One injection to profile the chemical composition and dual-antioxidation activities of Rosa chinensis Jacq.

An on-line high-performance liquid chromatography-diode-array-detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-total antioxidant capacity detection (HPLC-DAD-ESI-IT-TOF-MS-TACD) system was applied for the identification and evaluation of antioxidants in Rosa chinensis Jacq., an edible flower in food industry and a widely used traditional Chinese medicine. With the help of this platform, the HPLC fingerprint, mass fragmentations, and sample activity profiles against 1,1-diphenylpicryl-2-hydrazyl radical (DPPH•) and ferric reducing antioxidant power (FRAP) were recorded after one injection. Using this technique, 80 compounds were separated and identified by their LC/MS behaviors with the assistance of standard compounds. In addition, 11 different Rosa chinensis Jacq. samples were profiled and then quantified for their DPPH• and FRAP activities. Interestingly, a total of 52 compounds showed antioxidative effects against DPPH• and 61 were active against FRAP. The results demonstrated that the on-line system is a powerful technique for antioxidant discovery in Rosa chinensis Jacq. and other food resources.

Development of an on-line analytical platform to screen haptens in shuxuening injection based on high-performance liquid chromatography coupled with ion-trap multistage mass spectrometry and human serum albumin fluorescence detection.

Shuxuening injection (SXNI), one of the traditional Chinese medicine injections (TCMI), is widely used for the treatment of cardiovascular diseases in the clinic. However, its allergic reactions have impeded the clinical applications of SXNI, such adverse reactions have not been well understood due to the lack of methods for detecting haptens. In this study, a high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-human serum albumin-fluorescence detector (HPLC-DAD-MSn-HSA-FLD) system was established to identify and screen haptens for the first time. Flavones, flavonols and their glycosides in SXNI showed strong HSA binding ability in different degrees. Fifteen of these compounds were used to study the association of HSA binding ability and sensitizability using isothermal titration calorimetry (ITC) and fluorescence techniques, furthermore, RBL-2H3 cell experiments were conducted to verify the results. It was found that ginkgolides showed no sensitizability, while flavones and flavonol aglycones showed stronger sensitizability than their glycosides. The system was proven to be precise, stable and reproducible, which lays a foundation for screening haptens in SXNI and relevant samples.

An on-line analytical approach for detecting haptens in Shuang-huang-lian powder injection.

Shuang-huang-lian powder injection (SHLPI) is a traditional Chinese medicine injection (TCMI) frequently used in the clinical treatment of faucitis, bronchitis, and other viral and bacterial infections of upper respiratory tract. However, its allergenic reactions, being the main adverse effects (AEs) of SHLPI, have been a serious problem of its clinical safety. This problem has not been solved due to short of methods for detecting haptens in complex TCMIs. In this study, an on-line high-performance liquid chromatography-diode-array detector-mass spectrometry combined with bovine serum albumin-fluorescence detector (HPLC-DAD-MS-BSA-FLD) system was established for the first time, validated and applied for identification of haptens in SHLPI. Fourteen of 35 identified compounds showed BSA binding activity, and they were six flavonoids, six caffeoylquinic acids (CQAs), and two phenylethanoid glycosides. The structure-activity relationships of 10 active components were studied, and their ability of sensitization together with that of two CQAs were further verified by ELISA assay. It was found that 10 compounds had sensitization, and flavonoids showed stronger sensitizability than CQAs while the diCQAs were slightly stronger than caffeoylquinic acids. The system was validated using 3-CQA as a positive control, and was proved to have good reproducibility, stability, precision (RSD<0.1%) and linearity (R2>0.9993). This online system is fast, sensitive and efficient for screening haptens in traditional Chinese medicine injection (TCMI), provides a new approach to reveal the chemical basis of haptens in TCMIs.

Rapid Screening of Potential Phosphodiesterase Inhibitors from the Roots of Ilex pubescens Hook. et Arn. Using a Combination of Ultrafiltration and LC-MS.

The cyclic nucleotide phosphodiesterase (PDE) plays an important role in regulating the levels of second messenger molecules cAMP and cGMP. Various PDE inhibitors have been successfully developed into drugs for targeted diseases. In addition, PDE inhibitors can also be found in different foods and natural medicines. In this study, ultrafiltration liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry (ultrafiltration LC-DAD-ESI-IT-TOF-MS) was applied to screen PDE inhibitors from the roots of Ilex pubescens Hook. et Arn. As a result, 11 major compounds were identified in I. pubescens roots, with nine compounds as potential PDE inhibitors, among which five were further confirmed to be active against PDEI and PDE5A dose-dependently in vitro, with ilexsaponin A1 and ilexsaponin B2 being the strongest. HPLC quantification of these bioactive compounds suggested that they are major components in the plant. The results demonstrate that ultrafiltration LC-DAD-ESI-IT-TOF-MS is an efficient method for rapid screening of PDE inhibitors from natural medicines.

Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples.

Rapid screening of transferrin-binders in the flowers of Bauhinia blakeana Dunn by on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-transferrin-fluorescence detection system.

Transferrin (Transferrin, TRF, TF) has drawn increasing attention in cancer therapy due to its potential applications in drug delivery. TF receptor, highly expressed in tumor cells, recognizes and transports Fe(3+)-TF into cells to release iron into cytoplasm. Thus, discovering TF-binding compounds has become an active research area and is of great importance for target therapy. In this study, an on-line analysis method was established for screening TF-binding compounds from the flowers of Bauhinia blakeana Dunn using a high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-transferrin-fluorescence detector (HPLC-DAD-MS(n)-TF-FLD) method. As a result, 33 of 80 identified or tentatively characterized compounds in the sample were TF-binding active. Twenty-five flavonol glycosides and eight phenolic acids were identified as TF-binders. Twelve of these active compounds together with six standard compounds were used to study the dose-response effects and structure-activity relationships of flavonoids and phenolic acids. The method was validated by vitexin with a good linearity in the range of concentrations used in the study. The limit of detection for vitexin was 0.1596 nmol. Our study indicated that the established method is simple, rapid and sensitive for screening TF-binding active compounds in the extract of Bauhinia blakeana Dunn, and therefore is important for discovering potential anti-cancer ingredients from complex samples for TF related drug delivery.

Rapid Identification and Assignation of the Active Ingredients in Fufang Banbianlian Injection Using HPLC-DAD-ESI-IT-TOF-MS.

Fufang Banbianlian Injection (FBI) is a well-known traditional Chinese medicine formula composed of three herbal medicines. However, the systematic investigation on its chemical components has not been reported yet. In this study, a high-performance liquid chromatography combined with diode-array detector, and coupled to an electrospray ionization with ion-trap time-of-flight mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method, was established for the identification of chemical profile in FBI. Sixty-six major constituents (14 phenolic acids, 14 iridoids, 20 flavonoids, 2 benzylideneacetone compounds, 3 phenylethanoid glycosides, 1 coumarin, 1 lignan, 3 nucleosides, 1 amino acids, 1 monosaccharides, 2 oligosaccharides, 3 alduronic acids and citric acid) were identified or tentatively characterized by comparing their retention times and MS spectra with those of standards or literature data. Finally, all constituents were further assigned in the individual herbs (InHs), although some of them were from multiple InHs. As a result, 11 compounds were from Lobelia chinensis Lour, 33 compounds were from Scutellaria barbata D. Don and 38 compounds were from Hedyotis diffusa Willd. In conclusion, the developed HPLC-DAD-ESI-IT-TOF-MS method is a rapid and efficient technique for analysis of FBI sample, and could be a valuable method for the further study on the quality control of the FBI.

Rapid screening and identification of compounds with DNA-binding activity from Folium Citri Reticulatae using on-line HPLC-DAD-MS(n) coupled with a post column fluorescence detection system.

To study the interactions between natural compounds and deoxyribonucleic acid (DNA), a method has been established combining a high-performance liquid chromatography-diode array detector-multi-stage mass spectrometer with a fluorescence detector (HPLC-DAD-MS(n)-FLD). The FLD was used to monitor fluorescence intensity of the ethidium bromide-DNA (EB-DNA) complex when a compound separated by HPLC was introduced. This novel method was used to simultaneously obtain the HPLC fingerprint, UV spectra, MS(n) fragments and DNA-binding activity profile of various components in Folium Citri Reticulatae. As a result, 35 compounds were identified, of which 25 were found in the extract of Folium Citri Reticulatae for the first time, and 33 compounds showed DNA-binding activities, with the most active being feruloylhexaric and p-coumaroylhexaric acids. In addition, the precision, stability and reproducibility of this method were validated by two positive controls, quercetin and hesperidin. This new on-line method is accurate, precise and reliable for further high-throughput screening of DNA-binding compounds from food samples and other complex matrices.

An on-line high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-deoxyribonucleic acid-4',6-diamidino-2-phenylindole-fluorescence detector system for screening the DNA-binding active compounds in Fufang Banbianlian Injection.

Fufang Banbianlian Injection (FBI), a well-known traditional Chinese medicine formula, has been recently approved and extensively used as a newly anti-inflammatory and anti-tumor drug. This prescription comprises an equal ratio of three traditional Chinese herbs, Lobelia chinensis Lour, Scutellaria barbata D. Don and Hedyotis diffusa Willd. The relationships between its chemical compositions and activities have not been understood well yet. To investigate the ingredients and their DNA-binding activities in FBI, an on-line high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-deoxyribonucleic acid-4',6-diamidino-2-phenylindole-fluorescence detector (HPLC-DAD-MS(n)-DNA-DAPI-FLD) system was developed using a combination of chromatographic, mass spectrometric and fluorescent detection techniques. 4',6-Diamidino-2-phenylindole (DAPI) specifically binds to three ATT base pairs on the DNA minor groove, and thus can be used as a fluorescent probe for screening active compounds that compete ATT sequences with DAPI. Using this system, 21 of 58 identified or tentatively characterized compounds in FBI showed DNA-binding activities, with most of the active compounds being flavone glycosides. In addition, the structure-activity relationships of these active compounds suggested that conjugated planar structures are favorable for DNA-binding activities, and adjacent hydroxyl groups in flavonoids can significantly improve their activities. This is, to the best of our knowledge, the first application of DAPI as a fluorescent probe for the screening of DNA-binding active compounds in complex samples.

Chemical Synthesis and Biological Activities of 20S,24S/R-Dihydroxyvitamin D3 Epimers and Their 1α-Hydroxyl Derivatives.

Bioactive vitamin D3 metabolites 20S,24S-dihydroxyvitamin D3 [20S,24S(OH)2D3] and 20S,24R-dihydroxyvitamin D3 [20S,24R(OH)2D3] were chemically synthesized and confirmed to be identical to their enzymatically generated counterparts. The absolute configurations at C24 and its influence on the kinetics of 1α-hydroxylation by CYP27B1 were determined. Their corresponding 1α-hydroxyl derivatives were subsequently produced. Biological comparisons of these products showed different properties with respect to vitamin D3 receptor activation, anti-inflammatory activity, and antiproliferative activity, with 1α,20S,24R(OH)2D3 being the most potent compound.

An on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-total antioxidant capacity detection system applying two antioxidant methods for activity evaluation of the edible flowers from Prunus mume.

An on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-total antioxidant capacity detection (HPLC-DAD-ESI-IT-TOF-MS-TACD) system was created for identification and evaluation of antioxidants in Prunus (P.) mume flowers. Applying this system, the HPLC fingerprint, ultraviolet (UV) spectra, mass fragmentations, active profiles against 1,1-diphenylpicryl-2-hydrazyl radical (DPPH•) scavenging activity and ferric reducing antioxidant power (FRAP) of each complex sample were obtained simultaneously after one injection. Synchronous structure identification and activities screening of complex samples were thus accomplished. In this study, 78 compounds were identified from P. mume flowers by their chromatographic behaviors, UV spectra and MS data with the assistance of standard compounds and literature reports. The DPPH and FRAP activity of 24 samples (23 different P. mume varieties and 1 related herbal medicine) were then quantified by their detailed activity profiles from the on-line system, and by the total activity of each sample extract from off-line 96-well plate method. As a result, 21 and 32 compounds in the on-line system showed anti-oxidative effects against DPPH and FRAP, respectively. The established on-line system is efficient, sensitive and reliable to tell the DPPH and FRAP antioxidant activities of individual compound in complex samples, and therefore would be a useful and promising technique for antioxidant screening from different food and medicinal matrices.

Comprehensive identification of 125 multifarious constituents in Shuang-huang-lian powder injection by HPLC-DAD-ESI-IT-TOF-MS.

A high-performance liquid chromatography-diode array detector-electrospray ionization-ion trap-time of flight-mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method was established for excellent separation and structural identification of constituents in Shuang-huang-lian powder injection (SHLPI). The typical ultraviolet absorptions, accurate empirical molecular formula and reasonable fragmentation mechanisms of these ingredients were used for their structural elucidation. In consequence, 125 constituents (33 phenolic acids, 29 flavonoids, 32 phenylethanoid glycosides, 15 iridoid glycosides, 8 lignans, 3 amino acids and 2 purines nucleosides, 2 quinoid glycosides and 1 alkylbenzene glycoside) were either unequivocally identified or tentatively characterized by comparing authentic standards or published data. The result showed that this study could provide valuable information for the quality control and further investigation of SHLPI formula.

An analysis method for simultaneous screening of deoxyribonucleic acid-binding active compounds and investigating their mechanisms by ultra-fast liquid chromatography tandem mass spectrometry coupled with fluorescence detection technology.

DNA has been known as the cellular target for many cytotoxic anticancer agents over the years. Discovering DNA-binding compounds has become an active research area, while various DNA-binding mechanisms make the drug discovery even more difficult. In this article, we present a novel analysis method to rapidly identify specific DNA-binding compounds from Pyrrosia lingua (Thunb.) using DNA-dual-fluorescent probes, ethidium bromide and Hoechst 33258, with the technology of ultra-fast liquid chromatography-diode array detector-tandem mass spectrometry and dual-wavelength fluorescence detector (UFLC-DAD-MS(n)-DFLD). Sixty-two compounds were identified, of which 22 were found to be active in DNA-binding. After investigation of their dose-response behaviors and structure-activity relationships, chlorogenic acids and flavonoid glycosides were found to be DNA-binders via both minor groove-binding and intercalation modes. The precision, reproducibility and stability of this method were validated by vitexin. The established system was sensitive, precise, and reliable to be used for both screening of DNA-binding compounds and investigating of their mechanisms.

Identification and quantitation of phenolic compounds from the seed and pomace of Perilla frutescens using HPLC/PDA and HPLC-ESI/QTOF/MS/MS.

INTRODUCTION: Perilla frutescens (L.) Britt., an essential traditional Asian crop and Chinese medicine, potentially exerts anti-oxidation effects through its phenolic compounds. These compounds have already been reported in perilla seed, however, little is reported in Perilla pomace, the primary waste during oil production of Perilla seed. OBJECTIVE: To investigate major phenolic compounds in perilla seeds and pomaces in order to check whether the pomace could be an alternative resource to the seed for nutritional and medical purposes. METHODS: Compounds in extracts of perilla seeds and pomaces were separated by high-performance liquid chromatography and detected by photodiode array, and by electrospray ionisation with quadrupole time-of-flight tandem mass spectrometry. Herb-markers selected by principal components analysis were then quantified in both seeds and pomaces. Moreover, a fingerprinting approach and multiple discriminant analysis were applied to screen the phenolic markers in 22 samples. RESULTS: Ten phenols were tentatively identified, among which four (rosmarinic acid, luteolin, apigenin and rosmarinic acid-3-O-glucoside) were selected as herb-markers. Perilla seeds and pomaces showed similar phenol profiles, however, the pomaces contained almost two times the amount of the four herb-markers than the seeds. CONCLUSION: The results indicated perilla pomace is a promising alternative source of phenolic compounds that could be recovered and potentially used as natural anti-oxidants.

Ultra-performance LC separation and quadrupole time-of-flight MS identification of major alkaloids in Plumula Nelumbinis.

INTRODUCTION: As an essential medicine and tea source in many countries, Plumula Nelumbinis potentially exerts its major biological activities through its alkaloids. However, the activities of Plumula Nelumbinis are not fully understood due to the lack of studies on its chemical components. OBJECTIVE: To establish an ultra-performance liquid chromatography combined with diode-array detector (UPLC/DAD) method, coupled to an electrospray ionisation with quadrupole time-of-flight mass spectrometry (ESI/QTOF/MS) method, for the separation and identification of Plumula Nelumbinis alkaloids. METHODS: The eluant from an UPLC separation of an ethanol extract of Plumula Nelumbinis was directly infused into an ESI/QTOF/MS system. Both positive and negative ion modes of ESI with low and high collision energy (CE) were used to obtain sufficient MS information. RESULTS: Twenty-one alkaloids were tentatively identified based on their chromatographic characteristics, UV spectra, exact mass, MS fragments and literature reports. They consist of six bis-1-benzyltetrahydroisoquinoline, eleven benzyltetrahydroisoquinoline (including two glycoalkaloids and two quaternary ammoniums), two aporphine, one proaporphine and one indole alkaloids. Eleven were identified in Plumula Nelumbinis for the first time and seven were first reported in Nelumbo nucifera Gaertn. Five compounds, namely norcoclaurine-4'-O-glucoside, norcoclaurine-6-O-glucoside, isolotusine, 6-demethyl-4-demethylN-methylcoclaurine and N-norisoliensinine, were characterised and proposed as new compounds. CONCLUSION: The established UPLC/DAD - ESI/QTOF/MS method is efficient for systematic identification of the alkaloids in Plumula Nelumbinis extract.

Novel vitamin D analogs as potential therapeutics: metabolism, toxicity profiling, and antiproliferative activity.

AIM: To discover novel [20(OH)D3] analogs as antiproliferative therapeutics. MATERIALS AND METHODS: We studied in vitro liver microsome stability, in vivo toxicity using mice, vitamin D receptor (VDR) translocation, in vitro antiproliferative effect, CYP enzyme metabolism. RESULTS: 20S- and 20R(OH)D3 had reasonable half-lives of 50 min and 30 min (average) respectively in liver microsomes. They were non-hypercalcemic at a high dose of 60 µg/kg. Three new 20(OH)D3 analogs were designed, synthesized and tested. They showed higher or comparable potency for inhibition of proliferation of normal keratinocytes and in the induction of VDR translocation from cytoplasm to nucleus, compared to 1,25(OH)2D3. These new analogs demonstrated different degrees of metabolism through a range of vitamin D-metabolizing CYP enzymes. CONCLUSION: Their lack of calcemic toxicity at high doses and their high biological activity suggest that this novel 20(OH)D3 scaffold may represent a promising platform for further development of therapeutically-useful agents.

Identification of antioxidants in Fructus aurantii and its quality evaluation using a new on-line combination of analytical techniques.

A new on-line method for simultaneous identification and monitoring of antioxidants in Fructus aurantii was established by coupling high performance liquid chromatography-diode array detector-electrospray ionisation-ion trap-time of flight-mass spectrometry with post-column derivatisation and luminol-potassium ferricyanide chemiluminescence (HPLC-DAD-ESI-IT-TOF-MS-PCD-LPFCL). While the HPLC fingerprint, structural identification and radical scavenging profile were rapidly obtained by an on-line assay using ultraviolet (UV) absorption, MS and LPFCL, details of the precise substitution patterns of various structures were achieved through UV absorption using PCD addition of shift reagents. Twenty-five flavonoids were identified by either their PCD and MS data or comparison with reference substances. Data collected both from chromatograms and activity profiles of 12 samples revealed significant differences among samples from different habitats. The results showed that this method was rapid and precise, and therefore would be an effective and sensitive method for biocompounds analysis and quality evaluation for complex food and medicinal samples.