New insights into the chemistry and anticancer activity of Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis (O. xuefengensis), the sister taxon of Ophiocordyceps sinensis (O. sinensis), is consumed as a "tonic food" due to its health benefits. However, little is known regarding the chemistry and bioactivity of O. xuefengensis. In this study, we characterized 80 indole-based alkaloids in the ethyl acetate fraction of O. xuefengensis by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS/MS), of which 54 indole-based alkaloids were identified as possibly new compounds. Furthermore, 29 of these compounds were established as potential anti-cancer compounds by ligand fishing combined with HPLC-Q-TOF-MS/MS. Moreover, molecular docking identified the NH- and OH- groups of these compounds as the key active groups. The present study has expanded the knowledge on the characteristic indole-based alkaloids and anti-cancer activity of O. xuefengensis.

Threaded 3D microfluidic paper analytical device-based ratiometric fluorescent sensor for background-free and visual detection of organophosphorus pesticides.

With the increasing concerns of food safety and environmental protection, it is desirable to develop reliable, effective, and portable sensors for detection of organophosphorus pesticides (OPs). Here, a cascade reaction system integrated with threaded 3D microfluidic paper analytical device (3D µPAD) was firstly developed for background-free and visual detection of OPs in agricultural samples. Butyrylcholinesterase (BChE) hydrolyzed acetylcholine into thiocholine (TCh), which reduced MnO2 nanosheets into Mn2+. With addition of OPs, BChE activity was irreversibly inhibited, and the generation of TCh and the reduction of MnO2 nanosheets were prevented. Then the remaining MnO2 nanosheets oxidized o-phenylenediamine into 2,3-diaminophenazine with yellow-emission fluorescence, which quenched the fluorescence intensity of red-emission carbon dots (RCDs) via inner-filter effect. Based on above mechanism, a ratiometric fluorescent system was established for OPs detection. Threaded 3D µPAD consisted of 4 layers, which allowed to load and/or add reagents to trigger the cascade reaction system for OPs detection. The fluorescent images presented distinguishable color variations from red to yellow with dichlorvos concentrations ranging from 2.5 to 120 µg L-1, and the limit of detection was 1.0 µg L-1. In the practical samples testing, threaded 3D µPAD can eliminate background influence on fluorescent signal for OPs detection. Threaded 3D µPAD integrated with ratiometric sensing platform has merits of accuracy response, facile operation, and background-free detection, which supplies a new alternative approach for on-site pesticide detection.

Screening of potent α-glucosidase inhibitory and antioxidant polyphenols in Prunella vulgaris L. by bioreaction-HPLC-quadrupole-time-of-flight-MS/MS and in silico analysis.

Prunella vulgaris L. is a well-known traditional Chinese medicine for blood glucose homeostasis and antioxidant potential. Ethyl acetate fraction of P. vulgaris L. demonstrated higher phenolic content (85.53 ± 6.74 mg gallic acid equivalents per gram dry weight), α-glucosidase inhibitory (IC50 , 69.13 ± 2.86 µg/ml), and antioxidant (IC50 , 8.68 ± 1.01 µg/ml) activities. However, the bioactive polyphenols responsible for the beneficial properties remain unclear. Here, bioreaction-HPLC-quadrupole-time-of-flight-MS/MS method was developed for rapid, accurate, and efficient screening and identification of polyphenols with α-glucosidase inhibitory and antioxidant activities from P. vulgaris L. Bioactive polyphenols can specifically bind with α-glucosidase or react with 1,1-diphenyl-2-picryl-hydrazyl radical, which was easily discriminated from nonactive compounds. Subsequently, 20 bioactive polyphenols (16 phenyl propionic acid derivatives and four flavonoids) were screened and identified. Furthermore, molecular docking analysis revealed that screened 20 polyphenols bind with the active sites of α-glucosidase through hydrogen bonding and π-π stacking. Density functional theory calculations demonstrated their electron transport ability and chemical reactivity. The in silico analysis confirmed the screened results. In summary, this study provided a valuable strategy for rapid discovering bioactive compounds from complex natural products and offered scientific evidence for further development and application of P. vulgaris L.

Rapid profiling of potential antitumor polymethoxylated flavonoids in natural products by integrating cell biospecific extraction with neutral loss/diagnostic ion filtering-based high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry.

INTRODUCTION: Citri Reticulatae Pericarpium Viride (CRPV, Qing Pi in Chinese) has been widely used in traditional Chinese medicine. Polymethoxylated flavonoids (PMFs), which are a special group of flavonoids with strong antitumor activity, are broadly distributed in citrus peels. However, systematic investigation of antitumor PMFs in CRPV has received little attention to date. OBJECTIVES: An MCF-7 cell biospecific extraction method integrated with neutral loss/diagnostic ion filtering-based HPLC-QTOF-MS/MS strategy was developed for rapid and specific profiling of antitumor PMFs and systematic identification of PMFs in CRPV. METHODOLOGY: By incubating MCF-7 cells with CRPV extract, potential antitumor PMFs specifically bound to cells and were isolated. Then, by systematic investigation of fragmentation pathways, neutral loss and diagnostic ion filtering strategies were proposed to comprehensively and accurately identify PMFs. RESULTS: Sixteen antitumor PMFs were unambiguously or tentatively identified. Among them, minor compound 15 (5-hydroxy-6,7,8,3',4'-pentamethoxyflavone with a free hydroxyl group at C-5) exhibited excellent antitumor activity, with an IC50 value of 2.81 ± 0.76 µg/mL, which is lower than that of 5-fluorouracil (IC50 , 4.92 ± 0.83 µg/mL). Nobiletin (12) and tangeretin (16), two major PMFs, presented moderate antitumor activities with IC50 values of 13.06 ± 1.85 and 17.07 ± 1.18 µg/mL, respectively, and their contents were sensitively and precisely determined. CONCLUSIONS: To the best of our knowledge, this is the first report on the systematic investigation of antitumor PMFs in CRPV. The study will lay a foundation for the quality control and clinical application of CRPV.

Online extraction and enrichment coupling with high-speed counter-current chromatography for effective and target isolation of antitumor anthraquinones from seeds of Cassia obtusifolia.

Traditional bioassay-guided investigation of bioactive compounds from natural products comprises critical steps, such as extraction, repeated column separation, and activity assay. Thus, the development of facile, rapid, and efficient technology is critically important. Here, a HepG2 cell-based extraction method was first developed to rapidly screen potential antitumor compounds from the seeds ofCassia obtusifolia. Then, an online extraction and enrichment-high-speed counter-current chromatography (HSCCC) strategy was fabricated to facilely and efficiently isolate target antitumor compounds, which included direct extraction from solid C. obtusifolia, removal of polar interferences, enrichment of target compounds, and preparative isolation by HSCCC using flow rate stepwise increasing mode. After further purification by Sephadex LH-20 column, five antitumor anthraquinones, aurantio-obtusin, 1-desmethylaurantio-obtusin, chryso-obtusin, obtusin, and questin, were obtained for structural characterization and bioassay verification. The results may not only provide new perspectives for facile and rapid investigation of bioactive compounds from complex natural products, but also offer a scientific basis for the potential applications of C. obtusifolia.

Indole-based alkaloids from Ophiocordyceps xuefengensis.

Seven undescribed indole-based alkaloids, xuefengins A-D and xuefenglasins A-C, were isolated from natural Ophiocordyceps xuefengensis, along with six known alkaloids. Their structures were elucidated by comprehensive spectroscopy, with absolute configurations confirmed by comparison with calculated electronic circular dichroism spectra. Eleven of the isolates were tested for cytotoxicity against the U937, NB4, MCF-7, Hep G2, and A549 cancer cell lines. Two compounds exhibited moderate activities, with IC50 values of 2.83-25.68 µM and 1.54-12.16 µM. Further pharmacological studies showed that these two compounds inhibit cell proliferation by inducing apoptosis, and decreasing p38 and caspase-3 levels in A549 cells.

Profiling of tyrosinase inhibitors in mango leaves for a sustainable agro-industry.

Although mango leaves are the main ingredients in some traditional Chinese medicine preparations and folk tea, they with considerable quantities are usually discarded as agricultural waste. Thus, to extend their potential, reverse ultrafiltration-HPLC-DAD-QTOF-MS/MS combining with key ion filtering strategy was proposed to efficiently fish and systematically identify tyrosinase inhibitors in ethyl acetate fraction of mango leaves, which has the highest total phenolic content (40.00 ± 0.84 mg GAE/g DW) and tyrosinase inhibition activity (IC50, 17.62 ± 1.26 µg/mL). Finally, 36 polyphenolic tyrosinase inhibitors were unambiguously characterized or tentatively identified, and three of them were found in mango leaves for the first time. Results suggested that the proposed strategy was powerful for effective identification of bioactive compounds in complex mixtures (e.g. food, agricultural and sideline products), and the findings would lay a foundation for potential applications of mango leaves in pharmaceutical, cosmetic, and food industrial fields.

Cetyltrimethyl ammonium mediated enhancement of the red emission of carbon dots and an advanced method for fluorometric determination of iron(III).

Red-emissive carbon dots (CDs) were synthesized by one-step hydrothermal technique using citric acid (CA), and urea in N,N-dimethylformamide (DMF) solution. The CDs has an average diameter of 2.3 nm, excitation/emission maxima at 553/606 nm, and a low photoluminescence quantum yield (4%). Fluorescence is weakly quenched by the ions Fe3+, Hg2+, Cu2+, Co2+, Zn2+, Ca2+, Ni2+, and Pb2+. After addition of cetyltrimethyl ammonium ion (CTAB), electrostatic interaction between negatively charged CDs and CTAB causes the CDs to self-aggregate. The formation of CD/CTAB increases the average particle diameter to around 13 nm and enhances the quantum yield to 24%. The hydrophobic segments of CTAB twined into a network structure can selectively trap Fe3+ and then interact with surface groups of the CDs to cause quenching. The CD/CTAB nanoprobe enables fluorometric determination of Fe3+ with a linear response in the 0.10-10 µM concentration range and a 0.03 µM limit of detection. The probe was utilized for determination of Fe3+ in human serum samples, and satisfactory results were obtained. Graphical abstractSchematic representation of fluorometric analysis of Fe(III) ion by cetyltrimethyl ammonium ion (CTAB) mediated red emission carbon dots (CDs). The hydrophobic segments of CTAB twined into a network structure can selectively trap Fe(III) and then interact with surface groups of the CDs to cause quenching.

Online extraction-HPLC-FRAP system for direct identification of antioxidants from solid Du-zhong brick tea.

Du-zhong brick tea (DBT), a new post-fermented functional tea, is marketed in China for its prevention of diseases from oxidative stress. However, antioxidant compounds in DBT are not clear. Here, a novel online extraction-HPLC-ferric reducing antioxidant power (OLE-HPLC-FRAP) has been developed to screen antioxidants in DBT. For OLE, guard column is packed with DBT (1.0 mg), then compounds are extracted directly by HPLC mobile phase for HPLC-FRAP analysis without sample pretreatment procedures. Eleven antioxidants have been screened and identified, three of which are detected in Du-zhong for the first time. Contributions of eleven antioxidant compounds to total antioxidant activity are evaluated using chlorogenic acid as antioxidant marker. Results indicate that the developed OLE-HPLC-FRAP system is a simple and powerful tool to analyze antioxidant compounds from solid complex mixtures, which also contribute to the enhancement of the value of DBT as a good functional tea.

Multielement analysis of Zanthoxylum bungeanum Maxim. essential oil using ICP-MS/MS.

The concentrations of trace elements (Cr, Ni, As, Cd, Hg, and Pb) in Zanthoxylum bungeanum Maxim. essential oil (ZBMEO) were determined by inductively coupled plasma tandem mass spectrometry. The ZBMEO sample was directly analyzed after simple dilution with n-hexane. Aiming for a relatively high vapor pressure of n-hexane and its resultant loading on plasma, we used a narrow injector torch and optimized plasma radio frequency power and carrier gas flow to ensure stable operation of the plasma. An optional gas flow of 20% O2 in Ar was added to the carrier gas to prevent the incomplete combustion of highly concentrated organic carbon in plasma and the deposition of carbon on the sampling and skimmer cone orifices. In tandem mass spectrometry mode, O2 was added to the collision/reaction cell to eliminate the interferences. The limits of detection for Cr, Ni, As, Cd, Hg, and Pb were 2.26, 1.64, 2.02, 1.35, 1.76, and 0.97 ng L-1, respectively. After determination of 23 ZBMEO samples from different regions in China, we found that the average concentration ranges of trace elements in the 23 ZBMEO samples were 0.72-6.02 ng g-1, 0.09-2.87 ng g-1, 0.21-5.84 ng g-1, 0.16-2.15 ng g-1, 0.13-0.92 ng g-1, and 0.17-0.73 ng g-1 for Cr, Ni, As, Cd, Hg, and Pb, respectively. The trace elements in ZBMEO differed significantly when different extraction technologies were used. The study revealed that the contents of the toxic elements As, Cd, Hg, and Pb were extremely low, and hence they are unlikely to pose a health risk following ZBMEO ingestion. Graphical abstract The working mechanism of sample analysis by ICP-MS/MS.

Use of an online extraction liquid chromatography quadrupole time-of-flight tandem mass spectrometry method for the characterization of polyphenols in Citrus paradisi cv. Changshanhuyu peel.

Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC-DAD-QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction of Citrus paradisi cv. Changshanhuyu (Changshanhuyu) peel, OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of twenty-two secondary metabolites were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and nine of them were discovered in Changshanhuyu peel for the first time to our knowledge. It is concluded that the developed OLE-HPLC-DAD-QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products.

Antioxidant profiling of vine tea (Ampelopsis grossedentata): Off-line coupling heart-cutting HSCCC with HPLC-DAD-QTOF-MS/MS.

Vine tea with strong antioxidant activity is commonly consumed as healthy tea/beverage. However, detailed information about its antioxidants is incomplete. Here, off-line hyphenation of heart-cutting high-speed countercurrent chromatography (HSCCC) with high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS) were described for systematic profiling antioxidants in vine tea. At first, antioxidants were rapidly screened by 1,1-diphenyl-2-picryl-hydrazyl radical-high performance liquid chromatography (DPPH-HPLC). Subsequently, stepwise HSCCC using petroleum ether-ethyl acetate-methanol-water (4:9:4:9, v/v/v/v) and (4:9:5:8, v/v/v/v) as solvent systems was optimized to fractionate and enrich antioxidants from ethyl acetate fraction of vine tea. Finally, heart-cutting mode was used to collect five interesting HSCCC fractions for HPLC-DAD-QTOF-MS/MS analysis. Desirable orthogonality between HSCCC and HPLC led to identification of fifteen antioxidant flavonoids, while four minor flavonoids were first reported in vine tea. Results showed that the developed system is efficient to comprehensively explore antioxidants from complex natural herbs.

Antioxidant homoisoflavonoids from Polygonatum odoratum.

Polygonatum odoratum is widely used as a traditional food supplement and herbal medicine with strong antioxidant activity. However, systematic investigation of its antioxidants was limited. Ethanol extract of P. odoratum was fractioned on macroporous absorptive resin (D101) column. A bioassay-guided purification of flavonoid-rich fraction (IC50 value at 74.1 ± 11.9 µg/mL for DPPH scavenging) was realised via high-speed counter-current chromatography (HSCCC) using petroleum ether-ethyl acetate-methanol-water (2:3:3:2, v/v/v/v) as the solvent system combination with Sephadex LH-20 column chromatography (CC) eluting with MeCN-MeOH (1:1, v/v). Three novel homoisoflavonoids (1-3), along with eight homoisoflavonoids (4-11), were isolated. Their structures were elucidated by interpretating various spectroscopic data. All the isolated homoisoflavonoids showed potent antioxidant activities, while compounds 1, 4, and 6 with dihydroxylated B-rings exhibited stronger antioxidant activities (IC50 values at 3.8 ± 0.5, 4.9 ± 0.3 and 3.9 ± 0.4 µg/mL) than ascorbic acid (IC50 value at 5.3 ± 0.6 µg/mL).

Sensitive characterization of polyphenolic antioxidants in Polygonatum odoratum by selective solid phase extraction and high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry.

The complexity of natural products always leads to the co-elution of interfering compounds with bioactive compounds, which then has a detrimental effect on structural elucidation. Here, a new method, based on selective solid phase extraction combined with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking and high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS), is described for sensitive screening, selective extraction and identification of polyphenolic antioxidants in Polygonatum odoratum. First, 25 polyphenolic antioxidants (1-25) were screened by DPPH spiking with HPLC. Second, polydopamine coated Fe3O4 microspheres (Fe3O4@PDA) were prepared to selectively extract target antioxidants with extraction efficiency from 55% to 100% when the amount of Fe3O4@PDA, extraction time, desorption solvent and time were 10mg, 20 min, acetonitrile, and 5 min. Third, 25 antioxidants (10 cinnamides and 15 homoisoflavanones) were identified by HPLC-DAD-QTOF-MS/MS. Furthermore, the DPPH scavenging activities of purified compounds (IC50, 1.6-32.8 µg/mL) validated the method. Among the identified antioxidants, four of them (12, 13, 18 and 19) were new compounds, four of them (2, 4, 8 and 14) were first obtained from family Liliaceae, five of them (1, 3, 5, 7 and 9) were first reported in genus Polygonatum, while one compound (24) was first identified in this species. The results indicated that the proposed method was an efficient and sensitive approach to explore polyphenolic antioxidants from complex natural products.

Magnetic ligand fishing combination with high-performance liquid chromatography-diode array detector-mass spectrometry to screen and characterize cyclooxygenase-2 inhibitors from green tea.

Cyclooxygenase-2 (COX-2) inhibitors may be used to efficiently treat inflammation or cancer diseases. In the present study, we established a new screening assay based on magnetic Fe3O4@SiO2-COX-2 ligand fishing combination with high-performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MSn) to screen and identify COX-2 inhibitors from green tea. Optimized conditions (pH at 7.4, temperature at 30°C, and incubation time for 30min) for fishing out COX-2 inhibitors were achieved by testing positive control, celecoxib, with active and inactive COX-2. Notably, immobilized COX-2 showed high stability (remained 94.7% after ten consecutive cycles), reproducibility (RSD<10% for batch-to-batch evaluation). Finally, eight catechins with COX-2 binding activity were screened in green tea, and their structures were characterized by ultraviolet (UV), accurate molecular weight, diagnostic fragment ions and nuclear magnetic resonance (NMR). Particularly, the COX-2 inhibitory activities of two rare catechins, [(-)-epigallocatechin-3-(3″-O-methyl)-gallate (3″-O-methyl-EGCG, IC50=0.17±0.03µM 0.16±0.01), (-)-epicatechin-3-(3″-O-methyl)-gallate (3″-O-methyl-ECG, IC50=0.16±0.02µM)], were reported for the first time. The results indicated that the proposed method was a simple, robust and reproducible approach for the discovery of COX-2 inhibitors from complex matrix.

Functionalized magnetic nanoparticles coupled with mass spectrometry for screening and identification of cyclooxygenase-1 inhibitors from natural products.

Development of simple and effective methods for high-throughput, high-fidelity screening and identification of cyclooxygenase-1 (COX-1) inhibitors from natural products are important for drug discovery to treat inflammation and carcinogenesis. Here, we developed a new screening assay based on cyclooxygenase-1 (COX-1) functionalized magnetic nanoparticles (i.e. Fe3O4@SiO2-COX-1) for solid phase ligand fishing, and then mass spectrometry (MS) was applied for structural identification. Incubation conditions were optimized. High specificity for isolating COX-1 inhibitors was achieved by testing positive control, indomethacin, with active and inactive COX-1. Moreover, high stability of immobilized COX-1 (remained 95.3% after ten consecutive cycles) allows the analysis reproducible. When applied to turmeric extract, four curcuminoids (i.e. curcumin, demethoxycurcumin, bisdemethoxycurcumin, and 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-(1E,6E)-1,6-heptadiene-3,5-dione), difficult to be distinguished from original MS spectrum of turmeric extract, were isolated as main COX-1 inhibitors. Their structures were characterized based on their accurate molecular weight and diagnostic fragment ions. The results indicated that the proposed method was a simple, robust and reproducible approach for the discovery of COX-1 inhibitors from complex matrixes.

Taraxacum mongolicum extract exhibits a protective effect on hepatocytes and an antiviral effect against hepatitis B virus in animal and human cells.

In order to validate the antiviral effect against hepatitis B virus (HBV) of Taraxacum mongolicum (T. mongolicum), the protective effect on hepatocytes, and antiviral properties against duck hepatitis B virus (DHBV) and HBV of T. mongolicum extract (TME) were evaluated in chemically-injured neonatal rat hepatocytes, DHBV-infected duck fetal hepatocytes and HBV-transfected HepG2.2.15 cells, respectively. The results demonstrated that TME at 50-100 µg/ml improved D-galactosamine (D-GalN), thioacetamide (TAA) and tert-butyl hydroperoxide (t-BHP)-injured rat hepatocytes, and produced protection rates of 42.2, 34.6 and 43.8% at 100 µg/ml, respectively. Furthermore, TME at 1-100 µg/ml markedly inhibited DHBV DNA replication. Additionally, TME at 25-100 µg/ml reduced HBsAg and HBeAg levels and produced inhibition rates of 91.39 and 91.72% at 100 µg/ml, respectively. TME markedly inhibited HBV DNA replication at 25-100 µg/ml. The results demonstrate the potent antiviral effect of T. mongolicum against HBV effect. The protective of TME effect on hepatocytes may be achieved by its ability to ameliorate oxidative stress. The antiviral properties of TME may contribute to blocking protein synthesis steps and DNA replication. Furthermore, major components of TME were quantificationally analyzed. These data provide scientific evidence supporting the traditional use of TME in the treatment of hepatitis.

Inhibition of the Fe(III)-catalyzed dopamine oxidation by ATP and its relevance to oxidative stress in Parkinson's disease.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity.

Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong) leaves using HSCCC guided by DPPH-HPLC experiment.

Seven antioxidants were purified from Eucommia ulmoides Oliv. leaves using HSCCC guided by DPPH-HPLC experiment. HSCCC was successfully used to separate target antioxidants by three runs with different solvent systems after D101 column chromatography fractionation. Ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected as the optimum solvent system to purify geniposidic acid. Ethyl acetate-ethanol-water (4:1:5, v/v/v) was used to isolate caffeic acid, chlorogenic acid and ferulic acid. While three flavonoids, quercetin-3-O-sambubioside, rutin and isoquercitrin were purified by petroleum ether-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). The structures were identified by MS and NMR. Antioxidant activities were assessed, and compounds 2-7 showed strong antioxidant activities. This is the first report about separation of antioxidants from E. ulmoides leaves by HSCCC. The results indicated that the combinative methods using DPPH-HPLC and HSCCC could be widely applied for screening and isolation of antioxidants from complex extracts.

Separation of intermediates of iron-catalyzed dopamine oxidation reactions using reversed-phase ion-pairing chromatography coupled in tandem with UV-visible and ESI-MS detections.

Reversed-phase ion-pairing chromatography (RP-IPC) is coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) through an interface comprising a four-way switch valve and an anion exchange column. Regeneration of the anion exchange column can be accomplished on-line by switching the four-way switch valve to interconnect the column to a regeneration solution. Positioning the anion exchange column between the RP-IPC and ESI-MS instruments allows the ion-pairing reagent (IPR) sodium octane sulfonate to be removed. The IPC-ESI-MS method enabled us to separate and detect four intermediates of the Fe(III)-catalyzed dopamine oxidation. In particular, 6-hydroxydopamine, which is short-lived and highly neurotoxic, was detected and quantified. Together with the separation of other intermediates, gaining insight into the mechanism and kinetics of the Fe(III)-catalyzed dopamine oxidation becomes possible.