Screening and identification of tyrosinase inhibitors in edible plant materials by on-line UPLC-enzyme reactor coupled with UHPLC-FTMS.

Tyrosinase plays a primary role in melanin biosynthesis and enzymatic browning of freshly cut fruits and vegetables. Herein, an on-line ultraperformance liquid chromatography diode array detector biochemical detection (UPLC-DAD-BCD) method was established to identify trace amount potent tyrosinase inhibitors and antibrowning agents in complex mixtures. The tyrosinase inhibition activities of some representative compounds were evaluated by using the established method and their chromatography-activity relationships were obtained. Then the proposed UPLC-DAD-BCD method was applied to screen tyrosinase inhibitors in edible herbal extracts and identified two tyrosinase inhibitors in green tea and three in cinnamon. The above active ingredients were determined by ultra-high-performance liquid chromatography linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-FTMS). The on-line UPLC-DAD-BCD in combination with UHPLC-FTMS was confirmed to be a powerful technique to screen and elucidate the active ingredients in complex matrixes and could be applied to evaluate the integrated effects of multiple ingredients against corresponding targets.

Simultaneous screening, identification, quantitation, and activity evaluation of six acetylcholinesterase (AChE) inhibitors in Coptidis Rhizoma by online UPLC-DAD coupled with AChE biochemical detection.

Acetylcholinesterase (AChE) inhibition is a common treatment in the early stages of dementia and neurodegenerative diseases, including Alzheimer's disease (AD). Coptidis Rhizoma (CR), a traditional Chinese medicine (TCM), contains numerous isoquinoline alkaloids that substantially inhibit AChE played neuroprotective effects in the treatment of cognitive diseases. We established a method using an ultra-high-performance liquid chromatography-diode array detector coupled with AChE biochemical detection (UPLC-DAD-AChEBCD) to screen and identify AChE inhibitors (AChEIs), measure AChEIs content and activity, and evaluate the quality of CR derived from different plant species and growth year. The chromatographic fingerprint and AChEIs activity profiles of CR were simultaneously obtained by UPLC-DAD-AChEBCD, and six alkaloids including groenlandicine, coptisine, epiberberine, jatrorrhizine, berberine, and palmatine, were identified by UPLC-electrospray ionization-tandem mass spectrometry. Data analysis based on AChEIs content and total activity of 12 batches of CR indicated differences among different species and growth year. Therefore, the online method could be used to rapidly identify AChEIs in complex matrixes and screen potential agents for neurodegenerative prevention and treatment, as well as provide information for the identification and quantitation of active markers directly associated with herbal medicine quality.

"Quantity-effect" research strategy for comparison of antioxidant activity and quality of Rehmanniae Radix and Rehmannia Radix Praeparata by on-line HPLC-UV-ABTS assay.

BACKGROUND: Quantitation analysis and chromatographic fingerprint of multi-components are frequently used to evaluate quality of herbal medicines but fail to reveal activity of the components. It is necessary to develop a rational approach of chromatography coupled with activity detection for quality assessment of herbal medicines. METHODS: An on-line HPLC-ultraviolet detection-2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging (HPLC-UV-ABTS) method was developed to obtain the chromatographic fingerprints and ABTS+• inhibition profiles (active fingerprints) of Rehmanniae Radix (Dihuang) and Rehmannia Radix Praeparata (Shu Dihuang). Eighteen compounds showing ABTS+• inhibition activity were identified by HPLC-fourier-transform mass spectrometry (HPLC-FTMS). Verbascoside was used as a positive control to evaluate the total activities of the samples and the contribution rate of each compound. The similarities of the chromatographic and active fingerprints were estimated by the vectorial angle cosine method. RESULTS: The results showed that the HPLC-UV-ABTS method could efficiently detect antioxidant activity of the herbal medicine samples. The antioxidants were different between the two herbs and several new antioxidants were identified in Shu Dihuang. A function equation was generated in terms of the negative peak area (x) and the concentrations of verbascoside (y, µg/mL), y = 2E-07 × 4 - 8E-05 × 3 + 0.0079 × 2 + 0.5755x + 1.4754, R2 = 1. Iridoid glycosides were identified as main antioxidants and showed their higher contributions to the total activity of the samples. The total contributions of the three main active components in the Dihuang and Shu Dihuang samples to the total activity, such as echinacoside, verbascoside and an unknown compound, were 39.2-58.1% and 55.9-69.4%, respectively. The potencies of the main active components in the Shu Dihuang samples were two to ten times those in the Dihuang samples. Similarity values for S12 in the chromatographic fingerprints and S03, S12 and P03 in the active fingerprints were less than 0.9. The three batches of samples might show their different quality with the other samples. CONCLUSIONS: The results suggested that the combination of "quantity-effect" research strategy and the HPLC-UV-ABTS analysis method could comprehensively evaluate the active components and quality of Dihuang and Shu Dhuang.

On-line high-performance liquid chromatography coupled with biochemical detection method for screening of α-glucosidase inhibitors in green tea.

An on-line high-performance liquid chromatography-biochemical detection (HPLC-BCD) method, in which compounds separated by HPLC were on-line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α-glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on-line HPLC-BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α-glucosidase and p-nitrophenyl α-d-glucopyranoside (PNPG). PNPG (1.25 mm), α-glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α-glucosidase inhibitors in green tea. Four components in green tea showed α-glucosidase inhibition action and three of them were identified as HHDP-galloyl glucose, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate by HPLC-fourier-transform mass spectrometry (HPLC-FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on-line HPLC-BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.

Multiple on-line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection.

On-line high performance liquid chromatography (HPLC) coupled with three biochemical detection (BCD) methods was applied to evaluate bioactive components in Danshen injection. On-line HPLC-photo-diode array-fluorescence detection based on the fluorogenic substrate 7-acetoxy-1-methyl quinolinium iodide, was built to search acetylcholinesterase (AChE) inhibitors in Danshen injection. On-line HPLC coupled with the scavenging assay of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals was developed to screen antioxidants. The three active profiles were obviously different. Radical scavenging profiles revealed seven strong peaks in the chromatographic fingerprint possessing obvious free radical inhibition effects, while some minor peaks exhibited stronger AChE inhibition activities. The main radical scavengers and AChE inhibitors were identified by HPLC-MS. Several unknown ingredients showing strong AChE inhibition activities needed further identification except protocatechuic aldehydrate, salvianolic acid H or I and lithospermic acid. The on-line multiple on-line HPLC-BCD methods will provide powerful tools in the field of pharmacognosy for fast-track identification of interesting and/or novel bioactive compounds.

Huachansu mediates cell death in non-Hodgkin's lymphoma by induction of caspase-3 and inhibition of MAP kinase.

Huachansu (HCS), a hot water extract of the skin glands of Bufo gargarizans (B. melanostictus), has been used extensively in the treatment of various solid tumors in Asia, particularly in China. However, its effect on the growth of malignancies of hematopoietic origin, particularly lymphomas, is limited. Here we investigated the antiproliferative effect and molecular mechanisms of HCS using non-Hodgkin's lymphoma (NHL) Raji, Ramos, and Namalwa cells and the mantle cell lymphoma cells SP53. HCS inhibited proliferation in these cell lines with an IC50 ranging from 3.1 to 25 µl/ml. At a concentration of 25 µl/ml, HCS triggered a sub-G1 arrest in Ramos cells and induced early to late apoptotic cell death. Cleaved caspase-3 was formed in a concentration-dependent manner in Ramos cells following treatment with HCS for 24 h. Intriguingly, when the Ramos cells were treated with the caspase inhibitor ZDEVD, the apoptotic activity of HCS was partially blocked. Furthermore, HCS also blocked the expression of survivin and pRB proteins in a concentration-dependent manner in Ramos cells. Mechanistically, HCS downregulated both the MAPK gene and proteins in Ramos cells. Collectively, our data suggest that HCS is effective in inducing cell death and apoptosis, in part, by activating caspase-3 activity and suppressing MAP kinase in NHL cells.

Risk factors and precautions of inpatient suicide from the perspective of nurses: A qualitative study.

The risk factors and precautions of inpatient suicide were explored. Thirty suicide victims were drawn from the adverse event reports of suicidal act during hospitalization in a general hospital from 2008 to 2014. Data were gathered from the focus group interviews of twelve nurses who had experienced inpatient suicide. The data were analyzed by using analytical technique based on grounded theory, and software QSR NVIVO8 was used to aid the collation of data. Three main themes of risk factors about inpatient suicide emerged from the analysis: individual value, social factors and environmental factors. The individual value was categorized into different groups such as sense of guilt, hopelessness and low self-esteem. Social factors included two aspects of negative life events and social support. Three themes of precautions about inpatient suicide appeared in this study: evaluation, nursing and information exchange. Evaluation was elaborated from both physical and psychological assessments. This finding extends existing work of risk factors and precautions about inpatient suicide and brings new knowledge about the reasons why inpatients commit suicide.

Diagnosis of recurrent uterine cervical cancer: PET versus PET/CT: a systematic review and meta-analysis.

OBJECTIVE: The aim of this work was to assess and compare the overall value of stand-alone FDG PET and PET/CT in diagnosing recurrent cervical cancer with a meta-analysis. METHODS: All the English published studies which addressed the use of PET whether interpreted with or without the use of CT for the diagnosis of recurrent cervical cancer were collected. Methodological quality of the included studies was evaluated. Pooled sensitivity and specificity were calculated, summary receiver operating characteristics (SROC) curve analysis was used to compare the diagnostic ability of stand-alone PET and PET/CT. RESULT: A total of 18 studies were included in this meta-analysis, with a total of 762 subjects. Pooled sensitivity and specificity of PET and PET/CT were 0.91 (95 % CI 0.87-0.94) and 0.94 (95 % CI 0.89-0.97), and 0.92 (95 % CI 0.91-0.94) and 0.84 (95 % CI 0.74-0.91), respectively. The areas under the SROC curve (AUCs) of PET and PET/CT were 0.9610 and 0.9491, respectively. There was no statistical significance between the AUC of PET and PET/CT (P > 0.05). CONCLUSION: Both PET and PET/CT have good performance in the detection of recurrent cervical cancer. However, interpreted CT images may have limited additional value on PET in detecting recurrent cervical cancer.

The Spectrum-Effect integrated fingerprint of Polygonum cuspidatum based on HPLC-diode array detection-flow injection-chemiluminescence.

AIM: To establish the Spectrum-Effect integrated fingerprint of Polygonum cuspidatum to evaluate the quality of P. cuspidatum. METHODS: An on-line HPLC-DAD-flow injection chemiluminescence (FICL) method was developed to investigate the quality of P. cuspidatum from different habitats based on the established Spectrum-Effect integrated fingerprint. RESULTS: Nineteen batches of samples of P. cuspidatum were evaluated for the similarity of their chromatographic and free radical scavenging fingerprints, and the results compared. Main antioxidants were estimated by regression analysis between peak areas of thirteen compounds and their activities. Some active compounds were identified by HPLC-ESI-MS. CONSULSIONS: The results indicated that main antioxidants in P. cuspidatum could be rapidly screened by the established Spectrum-Effect integrated fingerprint based on on-line HPLC-DAD-FICL, and would be more efficient and objective method to evaluate the quality of P. cuspidatum.

Identification and determination of the major triterpenes in Rhizoma Alismatis by HPLC-evaporative light scattering detection and HPLC/electrospray ionization-MS.

A simple and reliable HPLC-evaporative light scattering detection method was established for the determination of seven triterpenes in Rhizoma Alismatis, a commonly used herbal medicine. HPLC coupled with electrospray ionization-MS was applied to identify the triterpenes. The positive ion mode was used in MS detection, and the fragmentation patterns of the analytes were proposed. The quantitative method was validated for linearity, sensitivity, precision, and accuracy. All calibration curves showed good linearity (r2 > 0.9943) within the test ranges. This method showed good reproducibility with intraday and interday variations of less than 3.39 and 5.20%, respectively. The mean recoveries ranged from 96.06 to 103.5%. The proposed method was successfully applied for the quantitative analysis of the triterpenes in samples from four different habitats. The results indicated great variation of the contents of these components among the samples, and the developed assay could be considered as a suitable quality evaluation method for Rhizoma Alismatis.

Changes in cancer cell metabolism revealed by direct sample analysis with MALDI mass spectrometry.

Biomarker discovery using mass spectrometry (MS) has recently seen a significant increase in applications, mainly driven by the rapidly advancing field of metabolomics. Instrumental and data handling advancements have allowed for untargeted metabolite analyses which simultaneously interrogate multiple biochemical pathways to elucidate disease phenotypes and therapeutic mechanisms. Although most MS-based metabolomic approaches are coupled with liquid chromatography, a few recently published studies used matrix-assisted laser desorption (MALDI), allowing for rapid and direct sample analysis with minimal sample preparation. We and others have reported that prostaglandin E3 (PGE3), derived from COX-2 metabolism of the omega-3 fatty acid eicosapentaenoic acid (EPA), inhibited the proliferation of human lung, colon and pancreatic cancer cells. However, how PGE3 metabolism is regulated in cancer cells, particularly human non-small cell lung cancer (NSCLC) cells, is not fully understood. Here, we successfully used MALDI to identify differences in lipid metabolism between two human non-small-cell lung cancer (NSCLC) cell lines, A549 and H596, which could contribute to their differential response to EPA treatment. Analysis by MALDI-MS showed that the level of EPA incorporated into phospholipids in H596 cells was 4-fold higher than A549 cells. Intriguingly, H596 cells produced much less PGE3 than A549 cells even though the expression of COX-2 was similar in these two cell lines. This appears to be due to the relatively lower expression of cytosolic phospholipase A2 (cPLA2) in H596 cells than that of A549 cells. Additionally, the MALDI-MS approach was successfully used on tumor tissue extracts from a K-ras transgenic mouse model of lung cancer to enhance our understanding of the mechanism of action of EPA in the in vivo model. These results highlight the utility of combining a metabolomics workflow with MALDI-MS to identify the biomarkers that may regulate the metabolism of omega-3 fatty acids and ultimately affect their therapeutic potentials.

MTHFR C677T polymorphism and ovarian cancer risk: a meta-analysis.

BACKGROUND: Many studies have investigated possible association between the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and ovarian cancer risk, but the impact is still unclear owing to the obvious inconsistencies. This study was performed to quantify the strength of the association with a meta- analysis. METHODS: We searched the PubMed, Embase, and CNKI databases for studies relating the association between MTHFR C677T polymorphism and ovarian cancer risk and estimated summary odds ratios (ORs) with confidence intervals (CIs) for assessment. RESULTS: Finally, eight studies with a total of 3,379 ovarian cancer cases and 4,078 controls were included into this meta-analysis. Overall the showed that MTHFR C677T polymorphism was not associated with ovarian cancer risk under all genetic models (ORT versus C = 1.03, 95%CI 0.90-1.18; ORTT versus CC = 1.08, 95%CI 0.79-1.47; ORTT versus TC+CC = 1.05, 95%CI 0.80-1.37; ORTT +TC versus CC = 1.05, 95%CI 0.86-1.21). Meta-analyses of studies with confirmation of HWE also showed no significant association. Subgroup analyses by ethnicity showed there was no significant association in the Caucasians but MTHFR C677T polymorphic variant T contributed to increased risk of ovarian cancer in East Asians. No evidence of publication bias was observed. CONCLUSION: Meta-analyses of available data show that MTHFR C677T polymorphism is not associated with ovarian cancer risk in Caucasians, but the MTHFR polymorphic variant T may contribute to increased risk in East Asians.

Comparative study of Puerariae lobatae and Puerariae thomsonii by HPLC-diode array detection-flow injection-chemiluminescence coupled with HPLC-electrospray ionization-MS.

An on-line HPLC-diode array detection-flow injection chemiluminescence (HPLC-DAD-FICL) method was applied to estimate the difference of Puerariae lobatae and Puerariae thomsonii. Their chemical and active profiles could be obtained by HPLC-DAD-FICL in one run. Seventeen compounds in two species were tentatively identified by HPLC-electrospray ionization-MS (HPLC-ESI-MS) method. The main antioxidants were rapidly screened by active fingerprints coupled with MS data. Similarity and Hierarchical clustering analysis (HCA) were used to distinguish different samples. The results suggested that the chemical fingerprints of 16 batches of samples were similar by similarity evaluation, while HCA could discriminate the two species. The active fingerprints of Puerariae lobatae and Puerariae thomsonii were significantly different. More antioxidants were found in Puerariae lobatae than in Puerariae thomsonii. Main antioxidants, including 3'-hydroxypuerarin, genistein 8-C-glycoside-xyloside, puerarin, 6″-O-xylosylpuerarin, mirificin and daidzein in two species, may be reasonable markers for the discrimination of the two species. The integrated fingerprint based on the chemical and active characteristics may provide an objective quality evaluation for Puerariae lobatae and Puerariae thomsonii.

On-line high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry-chemiluminescence assay of radical scavengers in Epimedium.

An on-line analysis method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining HPLC with hydrogen dioxide radical scavenging and HPLC-DAD-MS-CL system. The structural identification and activity characteristics of various constituents could be rapidly achieved by the on-line assay of UV, MS and CL in one run. In 4 species of Epimedium studied 32 compounds, including phenolic acids, 8-isopentenyl-flavonoid glycosides and flavonoid glycosides containing a ortho-hydroxyl group, were identified by comparison with authentic standards and published mass data. Among these compounds, phenolic acids and flavonoid glycosides containing an ortho-hydroxyl group could obviously inhibit CL, which suggested their strong radical scavenging activity. These four species each exhibited different active properties, which might correlate to their respective quality. The results indicated that the on-line HPLC-DAD-MS-CL system would be a potential method to rapidly and sensitively screen radical scavengers in herbal medicines, and could display an integrated fingerprint based on different detectors.

Study on the radical scavengers in the traditional Chinese medicine formula shengmai san by HPLC-DAD coupled with chemiluminescence (CL) and ESI-MS/MS.

Shengmai San (SMS) has been used for the treatment of cardiovascular disease in Asia for long-time. It has been reported that SMS can protect against oxidative stress and tune immune response in patients. In this paper, a high performance liquid chromatography-diode array detection-chemiluminescence (HPLC-DAD-CL) method for on-line detection was successfully applied to screen antioxidants in complex SMS extracts. The H(2)O(2) scavenging activity of every fraction of SMS extracts was measured to evaluate each contribution to the total anti-oxidative activities of the prescription, followed by structures identification of the main active components by LC/MS/MS. HPLC-DAD-CL method was also applied for comparing the scavenging rates of main active components between the individual herbs and SMS. Interestingly, we found that the scavenging activities of main components detected in the individual herbs were different from those in SMS. The experimental data indicated that drug interactions during decocting process could result in the changes of the antioxidants' solubility. This study demonstrated that HPLC-DAD-CL on-line assay was a useful technique for rapidly screening and identifying bioactive components from complex multi-herbal prescription.

Potency fingerprint of herbal products Danshen injection for their quality evaluation.

The fingerprint technique has been studied frequently as a useful strategy for quality of traditional Chinese medicine. A novel potency fingerprint that can quantitatively analyze the antioxidant activity of individual constituent and provide the total antioxidant activities of the samples has been developed by high-performance liquid chromatography coupled with ultraviolet and pyrogallol-luminol chemiluminescence detection (HPLC-diode array detection (DAD)-PLD). Hierarchical clustering analysis has been used as a powerful pattern recognition tool to identify and classify Danshen injection from different factories. In addition, the combination use of the chromatographic fingerprint and potency fingerprint with principal component analysis was applied to quality control of Danshen injection. The results demonstrated that the proposed potency fingerprint was a useful means to control the quality and to clarify the possible mechanism of action of herbal products.

Quality control of flavonoids in Ginkgo biloba leaves by high-performance liquid chromatography with diode array detection and on-line radical scavenging activity detection.

Flavonoids in plants used for the treatment of various cardiovascular, cancer diseases have been reported to possess potential protective effects against oxidative injury. Ginkgo biloba leaves, known for their antioxidant activity, were chosen for this study. In this paper, 12 flavonoids in G. biloba leaves were identified by HPLC-diode array detection (DAD)-electrospray ionization MS. HPLC-DAD coupled with chemiluminescence detection was used to determine free radical scavenging activity of flavonoids. It was found that the flavonol glycosides could markedly inhibit the luminescent signal, which indicated that they are mainly responsible for the antioxidant activities of G. biloba leaves. Total antioxidant activity of these flavonoids was used to evaluate the differences of G. biloba leaves collected in 13 habitats. The combination of chemical and activity analysis can provide a valid method to quantify the bioactive components in G. biloba leaves, and this may be a more rational approach to the quality assessment of G. biloba leaves.

The antioxidant-activity-integrated fingerprint: an advantageous tool for the evaluation of quality of herbal medicines.

The activity-integrated fingerprints of Danshen injections have been established by high-performance liquid chromatography coupled with chemiluminescence detection (HPLC-CL) according to their antioxidants activity scavenging hydrogen peroxide. The authentication and validation of the activity-integrated fingerprints were performed. Then, a data-level information fusion method was employed to capture the chemical and antioxidant information encoded in HPLC-CL fingerprints. Finally, based on the fusion results, the quality of different batches of Danshen injections samples were further evaluated by similarity measure. In comparison with conventional fingerprints, the activity-integrated fingerprints, which simultaneously contained the chemical characteristics and antioxidant activities of constitutions could comprehensively and properly reveal the quality characteristics of the Danshen injections. In conclusion, the antioxidant-activity-integrated fingerprints were suitable for quality control of Danshen injections. This study could clearly demonstrate that the activity-integrated fingerprint was a powerful and meaningful tool to comprehensively improve the quality control of complex herbal medicines.

[Classification and application principles of optical-fibre transducer].

One of the new transducers is optical-fiber transducer, which has a wideying application prospect. The present paper describes the principles, classifications and advantages of the fiber sensor, and also reports the basic features and the outline of the application of the fiber sensors in types of intensity, phase and polarization modulation.