Ru(II) Phenanthroline-Based Oligothienyl Complexes as Phototherapy Agents.

Ru(II) polypyridyl complexes have gained widespread attention as photosensitizers for photodynamic therapy (PDT). Herein, we systematically investigate a series of the type [Ru(phen)2(IP-nT)]2+, featuring 1,10-phenanthroline (phen) coligands and imidazo[4,5-f][1,10]phenanthroline ligands tethered to n = 0-4 thiophene rings (IP-nT). The complexes were characterized and investigated for their electrochemical, spectroscopic, and (photo)biological properties. The electrochemical oxidation of the nT unit shifted by -350 mV as n = 1 → 4 (+920 mV for Ru-1T, +570 mV for Ru-4T); nT reductions were observed in complexes Ru-3T (-2530 mV) and Ru-4T (-2300 mV). Singlet oxygen quantum yields ranged from 0.53 to 0.88, with Ru-3T and Ru-4T being equally efficient (∼0.88). Time-resolved absorption spectra of Ru-0T-1T were dominated by metal-to-ligand charge-transfer (3MLCT) states (τTA = 0.40-0.85 µs), but long-lived intraligand charge-transfer (3ILCT) states were observed in Ru-2T-4T (τTA = 25-148 µs). The 3ILCT energies of Ru-3T and Ru-4T were computed to be 1.6 and 1.4 eV, respectively. The phototherapeutic efficacy against melanoma cells (SK-MEL-28) under broad-band visible light (400-700 nm) increases as n = 0 → 4: Ru-0T was inactive up to 300 µM, Ru-1T-2T were moderately active (EC50 ∼ 600 nM, PI = 200), and Ru-3T (EC50 = 57 nM, PI > 1100) and Ru-4T (EC50 = 740 pM, PI = 114,000) were the most phototoxic. The activity diminishes with longer wavelengths of light and is completely suppressed for all complexes except Ru-3T and Ru-4T in hypoxia. Ru-4T is the more potent and robust PS in 1% O2 over seven biological replicates (avg EC50 = 1.3 µM, avg PI = 985). Ru-3T exhibited hypoxic activity in five of seven replicates, underscoring the need for biological replicates in compound evaluation. Singlet oxygen sensitization is likely responsible for phototoxic effects of the compounds in normoxia, but the presence of redox-active excited states may facilitate additional photoactive pathways for complexes with three or more thienyl groups. The 3ILCT state with its extended lifetime (30-40× longer than the 3MLCT state for Ru-3T and Ru-4T) implicates its predominant role in photocytotoxicity.

Screening of Phenanthroquinolizidine Alkaloid Derivatives for Inducing Cell Death of L1210 Leukemia Cells with Negative and Positive P-glycoprotein Expression.

We describe the screening of a set of cryptopleurine derivatives, namely thienoquinolizidine derivatives and (epi-)benzo analogs with bioactive phenanthroquinolizidine alkaloids that induce cytotoxic effects in the mouse lymphocytic leukemia cell line L1210. We used three variants of L1210 cells: i) parental cells (S) negative for P-glycoprotein (P-gp) expression; ii) P-glycoprotein positive cells (R), obtained by selection with vincristine; iii) P-glycoprotein positive cells (T), obtained by stable transfection with a human gene encoding P-glycoprotein. We identified the most effective derivative 11 with a median lethal concentration of ≈13 µM in all three L1210 cell variants. The analysis of the apoptosis/necrosis induced by derivative 11 revealed that cell death was the result of apoptosis with late apoptosis characteristics. Derivative 11 did not induce a strong alteration in the proportion of cells in the G1, S or G2/M phase of the cell cycle, but a strong increase in the number of S, R and T cells in the subG1 phase was detected. These findings indicated that we identified the most effective inducer of cell death, derivative 11, and this derivative effectively induced cell death in S, R and T cells at similar inhibitory concentrations independent of P-gp expression.

Multifunctional mesoporous curcumin encapsulated iron-phenanthroline nanocluster: A new Anti-HIV agent.

A new strategy to encapsulating the drug curcumin into the hydrophobic core of the iron-phenanthroline nanocomplex (NIP) and eventually its release is signified. NIP was prepared via coordinate interaction between Fe2+ and the lone pairs present on the N atoms of the bidentate phenanthroline ligand (spherical morphology, diameter 18.8 nm, mesoporous with pore size 2.443 nm, amorphous). Thereafter, curcumin was successfully encapsulated (NCIP) in NIP, resulting in its enhanced stability (spherical morphology, diameter 46.8 nm). The nanocomplex NIP was used for drug delivery applications. We evaluated the anti-HIV effects of NCIP in vitro on cultures of HIV infected human microglia. The treatment of HIV-1 infected microglia with NCIP significantly decreased the expression of HIV-p24 by 41% and pro-inflammatory mediators TNF-α, IL-8 and NO by 61.2%, 41% and 50.2%, respectively, compared to NIP. Flow cytometry data also support the decrease in TNF-α and IL-8 expression in case of NCIP. NCIP induced antioxidative effects by increasing the gene expression of catalase (CAT) and simulatenously decreasing hemeoxygenase-1 (HMOX-1) gene expression, thereby maintaining homeostasis which reduces neuroinflammation. These results support our premise that NCIP may be a significant adjuvant when used with traditional anti-retroviral regimens and may ameliorate HIV-1 associated neurotoxicity.

DNA binding, prominent photonuclease activity and antibacterial PDT of cobalt(II) complexes of phenanthroline based photosensitizers.

The chemistry of Co(II) complexes showing efficient light induced DNA cleavage activity, binding propensity to calf thymus DNA and antibacterial PDT is summarized in this article. Complexes of formulation [Co(mqt)(B)2]ClO4 1-3 where mqt is 4-methylquinoline-2-thiol and B is N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq 2) and dipyrido[3,2-a:2',3'-c]phenazine (dppz 3) have been prepared and characterized. The DNA-binding behaviors of these three complexes were explored by absorption spectra, viscosity measurements and thermal denaturation studies. The DNA binding constants for complexes 1, 2 and 3 were determined to be 1.6 × 103 M-1, 1.1 × 104 M-1 and 6.4 × 104 M-1 respectively. The experimental results suggest that these complexes interact with DNA through groove binding mode. The complexes show significant photocleavage of supercoiled (SC) DNA proceeds via a type-II process forming singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was studied using photodynamic antimicrobial chemotherapy (PACT) assay against E. coli and all complexes exhibited significant reduction in bacterial growth on photoirradiation.

Gas-Phase Ion/Ion Reactions Involving Tris-Phenanthroline Alkaline Earth Metal Complexes as Charge Inversion Reagents for the Identification of Fatty Acids.

Fatty acids (FA) play vital biological roles as energy sources, signaling molecules and key building blocks of complex lipids in cell membranes. Modifications to FA structure and composition are associated with the onset and progression of a number of chronic diseases. Consequently, the sensitive detection and unambiguous structure elucidation of FA is integral to the advancement of biomedical sciences. Recent advances in FA analysis have taken advantage of wet chemical derivatization to enhance detection and drive unique fragmentation in tandem mass spectrometry protocols. Here, we significantly further this approach through demonstrating gas-phase charge inversion of singly deprotonated FA ions, [M - H]-, using doubly charged tris-phenanthroline alkaline earth metal complexes, [Cat(Phen)3]2+ (Cat = Mg2+, Ca2+, Sr2+, or Ba2+). Metal cationized FA, [M - H + Cat]+ are obtained after the gas-phase ion/ion reaction. Low-energy collision-induced dissociation (CID) of the [M - H + Cat]+ cations facilitates double bond localization for a variety of monounsaturated and polyunsaturated FAs. Ultimately, detailed characterization presented unambiguous distinction among FA double bond positional isomers, such as n-3 and n-6 isomers. The method was successfully used to identify the FA profile of corn oil, including double bond localization for unsaturated FAs present.

Heparin-stabilized gold nanoparticles-based CUPRAC colorimetric sensor for antioxidant capacity measurement.

Sensing of total antioxidant capacity (TAC) as an integrated parameter showing the collective action of various antioxidants is an important challenge in food, biochemical and drug analysis. A novel heparin-stabilized gold nanoparticles (AuNPs)-based 'cupric reducing antioxidant capacity' (CUPRAC) colorimetric sensor was designed for TAC measurement. Heparin, a sulfated polysaccharide, was both the reducing and stabilizing agent for distinct negatively-charged AuNPs synthesis. The stabilized AuNPs were added to the copper(I)-neocuproine (Cu(I)-Nc) solution formed from the reaction of Cu(II)-Nc with antioxidants, and the absorbance of the resulting Cu(I)-Nc-AuNPs (Cu(I)-Nc cationic chelate electrostatically adsorbed on gold nanoparticles) was measured at 455 nm. As opposed to other similar AuNPs-based sensors, the proposed nano-sensor exhibited excellent (1000-fold) tolerance toward inert electrolytes without aggregation. The linear range was wider than that of conventional CUPRAC, with lower LOD (0.2 µM for trolox) and higher molar absorptivity (8.36â€¯× 104 M-1 cm-1 for quercetin). The 'trolox equivalent antioxidant capacity' (TEAC) values and activity order for a number of antioxidants were in accordance with those of the reference CUPRAC assay. Antioxidant additions to black tea extract gave recoveries of 93-97% and RSD 2-6%. This green sensor significantly reduced reagent consumption, and operated in complex food samples with a simple, reliable and robust methodology.

Comparisons of the spectroscopic and microbiological activities among coumarin-3-carboxylate, o-phenanthroline and zinc(II) complexes.

Coumarins (2H-chromen-2-one) are oxygen-containing heterocyclic compounds that belong to the benzopyranones family. In this work we have synthesized different coordination complexes with coumarin-3-carboxylic acid (HCCA), o-phenanthroline (phen) and zinc(II). In the reported [Zn(CCA)2(H2O)2] complex, coumarin-3-carboxylate (CCA) is acting as a bidentate ligand while in the two prepared complexes, [Zn(phen)3]CCA(NO3) (obtained as a single crystal) and [Zn(CCA)2phen].4H2O, CCA is acting as a counterion of the complex cation [Zn(phen)3]+2 or coordinated to the metal center along with phen, respectively. These compounds were characterized on the basis of elemental analysis and thermogravimetry. NMR, FTIR and Raman spectroscopies of the compounds and the CCA potassium salt (KCCA) allow to determine several similarities and differences among them. Finally, their behavior against alkaline phosphatase enzyme and their antimicrobial activities were also measured.

Probing the Interactions of Cytotoxic [Pt(1S,2S-DACH)(5,6-dimethyl-1,10-phenanthroline)] and Its PtIV Derivatives with Human Serum.

The discrepancy between the in vitro cytotoxic results and the in vivo performance of Pt56MeSS prompted us to look into its interactions and those of its PtIV derivatives with human serum (HS), human serum albumin (HSA), lipoproteins, and serum-supplemented cell culture media. The PtII complex, Pt56MeSS, binds noncovalently and reversibly to slow-tumbling proteins in HS and in cell culture media and interacts through the phenanthroline group with HSA, with a Kd value of ∼1.5×10-6 m. All PtIV complexes were found to be stable toward reduction in HS, but those with axial carboxylate ligands, cct-[Pt(1S,2S-DACH)(5,6-dimethyl-1,10-phenantroline)(acetato)2 ](TFA)2 (Pt56MeSS(OAc)2 ) and cct-[Pt(1S,2S-DACH)(5,6-dimehtyl-1,10-phenantroline)(phenylbutyrato)2 ](TFA)2 (Pt56MeSS(PhB)2 ), were spontaneously reduced at pH 7 or higher in phosphate buffer, but not in Tris buffer (pH 8). HS also decreased the rate of reduction by ascorbate of the PtIV complexes relative to the reduction rates in phosphate buffer, suggesting that for this compound class, phosphate buffer is not a good model for HS.

The Antibacterial Activity of Metal Complexes Containing 1,10- phenanthroline: Potential as Alternative Therapeutics in the Era of Antibiotic Resistance.

The "antibiotic era", characterized by the overuse and misuse of antibiotics, over the last half-century has culminated in the present critical "era of resistance". The treatment of bacterial infections is challenging because of a decline in the current arsenal of useful antibiotics and the slow rate of new drug development. The discovery of a new gene (mcr-1) in 2015, which enables bacteria to be highly resistant to polymyxins (such as colistin), the last line of antibiotic defence left, heralds a new level of concern as this gene is susceptible to horizontal gene transfer, with alarming potential to be spread between different bacterial populations, suggesting that the progression from "extensive drug resistance" to "pan-drug resistance" may be inevitable. Clearly there is a need for the development of novel classes of anti-bacterial agents capable of killing bacteria through mechanisms that are different to those of the known classes of antibiotics. 1,10-phenanthroline (phen) is a heterocyclic organic compound which exerts in vitro antimicrobial activity against a broad-spectrum of bacteria. The antimicrobial activity of phen can be significantly modulated by modifying its structure. The development of metal-phen complexes offers the medicinal chemist an opportunity to expand such structural diversity by controlling the geometry and varying the oxidation states of the metal centre, with the inclusion of appropriate auxiliary ligands in the structure, offering the opportunity to target different biochemical pathways in bacteria. In this review, we summarize what is currently known about the antibacterial capability of metal-phen complexes and their mechanisms of action.

A total analytical system featuring a novel solid-liquid extraction chamber for solid sample flow analysis.

In this work, a total flow analysis system based on a novel solid-liquid extraction chamber is presented. This strategy enables all the main experimental procedures for the analysis of a solid sample to be performed automatically: enrichment of the liquid extract, sample treatment, filtration of the liquid extract from the solid sample, directing the extract towards detection, and finally cleansing of the chamber for the following solid sample to be analyzed. The chamber designed to be incorporated in the flow manifold presents two main features: it accommodates stirring bars for enhancing the extraction process, and it presents replaceable solid sample containers (a spare part of the solid-liquid extraction chamber) to easily replace the solid sample and therefore enhance sample analysis throughput. The chamber performance was assessed using two different solid samples, an ion exchanger resin and vegetable samples, focussing on proton and nitrate ion extraction, respectively. The main figures of merit achieved were relative standard deviation (RSD) and relative error values below 7 % for all determinations. The determination rate for vegetable samples was ca. 12 samples h-1. The proposed strategy may be exploited to perform automatically the analysis of solid samples as it embodies a simple automatic strategy of a very important but time-consuming and laborious analytical operation. Graphical abstract TAS for solid liquid extraction and nitrate potentiometric determination of vegetable samples.

[Cu(phen)2](2+) acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor.

We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)2](2+), where phen = 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)2](2+) acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)2](2+) were linear with the logarithm of the concentrations of complementary target DNA ranging from 1 × 10(-12) to 1 × 10(-6) M with a detection limit of 1.99 × 10(-13) M (signal/noise = 3). Moreover, the stability of the electrochemical DNA biosensor was also studied.

Development of an Analytical Method Based on Temperature Controlled Solid-Liquid Extraction Using an Ionic Liquid as Solid Solvent.

At the present paper, an analytical method based on temperature controlled solid-liquid extraction (TC-SLE) utilizing a synthesized ionic liquid, (N-butylpyridinium hexafluorophosphate, [BPy]PF6), as solid solvent and phenanthroline (PT) as an extractant was developed to determine micro levels of Fe(2+) in tea by PT spectrophotometry. TC-SLE was carried out in two continuous steps: Fe(2+) can be completely extracted by PT-[BPy]PF6 or back-extracted at 80 °C and the two phases were separated automatically by cooling to room temperature. Fe(2+), after back-extraction, needs 2 mol/L HNO3 as stripping agent and the whole process was determined by PT spectrophotometry at room temperature. The extracted species was neutral Fe(PT)mCl2 (m = 1) according to slope analysis in the Fe(2+)-[BPy]PF6-PT TC-SLE system. The calibration curve was Y = 0.20856X - 0.000775 (correlation coefficient = 0.99991). The linear calibration range was 0.10-4.50 µg/mL and the limit of detection for Fe(2+) is 7.0 × 10(-2) µg/mL. In this method, the contents of Fe(2+) in Tieguanyin tea were determined with RSDs (n = 5) 3.05% and recoveries in range of 90.6%-108.6%.

Sagitol D, a New Thiazole Containing Pyridoacridine Alkaloid from a Vietnamese Ascidian.

A new thiazole containing pyridoacridine alkaloid, named sagitol D (1), and five known alkaloids kuanoniaminesA (2), C (3), D (4), E (5), and F (6), have been isolated from an unidentified Vietnamese ascidian. The structure of the new compound was established from NMR spectroscopic data. Kuanoniamines C, D, E, and F showed moderate antioxidant activity in the DPPH (IC50 36 µM) and ABTS assays (TE = 0.5), while sagitol D showed weak activity (IC50 92 M;TE = 0.10), and kuanoniamine A was inactive.

Europium, uranyl, and thorium-phenanthroline amide complexes in acetonitrile solution: an ESI-MS and DFT combined investigation.

The tetradentate N,N'-diethyl-N,N'-ditolyl-2,9-diamide-1,10-phenanthroline (Et-Tol-DAPhen) ligand with hard-soft donor atoms has been demonstrated to be promising for the group separation of actinides from highly acidic nuclear wastes. To identify the formed complexes of this ligand with actinides and lanthanides, electrospray ionization mass spectrometry (ESI-MS) combined with density functional theory (DFT) calculations was used to probe the possible complexation processes. The 1 : 2 Eu-L species ([EuL2(NO3)](2+)) can be observed in ESI-MS at low metal-to-ligand ([M]/[L]) ratios, whereas the 1 : 1 Eu-L species ([EuL(NO3)2](+)) can be observed when the [M]/[L] ratio is higher than 1.0. However, ([UO2L(NO3)](+)) is the only detected species for the uranyl complexes. The [ThL2(NO3)2](2+) species can be observed at low [M]/[L] ratios; the 1 : 2 species ([ThL2(NO3)](3+)) and a new 1 : 1 species ([ThL(NO3)3](+)) can be detected at high [M]/[L] ratios. Collision-induced dissociation (CID) results showed that Et-Tol-DAPhen ligands can coordinate strongly with metal ions, and the coordination moieties remain intact under CID conditions. Natural bond orbital (NBO), molecular electrostatic potential (MEP), electron localization function (ELF), atoms in molecules (AIM) and molecular orbital (MO) analyses indicated that the metal-ligand bonds of the actinide complexes exhibited more covalent character than those of the lanthanide complexes. In addition, according to thermodynamic analysis, the stable cationic M-L complexes in acetonitrile are found to be in good agreement with the ESI-MS results.

Physicochemical properties of ternary oxovanadium(IV) complexes with oxydiacetate and 1,10-phenanthroline or 2,2'-bipyridine. Cytoprotective activity in hippocampal neuronal HT22 cells.

The aim of this work was to find a relationship between physicochemical properties of the oxovanadium(IV) complexes, namely [VO(ODA)(H2O)2], [VO(ODA)(phen)]·1.5H2O and [VO(ODA)(bipy)]·2H2O (ODA = oxydiacetate) as well as [VO(H2O)5](2+), and their biological activity. A potentiometric titration method has been used to characterize the stability of the complexes in aqueous solutions. Furthermore, the reactivity of the complexes towards superoxide free radicals was assessed by employing the NBT assay as well as a cyclic voltammetry (CV) technique. Additionally, the investigations of the antioxidant properties of the complexes were complemented by studying their reactivity towards organic radicals (the ABTS and DPPH tests). Finally, the biological properties of the complexes were investigated in relation to their cytoprotective activity against the oxidative damage generated exogenously by using hydrogen peroxide in the Hippocampal neuronal cell line HT22 (the MTT and LDH tests). The obtained results showed that all the compounds under study display antioxidant properties but a concentration-depended protective effect against the oxidative damage was found for [VO(ODA)(bipy)]·2H2O only.

Quantum chemistry study of uranium(VI), neptunium(V), and plutonium(IV,VI) complexes with preorganized tetradentate phenanthrolineamide ligands.

The preorganized tetradentate 2,9-diamido-1,10-phenanthroline ligand with hard-soft donors combined in the same molecule has been found to possess high selectivity toward actinides in an acidic aqueous solution. In this work, density functional theory (DFT) coupled with the quasi-relativistic small-core pseudopotential method was used to investigate the structures, bonding nature, and thermodynamic behavior of uranium(VI), neptunium(V), and plutonium(IV,VI) with phenanthrolineamides. Theoretical optimization shows that Et-Tol-DAPhen and Et-Et-DAPhen ligands are both coordinated with actinides in a tetradentate chelating mode through two N donors of the phenanthroline moiety and two O donors of the amide moieties. It is found that [AnO2L(NO3)](n+) (An = U(VI), Np(V), Pu(VI); n = 0, 1) and PuL(NO3)4 are the main 1:1 complexes. With respect to 1:2 complexes, the reaction [Pu(H2O)9](4+)(aq) + 2L(org) + 2NO3(-)(aq) → [PuL2(NO3)2](2+)(org) + 9H2O(aq) might be another probable extraction mechanism for Pu(IV). From the viewpoint of energy, the phenanthrolineamides extract actinides in the order of Pu(IV) > U(VI) > Pu(VI) > Np(V), which agrees well with the experimental results. Additionally, all of the thermodynamic reactions are more energetically favorable for the Et-Tol-DAPhen ligand than the Et-Et-DAPhen ligand, indicating that substitution of one ethyl group with one tolyl group can enhance the complexation abilities toward actinide cations (anomalous aryl strengthening).

Evaluation of total reducing power of edible oils.

The lipophilicity of untreated edible oils narrows the application of most published methods for the determination of antioxidant activity to hydrophilic extracts of oils. This research addresses the issue of the estimation of the total antioxidant properties of untreated edible oils by modifying two widely applied analytical methods, the Fe-Phenanthroline and the CUPRAC assays, to be used in untreated oils. The modifications pertain to the selection of mixture of solvents (ethanol-butanol in 3:1 v/v ratio), and the optimization of the reaction conditions (reagents concentration and reaction time). The developed methods were applied to a number of hydrophilic and lipophilic standard compounds and different types of commercial edible oils, as well as their corresponding aqueous or organic extracts. This implementation elucidated the differences in the antioxidant content of edible oils. All the results were compared to those of the DPPH and Folin-Ciocalteu methods and the analytical figures of merit for the methods have been estimated. Lastly, it was concluded that the modified CUPRAC assay has higher sensitivity compared to the Fe-Phenanthroline assay.

Design, synthesis, anti-tobacco mosaic virus (TMV) activity, and SARs of 7-methoxycryptopleurine derivatives.

A series of 7-methoxycryptopleurine derivatives 2-23 were prepared and evaluated for their antiviral activity against tobacco mosaic virus (TMV) for the first time. The bioassay results showed that most of these compounds exhibited excellent in vivo anti-TMV activity, of which 7-methoxycryptopleurine salt derivatives 16, 19, and 23 displayed significantly higher activity than 7-methoxycryptopleurine (1) and commercial ribavirin and ningnanmycin. Salification, the most commonly employed method for modifying physical-chemical properties, did significantly increase antiviral activity, and different salt forms displayed different antiviral effect. This study provides fundamental support for development and optimization of phenanthroquinolizidine alkaloids as potential inhibitors of plant virus.

Optimization of liquid chromatographic method for the separation of nine hydrophilic and hydrophobic components in Salviae miltiorrhizae Radix et Rhizoma (Danshen) using microemulsion as eluent.

In this study, we have proposed and developed a novel, environmental-friendly and simple method for separation of nine hydrophilic and hydrophobic components in Danshen using microemulsion liquid chromatography. The proposed method was optimized via the preliminary screening experiment and the experimental design. The following factors were investigated in preliminary screening experiment: pH of mobile phase, column type, the nature of surfactant, the nature of oil phase and additives. In order to simultaneously optimize resolution and analysis time, the chromatographic optimization function (COF) was adopted to evaluate chromatograms. The central composite design (CCD) was used to create the matrix of experiments for mapping the chromatographic response surface. Finally, the COF values were fitted into a second order polynomial model and the response surface methodology (RSM) was employed to find the optimal eluent constituents. The reliability of the established model was confirmed by the good agreement obtained between experimental data and predictive values. Based on the results from the preliminary screening experiment and the CCD optimization, the optimal mobile phase was identified as a solution consisting of 6.68% (w/w) polyoxyethylene lauryl ether (Brij35), 0.84% (w/w) cyclohexane, 6.92% (w/w) n-butanol, 85.56% (w/w) phosphate buffer (pH 6.60) and 8mM cetyltrimethyl ammonium bromide (CTAB).

Identification and anti-oxidant capacity determination of phenolics and their glycosides in elderflower by on-line HPLC-CUPRAC method.

INTRODUCTION: Development and application of an on-line cupric reducing anti-oxidant capacity (CUPRAC) assay coupled with HPLC for separation and on-line determination of phenolic anti-oxidants in elderflower (Sambucus nigra L.) extracts for their anti-oxidant capacity are significant for evaluating health-beneficial effects. Moreover, this work aimed to assay certain flavonoid glycosides of elderflower that could not be identified/quantified by other similar on-line HPLC methods (i.e. 2,2-diphenyl-1-picrylhdrazyl and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid). OBJECTIVE: To identify anti-oxidant constituents in elderflower by HPLC and to evaluate their individual anti-oxidant capacities by on-line HPLC-CUPRAC assay with a post-column derivatisation system. METHODS: The separation and UV detection of polyphenols were performed on a C18 -column using gradient elution with two different mobile phase solutions, that is acetonitrile and 1% glacial acetic acid, with detection at 340 nm. The HPLC-separated anti-oxidant polyphenols in column effluent react with copper(II)-neocuproine in a reaction-coil to reduce the latter to copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. RESULTS: The detection limits of tested compounds at 450 nm after post-column derivatisation were compared with those of at 340 nm UV-detection without derivatisation. LOD values (µg/mL) of quercetin and its glycosides at 450 nm were lower than those of UV detection at 340 nm. This method was applied successfully to elderflower extract. The flavonol glycosides of quercetin and kaempferol bound to several sugar components (glucose, rhamnose, galactose and rutinose) were identified in the sample. CONCLUSION: The on-line HPLC-CUPRAC method was advantageous over on-line ABTS and DPPH methods for measuring the flavonoid glycosides of elderflower.