, genistin, biochanin A, formononetin, puerarin, genistein, and daidzein, from pueraria by micellar electrokinetic chromatography (MEKC) with different surfactants. The following micellar systems of MEKC were systematically compared for the analysis of these isoflavones: (1) a single surfactant comprising the anionic surfactant sodium dodecyl sulfate (SDS), the cationic surfactant hexadecyltrimethylammonium bromide, the neutral surfactant polyoxyethylene sorbitan monolaurate (Tween 20), and the ionic liquid-type surfactant (also a cationic surfactant) 1-dodecyl-3-methylimidazolium tetrafluoroborate (C12MIMBF4); (2) different single surfactants with 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF4) as an additive (modifier); and (3) mixed micelles of SDS + Tween 20 and C12MIMBF4 + Tween 20. Both SDS with BMImBF4 as additive and mixed micelles of SDS + Tween 20 had the highest separation efficiency for the eight investigated compounds. Furthermore, the SDS with BMImBF4 as additive was more stable (good repeatability of retention time and peak shape of analytes) than mixed micelles of SDS + Tween 20, which may be the result of a stabilizing effect of BMImBF4. Therefore, the final analytical conditions were 15 mM SDS added with 50 mM BMImBF4 in 30 mM sodium tetraborate (STB, pH 9.5) as running buffer; applied voltage, 20 kV; injection, 50 mbar for 5 s; cartridge temperature, 25 °C; compounds were detected at 260 nm. The developed method was fully validated (limit of detection, limit of quantification, intraday precision, inter-day precision, and recovery) and successfully applied to determine the eight analytes in three Radix Puerariae samples. The present study indicated that SDS with ionic liquids as additive in MEKC was suitable for the analysis of isoflavones.

For a herbal medicine, harvesting at different growth stage affects its quality and efficiency. We hypothesized that fatty acid profiling might be used to discriminate herbal samples according to their growth stages. To test the hypothesis, fatty acids of Pterocephalus hookeri samples collected at the flowering stage (FS) and non-flowering stages (NFS) were characterized and compared using gas chromatography-mass spectrometry (GC-MS) and followed by multivariate statistical analysis. A total of 14 fatty acids were identified and quantified in all the P. hookeri samples. Both the relative and absolute compositions of the 14 fatty acids varied greatly between the FS and NFS groups, suggesting each group has its own fatty acid pattern. Orthogonal partial least squares discriminant analysis (OPLS-DA) and hierarchical clustering analysis (HCA) based on data sets of relative and absolute composition of fatty acids showed that 13 tested samples could be clearly classified into two clusters, in terms of their growth stages. More importantly, α-linolenic acid, a plant-derived n-3 polyunsaturated fatty acid (PUFA), was identified as the potential fatty acid biomarker for its greatest contribution to the group’s separation. In addition, to evaluate the quality of P. hookeri at the FS and NFS, oleanolic acid (OA) and ursolic acid (UA) were determined by HPLC, as described in the Chinese Pharmacopoeia (version 2010). A higher total concentration of OA and UA could be found in the P. hookeri samples at the flowering stage, which was suggested to be a better quality. These findings demonstrated that GC-MS-based fatty acid profiling, coupled with multivariate statistical analysis, provides a reliable platform to discriminate the herb collected at different growth stages, which is helpful for ensuring its efficacy.

Alkaline degradation of curcuminoids was observed when they were exposed to alkaline solution, which usually occurs during capillary electrophoresis (CE) analysis with alkaline buffers. In the present study, the stability of curcuminoids (curcumin, demethoxycurcumin, bis-demethoxycurcumin) in different solutions (a boric acid buffer, a microemulsion with n-octane as the oil phase and a boric acid buffer, and a microemulsion with an ionic liquid [BMIM]PF6 as the oil phase and a boric acid buffer) was investigated. The results indicated that the microemulsion systems have a significant protective effect on the analytes, even under alkaline conditions. Compared to the boric acid buffer only, the microemulsion with an ionic liquid showed the best protective effect. Therefore, a stable and rapid microemulsion electrokinetic chromatography (MEEKC) method, with an ionic liquid as the oil phase, was developed for the simultaneous determination of the three curcuminoids in the rhizome (Jianghuang) and tuberous root (Yujin) of Curcuma longa. The experimental parameters, including the microemulsion compositions (surfactant, co-surfactant and oil phase), pH, concentration of borate buffer and capillary temperature, were intensively investigated. Finally, the investigated curcuminoids were well separated within 9 min, using a running buffer containing 100 mM SDS, 1.6 M n-butanol and 20 mM [BMIM]PF6 in 10 mM borate buffer at pH 10.2. The developed method was fully validated (using LOD, LOQ, intra- and inter-day precision and recovery) and successfully applied to determine the content of the investigated curcuminoids in four real samples (Yujin and Jianghuang from Chengdu and Leshan). All of the results showed that MEEKC with an ionic liquid as the oil phase should be an ideal choice for the analysis of curcuminoids by CE.

Safety and efficacy are two key important aspects for the quality assessment of health-food products. Cordyceps sinensis, which has multiple pharmacological activities, is one of the most expensive traditional Chinese medicines (TCMs) and is used frequently in health-food products in China. Usually, nucleosides and their related compounds have been used as markers for evaluation of the quality of natural and/or cultured Cordyceps. However, heavy metals should also be considered as they may affect the safety of clinical use. In the present study, the contents of six nucleosides (adenosine, cytidine, guanosine, uridine, inosine and thymidine), six nucleobases (adenine, cytosine, guanine, uracil, hypoxanthine and thymine) and five nucleotides (adenosine-5′-monophosphate (AMP), cytidine-5′-monophosphate (CMP), guanosine-5′-monophosphate (GMP), uridine-5′-monophosphate (UMP), and thymidine-5′-monophosphate (TMP)) in natural C. sinensis samples from different collecting places were simultaneously determined by a validated HPLC-DAD method. At the same time, six heavy metals including Hg, As, Cr, Cd, Cu and Pb in natural C. sinensis and their related soil samples were determined by atomic absorption spectrometry (AAS). The results showed that the total content of nucleosides and their related compounds, as well as the individual levels of nucleotides, are much higher in fresh samples (collected in 2012) as compared to stored samples (collected in 2011). Therefore, the storage conditions may affect on the quality of natural C. sinensis. On the other hand, the total and individual contents of heavy metals vary in different Cordyceps and soil samples, but the levels of As and Cu in Cordyceps are correlated well with the soil samples from their collection locations. Therefore, the high levels of heavy metals, especially As and Cu, in natural Cordyceps originate from the soil of the producing areas.

In the present study, a rapid and repeatable microemulsion electrokinetic chromatography (MEEKC) method was developed for the simultaneous determination of three isomers (α-, β- and γ-asarone) in Acorus tatarinowii by using ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6) as oil phase. Experimental parameters including the microemulsion compositions (concentrations of surfactant, co-surfactant and oil phase), pH, concentration of borate buffer, capillary temperature and voltage were intensively investigated. Finally, the main compounds in the methanol extract of A. tatarinowii were well separated within 11 min using a running buffer composed of 40 mmol/L sodium dodecyl sulfonate (SDS), 2.0 mol/L n-propanol, 8 mmol/L [BMIM]PF6 in 10 mmol/L borate buffer of pH 9.5. The developed method was applied to determine the contents of α-, β- and γ-asarone in A. tatarinowii from five different producing areas in China (Anhui, Hebei, Sichuan, Zhejiang and Chongqing). The results indicated that the contents of three asarones are quite different in the investigated A. tatarinowii samples. On the other hand, the MEEKC with ionic liquid as oil phase should be a promising method for the analysis of volatile components especially isomers in medicinal herbs.Highlights► An MEEKC method with ionic liquid as oil phase was developed. ► Main asarone isomers in A. tatarinowii were simultaneously determined. ► Broaden the applications of MEEKC in the analysis of volatile isomers.

For a herbal medicine, harvesting at different growth stage affects its quality and efficiency. We hypothesized that fatty acid profiling might be used to discriminate herbal samples according to their growth stages. To test the hypothesis, fatty acids of Pterocephalus hookeri samples collected at the flowering stage (FS) and non-flowering stages (NFS) were characterized and compared using gas chromatography-mass spectrometry (GC-MS) and followed by multivariate statistical analysis. A total of 14 fatty acids were identified and quantified in all the P. hookeri samples. Both the relative and absolute compositions of the 14 fatty acids varied greatly between the FS and NFS groups, suggesting each group has its own fatty acid pattern. Orthogonal partial least squares discriminant analysis (OPLS-DA) and hierarchical clustering analysis (HCA) based on data sets of relative and absolute composition of fatty acids showed that 13 tested samples could be clearly classified into two clusters, in terms of their growth stages. More importantly, α-linolenic acid, a plant-derived n-3 polyunsaturated fatty acid (PUFA), was identified as the potential fatty acid biomarker for its greatest contribution to the group’s separation. In addition, to evaluate the quality of P. hookeri at the FS and NFS, oleanolic acid (OA) and ursolic acid (UA) were determined by HPLC, as described in the Chinese Pharmacopoeia (version 2010). A higher total concentration of OA and UA could be found in the P. hookeri samples at the flowering stage, which was suggested to be a better quality. These findings demonstrated that GC-MS-based fatty acid profiling, coupled with multivariate statistical analysis, provides a reliable platform to discriminate the herb collected at different growth stages, which is helpful for ensuring its efficacy.

Safety and efficacy are two key important aspects for the quality assessment of health-food products. Cordyceps sinensis, which has multiple pharmacological activities, is one of the most expensive traditional Chinese medicines (TCMs) and is used frequently in health-food products in China. Usually, nucleosides and their related compounds have been used as markers for evaluation of the quality of natural and/or cultured Cordyceps. However, heavy metals should also be considered as they may affect the safety of clinical use. In the present study, the contents of six nucleosides (adenosine, cytidine, guanosine, uridine, inosine and thymidine), six nucleobases (adenine, cytosine, guanine, uracil, hypoxanthine and thymine) and five nucleotides (adenosine-5′-monophosphate (AMP), cytidine-5′-monophosphate (CMP), guanosine-5′-monophosphate (GMP), uridine-5′-monophosphate (UMP), and thymidine-5′-monophosphate (TMP)) in natural C. sinensis samples from different collecting places were simultaneously determined by a validated HPLC-DAD method. At the same time, six heavy metals including Hg, As, Cr, Cd, Cu and Pb in natural C. sinensis and their related soil samples were determined by atomic absorption spectrometry (AAS). The results showed that the total content of nucleosides and their related compounds, as well as the individual levels of nucleotides, are much higher in fresh samples (collected in 2012) as compared to stored samples (collected in 2011). Therefore, the storage conditions may affect on the quality of natural C. sinensis. On the other hand, the total and individual contents of heavy metals vary in different Cordyceps and soil samples, but the levels of As and Cu in Cordyceps are correlated well with the soil samples from their collection locations. Therefore, the high levels of heavy metals, especially As and Cu, in natural Cordyceps originate from the soil of the producing areas.

Alkaline degradation of curcuminoids was observed when they were exposed to alkaline solution, which usually occurs during capillary electrophoresis (CE) analysis with alkaline buffers. In the present study, the stability of curcuminoids (curcumin, demethoxycurcumin, bis-demethoxycurcumin) in different solutions (a boric acid buffer, a microemulsion with n-octane as the oil phase and a boric acid buffer, and a microemulsion with an ionic liquid [BMIM]PF6 as the oil phase and a boric acid buffer) was investigated. The results indicated that the microemulsion systems have a significant protective effect on the analytes, even under alkaline conditions. Compared to the boric acid buffer only, the microemulsion with an ionic liquid showed the best protective effect. Therefore, a stable and rapid microemulsion electrokinetic chromatography (MEEKC) method, with an ionic liquid as the oil phase, was developed for the simultaneous determination of the three curcuminoids in the rhizome (Jianghuang) and tuberous root (Yujin) of Curcuma longa. The experimental parameters, including the microemulsion compositions (surfactant, co-surfactant and oil phase), pH, concentration of borate buffer and capillary temperature, were intensively investigated. Finally, the investigated curcuminoids were well separated within 9 min, using a running buffer containing 100 mM SDS, 1.6 M n-butanol and 20 mM [BMIM]PF6 in 10 mM borate buffer at pH 10.2. The developed method was fully validated (using LOD, LOQ, intra- and inter-day precision and recovery) and successfully applied to determine the content of the investigated curcuminoids in four real samples (Yujin and Jianghuang from Chengdu and Leshan). All of the results showed that MEEKC with an ionic liquid as the oil phase should be an ideal choice for the analysis of curcuminoids by CE.