ARTICLE-01
Synthesis and crystal structures of 5'-phenylspiro[indoline-3, 2'-pyrrolidin]-2-one derivatives
Background
1,3-Dipolar cycloaddition of azomethineylides to exocyclic olefins constitutes a versatile protocol for the construction of poly functionalized spiro-heterocycles viz. pyrrolidines [1] and pyrrolizines [2], which widely occur in natural products and biologically active compounds. The spiro- indole-pyrrolidine ring system is a frequently encountered structural motif in many biologically important and pharmacologically relevant alkaloids. Compounds with an indole/oxindole framework are promising pharmacophore which exhibit interesting applications in the biological and pharmacological arena [3]. The derivatives of spirooxindole ring systems are used as antimicrobial, antitumour agents and as inhibitors of the human NKI receptor besides being found in a number of alkaloids like horsifiline, spirotryprostatin and (+) elacomine [4]. The recently discovered small-molecule MDM2 inhibitor MI-219 and its analogues are in advanced preclinical development as cancer therapeutics [5]. Our interest in preparing pharmacologically active pyrrolidines led us to the compounds, 4'-Nitro-3',5'-diphenylspiro[indoline-3,2'-pyrrolidin]-2-one (I) and 3'-(4-Methoxyphenyl)- 4'-nitro -5'-phenylspiro[indoline-3, 2'-pyrrolidin]-2-one (II), and we have undertaken the X-ray crystal structure determination of these compounds in order to establish their conformations.Experimental
The spiro compounds reported in the present work were prepared (Scheme 1) by following our earlier literatures method [6-8]. A mixture of (E)-(2-nitrovinyl) benzene or (E)-1-methoxy-4-(2-nitrovinyl) benzene (1 mmol), isatin (1 mmol) and phenylglycine (1 mmol) was heated to reflux in methanol on a water-bath for 40 min. The progress of the reaction was monitored by thin layer chromatography (TLC). The starting materials vanished in the TLC indicating the completion of the reaction i.e, the azomethineylide (dipole) reacts with the substituted vinyl benzene (dipolarophile). Then, the reaction mixture was poured into crushed ice, the resulting solid filtered and washed with water to afford pure regio and stereoselective 3'-Phenyl-4'-nitro-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one or 3'-(4-Methoxyphenyl)-4'-nitro-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one in good yields. The synthesis scheme of 3'-(aryl)-4'-nitro-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one is shown below. For compound (I): Yield 80%; M.p. 239°C. For compound (II): Yield 78%; M.p. 231°C.
Scheme 1
Synthesis scheme of the compounds.
Results and Discussion
In both the molecules, the 2-oxyindole ring is planar (r.m.s deviation: 0.031 Å and 0.018 Å for I and II, respectively), which is common in spiro complexes [9,10]. The spiro rings of both molecules have the twisted envelope structure with the N atom at the flap position. The distance to the flap position from the mean plane of spiro carbon atoms, are 0.531(3) Å and 0.503(2) Å in compounds (I) and (II), respectively. The phenyl ring and methoxyphenyl rings are inclined by an angle of 31.45 (2)° in compound (II) which is similar to the inclination of the two phenyl rings in compound (I) (31.60(2)°). In compound (II), H9 and H8 have trans conformation with the torsion angle of 152.45(2)° (H9/C9/C8/H8) and H8 and H7 have cis conformation with the torsion angle of -5.43(2)° (H8/C8/C7/H7). In compound (I) also, similar conformation is found. The hydrogen conformation torsion angles in compound (I) are 152.81(3)° and 7.14(3)° for H9 & H8 and H8 & H7, respectively. Even though these conformations are similar, the directions in which the hydrogens are attached, are reciprocal in both the compounds. Figure 1, a superimposition of the planar 2-oxyindole rings, drawn using Mercury [11], clearly shows the reciprocal conformations of both the compounds. In both molecules, N-H···O hydrogen bonds make the R22 (8) ring motifs (Figure 2 and Figure 3). Further, the structures are stabilized by intermolecular hydrogen bonds.
Figure 1. Reciprocal conformations of both compounds, as seen from the superimposition of the planar 2-oxyindole rings.
Figure 2. Figure showing the intramolecular hydrogen bonds resulting in R22(8) motif in compound (I).
Figure 3. Figure showing the intramolecular hydrogen bonds resulting in R22(8) motif in compound (II).X-ray Crystallography
Single crystal X-ray intensity data for the compounds (Scheme 2) and (Scheme 3) were collected using a Nonius CAD-4 MACH 3 diffractometer with MoKα (0.71073 Å) radiation at room temperature (293 K). The data reduction was carried out using XCAD4 [12]. The absorption corrections were applied using the ψ-scan method [13]. The structures of both the compounds were solved by direct methods using SHELXS97 [14] and all the non-hydrogen atoms were refined anisotropically by full-matrix least-squares on F2 taking all the unique reflections using SHELXL97 [14]. The hydrogen atoms attached with carbon atoms were placed in their calculated positions and included in the isotropic refinement using the riding model with C-H = 0.93Å (-CH) or 0.97Å (-CH2) Å or 0.96Å (-CH3) Å with Uiso(H) = 1.2 Ueq (parent C atom) and amino bound hydrogen atoms were located from the difference Fourier map and include in the refinement isotropically. The crystal data, experimental conditions and structure refinement parameters for the compounds (I) and (II) are presented in Table 1. The re-crystallization of the compound (I) and repeated data collection with different crystal samples did not improve the R value and other statistical parameters. Crystals of better quality could not be obtained for the compound (I). Table 2 gives the geometry of the hydrogen bonds present in I and II. The molecular structures of compounds (I) and (II) showing the atom numbering scheme using ORTEP-3 [15] are given in Figures 4 and 5, respectively.
Scheme 2
Scheme showing the structural formula of compound (I).
Scheme 3
Scheme showing the structural formula of compound (II).
Table 1. The crystal data, experimental conditions and structure refinement parameters for the compounds (I) and (II)
Table 2. The geometry of the hydrogen bonds (Å, °)
Figure 4. The molecular structure of compound (I) showing the atom numbering scheme. Displacement ellipsoids are drawn at the 40% probability level, using ORTEP-3. Hydrogen atoms are drawn as spheres of arbitrary size.
Figure 5. The molecular structure of compound (II) showing the atom numbering scheme. Displacement ellipsoids are drawn at the 40% probability level, using ORTEP-3. Hydrogen atoms are drawn as spheres of arbitrary size.Conclusions
The title compounds were synthesized and the corresponding molecular crystal structures have been determined by single-crystal X-ray diffraction. In both the compounds, the R22(8) motif is present. Even though most of the conformational features are similar when seen separately, by super positioning the two structures it is found that the entire configuration is inverted with respect to the 2-oxyindole ring. This is due to the substitution of methoxyphenyl instead of phenyl ring in compound (I).Additional material
Crystallographic data (excluding structure factors) for the structures of compounds (I) and (II) reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication numbers, CCDC 802309 and CCDC 802308, respectively. Copies of the data can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1 EZ, UK. (fax: +44-(0)1223-336033 or email: deposit@ccdc.cam.ac.uk).Competing interests
The authors declare that they have no competing interests.Authors' contributions
JKS collected the X-ray data and solved the crystal structures under the guidance of SN. SMR and JS synthesized the title compounds under the guidance of SP. All the authors read and approved the final manuscript.Acknowledgements
One of the authors (JK) thanks the UGC for the RFSMS fellowship. SN thanks the CSIR for the funding provided under the Emeritus Scientist Scheme.References
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ARTICLE-02
Background
The use of cocoa has been documented for almost 4,000 years. The first population thought to consume the material was the Mesoamericans [1,2]. In the past decade there has been increasing interest and numerous publications on the putative health effects associated with the moderate consumption of cocoa and chocolate products [3-5]. In a parallel fashion, several groups initiated studies into the potential agents responsible for cardiovascular effects with the flavanols, (±)-catechin and (±)-epicatechin being candidate compounds [6].The growing interest in these compounds resulted in a plethora of methods for quantification in various foodstuffs including tea, wine, grapes and chocolate. While other analytical methods have been used, HPLC was the predominant method developed [7-13]. A thorough literature search using Google and Pubmed resulted in thousands of citations on polyphenol analysis and almost 900 citations on flavanol analysis by HPLC in chocolate, indicating the recent explosive growth in methods for these analytes. Considering the increased interest in the cocoa flavanols' potential cardiovascular effects, a standard quantification method would be pertinent for accurate determination of dose-response effects in clinical trials. There is both an ISO and an Institute for Nutraceutical Advancement (INA) method for flavanols in tea, but not yet a standard method for flavanol quantification in chocolate and cocoa [14,15].
With this as background, the National Confectioners Association (NCA) convened an analytical chemistry working group to develop a consensus HPLC method. This group conducted a collaborative study using samples provided by NCA to establish a method to quantify (±)-catechin and (±)-epicatechin in cocoa and chocolate and make it available to the industry.
Experimental
Scope and Applicability
This method is applicable for the analysis of (±)-epicatechin and (±)-catechin in cocoa powder, chocolate liquor and formulated chocolate products. This ring trial only included pure chocolate; any products containing inclusions (such as fruit or nuts) may not be appropriate for this method due to potential interference.A. Principle
This method determines the (±)-catechin and (±)-epicatechin content of cocoa and chocolate products. Fat is removed from the sample in order to prevent potential interference and protect the column by using multiple hexane extractions. Defatted samples are then dried for subsequent extraction of analytes. Defatted, dried samples are extracted, with sonication, at 40°C for 15 minutes using an acetone: water: acetic acid (70: 29.5: 0.5) solvent mixture. Extracted samples are then centrifuged to remove insoluble materials and brought up to a defined volume. The extracts are filtered into HPLC vials for chromatographic analysis. (±)-Catechin and (±)-epicatechin are separated by a reverse phase mechanism on a C18 column with an acidic acetonitrile-water mobile phase gradient. Analytes are detected and quantified by their fluorescence, with excitation at 280 nm and emission at 315 nm.B. Apparatus
(a) HPLC system: With solvent degasser, binary gradient pumping, gradient mixer, injector capable of 10 μL injection (either autosampler or manual), column oven, fluorescence detector and data analysis system
(b) Chromatography column: Reversed phase HPLC column octadecylsilane (ODS; C18) derivatized silica reversed phase HPLC column, pore size from 100 - 125 A, are recommended. Recommended column: Phenomenex Luna, 5 μm, C18(2), 100A, 250 × 3.0 mm (alternate columns may be used if they provide acceptable resolution)
(c) Analytical balance: Readability 0.1 mg or lower
(d) Pipettes: Capable of accurately delivering 20-1000 μL; 1-5 mL
(e) Vials: 2 mL, amber glass, screw cap, for storing Stock Standard solutions and for holding filtered HPLC sample prior to injection
(f) Test Tubes: Screw capped, with caps, capable of holding at least 10 mL
(g) Volumetric flasks: 10 mL, 20 mL, 50 mL and 100 mL, Class A, glass
(h) Centrifuge tubes: Plastic, for single use, 50 mL, screw cap (air tight)
(i) Vortex Mixer
(j) Ultrasonic Bath
(k) Flame-Proof Centrifuge: For centrifuging 50 mL tubes at 2500 × g
(l) Syringe filters: For filtering HPLC samples, 0.45 μm PVDF, PTFE or hydrophilic polypropylene, 13 or 25 mm diameter (Nylon filters are not recommended due to potential adsorption of metabolites)
(m) Syringe: All plastic, 1 mL to 5 mL as appropriate
(n) Glass beads: (approx. diameter 5 mm)
C. Reagents
(a) Water: High purity deionized water, filtered through a 0.45 μm or smaller pore filter
(b) Acetonitrile: HPLC grade
(c) Hexane: HPLC grade
(d) Acetone: HPLC grade
(e) Acetic Acid: Glacial
(f) Extraction Solvent: Mix 700 mL Acetone, 295 mL Water and 5 mL Acetic Acid
(g) Mobile Phase A: 0.2% Acetic Acid in Water. Add 2 mL Acetic Acid to 1 L Water.
(h) Mobile Phase B: 0.2% Acetic Acid in Acetonitrile. Add 2 mL Acetic Acid to 1 L Acetonitrile.
(i) Standards: (±)-Catechin hydrate, purity ≥ 98%, Sigma-Aldrich C1251-5G or equivalent; (±)-epicatechin, purity ≥ 98%, Sigma-Aldrich E4018-1G or equivalent. Certificate of analysis from supplier is required for purity correction of each new lot number.
D. Standards and Reagent Blank Preparation
(a) (i.) Stock standard solution A (approx 1000 μg/mL): Into a 50 mL volumetric flask, accurately weigh approximately 50 mg (±)-catechin hydrate and 50 mg (±)-epicatechin and record the weights. Add extraction solvent and mix or sonicate to dissolve. Bring to 50.00 mL with extraction solvent and mix. Label as Stock standard A.
(ii.) Stock standard solution B (approx 100 μg/mL): Pipette, using a Class A volumetric pipette, 5 mL of Stock standard A into a 50 mL volumetric flask and dilute to volume with extraction solvent. Label as Stock standard B.
(b) Obtain loss on drying (important for (±)-catechin hydrate): Crystal water is not stoichiometrically distributed; for (±)-epicatechin loss on drying normally equals 0) and HPLC purity of analyte from supplier's certificate of analysis for each new lot number to calculate purity: Purity (%) = [100 (%) - loss on drying (%)] * HPLC purity (%)/100 (%)
Calculate exact concentrations of each component in stock standard solution as shown below:
Stock standard B concentrations will require multiplication by an additional 1/10 factor.
(c) Fluorescence Detector Sensitivity Assessment: Stock Standard Selection (Stock Standard Solutions A, B): When running this method for the first time inject the appropriate injection volume, 10 μl, of Stock Standard A and B onto the HPLC system under the conditions provided in Section G. Examine the detector response for the two concentrations provided. Choose the most concentrated stock standard solution that does not saturate the detector as the Stock Standard with which to proceed. Discard the other stock standard solution. If proceeding with stock standard B, for future analysis note that the stock standard preparation procedure can be modified by preparing a stock standard solution of 0.1 mg/mL to save one dilution step.
(d) Reagent Blank: Use extraction solvent for the blank.
(e) Working standards: Prepare Working Standard Solutions from the chosen stock standard. Add indicated amounts of appropriate working standard (100 or 1000 μg/mL) to a 10 mL volumetric flask; bring to volume with the extraction solvent. Transfers should always be made with Class A volumetric pipettes. Alternatively test tubes can be used and the remaining extraction solvent for dilution to 10 mL can be added with Class A volumetric pipettes. See an example of the working standard dilution scheme in Table 1 below.
Table 1. Example of Working Standard Dilution Scheme(s)(f) Calculate the exact concentration of each component of the working standards as follows:
E. Lipid Removal from Cocoa and Chocolate Samples
(a) Accurately weigh approximately 2 grams of each finely divided/grated milk chocolate sample or 1 gram for cocoa powders/baking chocolate/dark chocolate samples into a labeled, tared 50 mL disposable centrifuge tube. Record the weight of the sample W SAMPLE.
(b) Add approx. 40 mL hexane (dispenser) and cap tightly.
(c) Mix until the sample is completely dispersed (check visually).
(d) Centrifuge for 5 minutes at 2500 × g.
(e) Carefully decant and dispose of the hexane phase immediately.
(f) Repeat defatting steps (b) to (e) one additional time.
(g) Remove the cap and allow the residual solvent to evaporate in an appropriate fume hood until remaining hexane has evaporated (e.g. over night). Alternately, a stream of nitrogen may be used to accelerate the drying process.
F. Preparation of Test Solutions
Continue with whole sample remaining in the centrifugation tube.
(a) Add 2 glass beads to the centrifuge tube containing the dried, defatted sample.
(b) Add 9 mL of extraction solvent (dispenser) and vigorously shake the sample to break centrifugation pellet. Sample does not need to be completely suspended yet. Shake headlong, if necessary gently tap several times.
(c) Place in an ultrasonic bath at 40°C for 15 minutes in total. After 5-10 minutes of sonication, remove sample from bath and handshake again until sample is completely suspended (check visually). Alternately, vortex sample.
(d) Remove the sample from the ultrasonic bath, centrifuge at 2500 × g for 5 minutes.
(e) Carefully and slowly decant the liquid portion into a 20 mL Class A volumetric flask (wide neck, if possible).
(f) Repeat the extraction steps (b) to (d) one additional time. Decant the liquid from the second extraction into the same 20 mL volumetric flask.
(g) Bring to volume with extraction solvent.
(h) Assemble a Syringe and Syringe Filter. Filter approximately 1 mL of sample into a HPLC Vial.
(i) Analyze by HPLC as described in Section G.
G. Chromatography
(a) Injection volume: 10 μL
(b) Flow rate: 0.65 mL/min for 3 mm i.d. column; Alter flow rate to maintain linear flow for other column dimensions.
(c) Detection: Fluorescence with excitation at 280 nm and emission at 315 nm
(d) Column temperature: 40°C
(e) Gradient Elution: See Table 2 for example gradient conditions. HPLC columns differ in their selectivity for these compounds. Gradient conditions should be altered as needed to achieve resolution of (±)-catechin and (±)-epicatechin from interfering peaks.
Table 2. HPLC Gradient Example(f) Concentration of analytes in sample extract: Check if concentration of analytes in sample extract lie within their calibration ranges. If necessary, dilute and re-run extraction solution.
(g) Check sample: Check sample by re-runing mid point calibration curve in middle and at end of sample sequence. Calculate the mean, standard deviation and coefficient of variation (%CV) of the peak areas.
H. Calculations
Integrate peak area for quantitation. If peak areas of analytes in sample extracts are above calibration curve, dilute sample extract solution with extraction solvent accordingly. If peak areas of analytes in sample extracts are below calibration curve, repeat sections E and F and increase sample weight accordingly.Construct standard curves, plotting calibration standard concentration of each standard against the area of the standard peak, using linear regression. Calculate the analytes (±)-catechin and (±)-epicatechin in the original sample as follows:
Analyte in sample [μg/g] = assay concentration of analyte [μg/mL] * × [mL]/Wsample[g]
WSample [g] = initial sample weight from section E (a)
× [mL] = 20 mL (volume of extraction solution in volumetric flask; section F)
I. HPLC System and Column Performance Criteria Qualification
An HPLC column which fully resolves the analytes of interest may be used for the method. Gradient slope, flow rates and injection volumes may be altered as appropriate to accommodate columns of differing dimensions.J. System Suitability
System suitability is a required procedure to ensure the HPLC system is working correctly. The following suitability tests are recommended to ensure correct system operation prior to initial use:Repeatability and carry-over: Before running any test solutions, demonstrate the repeatability and lack of carryover of the HPLC system as follows:
(a) System Artifacts: As the first two injections of the day, analyze the blank standard twice in succession. Inspect the two chromatograms for artifact peaks from the HPLC system. Artifacts in the first chromatogram, absent in the second, indicate a buildup of impurities on the system. Artifacts present in both runs indicate impurities expected in every run. If the first chromatogram shows artifact peaks but the second chromatogram does not, inject a blank solution as the first sample in every analytical set. The presence of artifact peaks indicates impurities in the HPLC solvents, the needle wash system, or carryover in the injection system. These problems, if present, should be corrected.
(b) Carryover: Inject Standard 5 and then the blank. Carefully examine the blank injection for carryover peaks. Calculate the carryover of any peaks seen in the blanks as a percentage of the concentration found in standard 5. Carryover of standard 5 to the blank injection should be less than 0.1%.
(c) Linearity of the standard curve: Analyze each of the 5 standards, and construct a standard curve. The R2 of each standard curve should be greater than 0.9990. If this linearity is not achieved, prepare fresh standards.
(d) System precision: Analyze five replicate analyses of standard number 3. Calculate the concentration of each analyte and calculate the mean, standard deviation and percent coefficient of variation (%CV) of the results. The %CV for all peaks should be ideally less than 2%.
K. Samples
Samples for analysis were prepared by the NCA Study Director and submitted as blind samples to five participating laboratories. Samples consisted of cocoa, dark chocolate, milk chocolate and NIST SRM [2384] Baking Chocolate having certified values for (±)-catechin and (±)-epicatechin. One dark chocolate samples was used as a blind duplicate to assess method repeatability.L. Quantification
Quantification was performed using the external calibration method as described in the method with all laboratories reporting regression coefficients in excess of 0.99 with the labs equally divided whether calibration was forced through zero.Results and Discussion
All results were submitted to the Study Director using the form that was provided with the samples and with all data statistically evaluated. Samples were run in duplicate or triplicate. Furthermore each data set was evaluated using the Q-test to test for outliers with some data being eliminated. The results can be seen in Table 3..
Figure 1. Chromatogram of Dark Chocolate Extract Analyzed Under Method Parameters. Peak at 11.964 is (±)-catechin while peak at 14.422 is (±)-epicatechin.Each laboratory also provided information about LOD (Limit of Detection) which was in pure solvent in the 40-50 ng/mL. The LOQ (Limit of Quantitation) ranged from 1- 2 μg/g Repeat injections of standards were also accomplished with %CVs reported in the 1-4% range.
The NIST (National Institute of Standard Technology) reference values for (±)-catechin and (±)-epicatechin in SRM (Standard Reference Material) 2384 are 245 +/- 51 (μg/g) and 1220 +/- 22 (μg/g) respectively with the data from this study indicating value of 254 +/- 23.5 (μg/kg) and 1137 +/- 68 (μg/kg) which are within the acceptable range of determinations established by NIST. While recovery studies have become a default method to assess method accuracy according to Swartz and Krull, the analysis of an established SRM is by itself a generally accepted method of validation [17]. Additionally, guidance from AOAC on methods validation indicates that spiking is not a desirable method to assess method accuracy as spiking solutions tend to be easily extractable hence the choice of the NIST standard to evaluate the method.
The sample labeled Milk Chocolate 1 is an example for a product containing very low amounts of the target analytes. With the analyte concentration in the sample extract at their lower limit of quantification and the chromatographic performance negatively affected by co-extracted matrix compounds the applied method operates at its limit. Hence the sample was not included in the statistical evaluation of the method. That being said, no issues related to complexation of polyphenols with milk reported by some researchers were seen [18].
The %CV ranged from 7-15% in this study. Laboratories used a column that satisfied the requirements of U.S. Pharmacopeia, previously described in methodology section [19]. The method was reviewed and compared with recommendations of Swartz and Krull. Finally, while the data in Table 3 may seem excessive to the casual observer, it is well within the parameters established by AOAC for another complex analyte [20].
The literature reports on the use of numerous solvents for the extraction of flavan-3-ols including mixtures of methanol, acetone, water and acid therefore the solvent combination used is within established parameters [21,22]. Furthermore, a variety of HPLC detector types have been used including UV, Diode Array, Mass Spec and fluorescence [23-27]. The choice of fluorescence detection is within established analytical parameters for this determination as it offers selectivity and sensitivity for these compounds with the identity of the peaks being established by the use of authentic standards.
Conclusion
The data from these studies indicate the proposed chocolate and cocoa method is suitable as an HPLC method for the determination of flavanol monomers, (±)-catechin and (±)-epicatechin in chocolate and cocoa. The method is the first such method developed by an industry group such as NCA for this purpose.Abbreviations
HPLC: High-Performance Liquid Chromatography; ISO: International Organization for Standardization; mL: Milliliter;°C: Degrees Celsius; nm: Nanometer; mm: Millimeter; μm: micrometer; PVDF: Polyvinylidene Fluoride; PTFE: Polytertrafluoroethylene; L: Liter; mg: Milligram; g: Gram; Min: Minutes; μg: Microgram; %: Percent; NIST: National Institute of Standards and Technology; SRM: Standard Reference Method; LOD: Limit of Detection; LOQ: Limit of Quantification; AOAC: Association of Official Analytical Chemists; UV -UltravioletCompeting interests
The authors declare that they have no competing interests.Authors' contributions
AB selected standard and sample preparation methods, chromatography parameters and suitable commercial samples. LS distributed samples to participating laboratories, collected and analyzed data and prepared the manuscript. All authors read and approved the final manuscript.Acknowledgements
The authors would like to thank the following company's laboratories for their participation in validating the method by testing commercial samples: The Hershey Company, Archer Daniels Midland Company, Kraft Europe, Kraft-Cadbury and Barry Callebaut. NCA is especially grateful to the following individuals for their commitment throughout this process: Jeff Hurst, Mark Payne, Lindo Groff, Mark Collison, Eva-Maria Berndt, Verena Jendreizik, Alison Branch and Olivier Nuytten.References
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ARTICLE-03
Salicylic acid functionalized silica-coated magnetite nanoparticles for solid phase extraction and preconcentration of some heavy metal ions from various real samples
1. Background
Heavy metals are released into the environment from industrial applications, including mining, refining and production of textiles, paints and dyes. These pollutants greatly threaten the health of human populations and the natural ecosystems even at low concentration. As they do not degrade biologically like organic pollutants, their presence in drinking water or industrial effluents is a public health problem due to their absorption and therefore possible accumulation in organisms [1-5]. The toxicities of heavy metals may be caused by the inhibition and reduction of various enzymes, complexation with certain ligands of amino acids and substitution of essential metal ions from enzymes [4-6]. Hence, their determination in industrial effluents, various water resources, environmental and biological samples is important, especially in the environment monitoring and assessment of occupational and environmental exposure to toxic metals.However, the direct determination of heavy metal ions at trace levels in real samples remains a challenging problem because of their low concentration and matrix effects even with frequently used sophisticated instrumental techniques such as inductively coupled plasma atomic emission spectrometry (ICP-AES), electrothermal atomic absorption spectrometry (ET-AAS) etc. without sample preconcentration and separation [7-10].
Flame atomic absorption spectrometry (F-AAS) is among the most widely used methods for the determination of the heavy metals at trace levels, but the sensitivity and selectivity of F-AAS is usually insufficient for the determination of heavy metals at trace concentrations in complex matrix environmental samples [11-13]. In trace analysis, therefore, preconcentration or separation of trace elements from the matrix is frequently necessary in order to improve their detection and selectivity by F-AAS [13-16]. Different techniques are used for the separation and preconcentration of metals in the solution. These include liquid-liquid extraction, precipitation, cation-exchange resins, cloud point extraction and solid phase extraction [2,17-20]. However, disadvantages such as significant chemical additives, solvent losses, complex equipments, large secondary wastes, prefiltration problems and time consuming procedures, limit the application of most of these techniques.
Solid phase extraction (SPE) addresses these problems. It can extend the detection limits and remove interfering constituents thereby improving the precision and accuracy of the analytical results. Activated carbon, polymeric fibers, Ambersorb, inorganic ion-exchanger, alumina and silica gel have been used to preconcentrate trace metal ions. However, they suffer from lack of selectivity, which leads to high interference of other existing species with the analyte metal ion and chemical stability [2,17].
Recently, using nanometer-sized materials in SPE as metals ions extractors has turned out to be an active area of research in the field of separation science because of their special properties. Magnetic nanoparticles, a new kind of nanometer-sized material, are widely used in the fields of biotechnology, biomedicine and as an efficient adsorbent with large specific surface area and small diffusion resistance [2,4,6,21-24]. The use of synthetic iron oxides is much more economical than commercial highly efficient activated carbon, in a 30:1 relative ratio depending on the particular kind of activated carbon [25]. The magnetic separation provides suitable route for online separation, where particles with affinity to target species are mixed with the heterogeneous solution. Upon mixing with the solution, the particles tag the target species. External magnetic fields are then applied to separate the tagged particles from the solution.
However, it should be pointed out that pure inorganic nanoparticles (such as Fe3O4 and Fe2O3) can easily form large aggregates, which may alter their magnetic properties [6,25]. Moreover, these nanometer-sized metal oxides are not target-selective and are unsuitable for samples with complicated matrices. Therefore, a suitable coating is essential to overcome such limitations. To overcome latter problem, chemical or physical modification of the sorbent surface with some organic compounds, especially chelating ones, is usually used to load the surface with some donor atoms such as oxygen, nitrogen, sulfur and phosphorus [2,6]. These donor atoms are capable of selective binding with certain metal ions. Salicylic acid (SA) is a commercial ligand with a carboxylic and a phenolic function site which can act as electron pair donors reacting with most of hard and intermediate cations. It has already been used, for example, as the modifier in chelating resins like Amberlite XAD-2-SA, Amberlite XAD-4-SA and silica gel-SA and it have shown good sorption capacity [26-28]. This may be due to the small size of ligand molecules that has facilitated extensive functionalization of the solid support matrices.
In this study, silica-coated magnetic nanoparticles modified with SA were synthesized by a sol-gel method. These magnetic nanoparticles were employed as an SPE adsorbent for separating and concentrating trace amounts of Cu(II), Cd(II), Ni(II) and Cr(III) ions from environmental and various other real matrices prior to their determination by F-AAS and was found to have superior preconcentration and metal loading ability compared to other adsorbents prepared using salicylic acid as the functional group. The propose method was validated by analyzing certified reference materials (both environmental and biological) and by performing recovery studies on water and food samples by F-AAS.
2. Experimental
2.1. Reagents and Apparatus
Chemicals used for experiments were all in analytical reagent grade. Aqueous solutions of chemicals were prepared with deionized water. The glass equipments kept in HNO3 10% (v/v) solution overnight and washed with deionized water several times, oven dried and kept in closed bags before use. Standard solutions of Cu(II), Cd(II), Ni(II) and Cr(III) ions were prepared from the nitrates of these elements each as 1000.0 mg L-1. Working solutions, as per the experimental requirements, were freshly prepared from the stock solution for each experimental run. pH adjustments were performed with 0.01-1.0 mol L-1 HCl and NaOH solutions.Certified reference materials such as vehicle exhaust particulates (NIES-8), human hair (NIES-5), tea leaves (NIES-7) and pepperbush (NIES-1) were obtained from the National Institute for Environment Studies (NIES). Zinc base die-casting alloy C (NBS-627) were provided by the Iron and Steel Institute of Japan (Tokyo, Japan) and the National Bureau of Standards, U.S. Department of Commerce, (Washington D.C., USA), respectively. A multivitamin capsule (bearing the commercial name Maxirich) was procured from Arjang pharmacy (Hamedan, Iran) and infant milk substitute, IMS, (commercially available as Lactogen 1), spinach, tomato and hydrogenated oil were obtained from the local market, Hamedan.
The concentration of metals was determined by atomic absorption spectrometry using a Varian model Spect AA 220 apparatus. The instrumental settings of the manufacturer were followed. Infrared spectra were recorded with a Fourier transform infrared spectrometer (FT-IR, Perkin Elmer, spectrum 100). Samples were gently ground and diluted in nonabsorbent KBr matrices to identify the functional groups and chemical bonding of the coated materials. Scanning electron microscopy (SEM) was performed to measure the particle size and shape (SEM-EDX, XL30 and Philips Netherland). The crystal structure of synthesized materials was determined by an X-ray diffractometer (XRD) (38066 Riva, d/G.Via M. Misone, 11/D (TN) Italy) at ambient temperature. Surface area and porosity were defined by N2 adsorption-desorption porosimetry (77 K) using a porosimeter (Bel Japan, Inc.). A Metrohm model 713 (Herisau, Switzerland) pH-meter with a combined glass electrode was used for pH measurements.
2.2. Preparation of samples
2.2.1. Natural and sewage water samples
The water samples, river water (collected from Alvand, Hamedan, Iran), canal water (collected from Yazd, Iran), sewage water (collected from area in the vicinity of local nickel electroplating industry, Hamedan) and tap water (collected from our faculty) were immediately filtered through Millipore cellulose membrane filter (0.45 μm pore size), acidified to pH 2 ± 0.01 with HNO3, and stored in precleaned polyethylene bottles. After then, pH of the sample was adjusted to 6.0 and the procedure described in section 2.5 has been carried out.2.2.2. Digestion of standard environmental, biological and metal alloy samples
Two certified reference materials (CRMs); vehicle exhaust particulates (NIES-8), pepperbush (NIES-1) and tea leaves (NIES-7), were analyzed. Approximately 0.50 g of this material, were weighed accurately into a Teflon cup, and dissolved in concentrated nitric acid (~10 mL), with heating in a water bath. The solution was cooled, diluted and filtered. The filtrate was made to 100.0 mL, with deionized water in a calibrated flask. An aliquot of the sample solution was taken, and the target metals ions were determined by the given procedure.The sample solutions of biological CRMs such as human hair (NIES-5) was prepared as proposed by International Atomic Energy Agency [29]. A 50.0 mg of each of the samples was agitated with 25 mL of acetone, and then washed three times with deionized water and with 25 mL of acetone. The washed samples were placed in a glass beaker individually and allowed to dry at room temperature. Decomposition of organic matter is an important part for determination of heavy metals in these samples. Therefore, each of the samples was dissolved in 10 mL of concentrated nitric acid. After adding 2.5 mL of 30% H2O2 the solution was boiled to dryness. The residue obtained was dissolved in minimum amount of 2% HCl and made up to a 50 mL volume in a calibrated flask. Then the procedure given in Section 2.5 was performed.
To dissolve the standard reference alloy, zinc based die-casting alloy C (NBS-627), 25 mg of the sample was taken into a beaker and dissolved in 10-50 mL of HCl:HNO3 mixture (3:1). The solution was boiled to near dryness. Finally, the residue was dissolved in minimum volume of 2% HCl and filtered through Whatman filter paper No. 1. The residue was washed with two 5 mL portions of hot 2% HCl. The solution was evaporated to dryness. The residue was dissolved in 5 mL of 2% HCl and make up to 50 mL with deionized water after its pH was adjusted to desired value.
2.2.3. Preparation of multivitamin capsule and food samples
Five multivitamin capsules (5.83 g) were taken in a beaker containing 25 mL of concentrated HNO3 and digested by slowly increasing the temperature of the mixture to 40 ± 0.2°C. The solution was gently evaporated on a steam bath until a residue was left. It was subsequently mixed with 50 mL of deionized water and HNO3 was then added drop wise until a clear solution was obtained on gentle heating.Powdered IMS food sample (200.0 mg) was heated in a beaker containing mixture of concentrated H2SO4 (20 mL) and HNO3 (10 mL) till a clear solution was obtained. It was allowed to cool and most of the acid was neutralized with NaOH. The total volume was made up to 50 mL with deionized water and kept as stock.
Hydrogenated oil (2.00 g) was taken in a beaker and dissolved in 15 mL of concentrated nitric acid with heating. The solution was cooled, diluted and filtered. The filtrate was made up to 50 mL with deionized water after adjusting its pH to the optimum value.
A 10 g sample of tomato sample was heated in silica crucible for 3 h on a hot plate and the charred material was transferred to a furnace for overnight heating at 650°C. The residue was cooled, treated with 10.0 mL concentrated nitric acid and 3 mL 30% H2O2 and again kept in a furnace for 2 h. The final residue was treated with 3 mL concentrated hydrochloric acid and 2-4 mL of 70% perchloric acid and evaporated to fumes. The solid residue was dissolved in water, filtered and the pH was adjusted to 6.0 by the addition of NaOH and HCl solutions. The preconcentration procedure given above in section 2.5 was then applied to these solutions.
0.1 g of vegetable sample was placed in a 100 mL beaker and 10 mL of concentrated HNO3 was added. The mixture was evaporated near to dryness on a hot plate at about 150°C. After cooling to room temperature, 3 mL of concentrated hydrogen peroxide was added. The mixture was again evaporated to dryness and the residue dissolved with 0.5 mol L-1 HNO3. It was filtered through a filter paper. The preconcentration procedure was applied to this sample solution.
2.3. Preparation of silica-coated magnetite nanoparticles
The magnetite nanoparticles (MNPs) were prepared by the conventional co-precipitation method with minor modifications [30]. In this method, ultrasonic vibration by an ultrasonic bath was used instead of magnetic stirring. FeCl3.6H2O (11.68 g) and FeCl2.4H2O (4.30 g) were dissolved in 200 mL deionized water under nitrogen gas in an ultrasonic bath at 85°C for a few minutes leading to smaller and more homogenized particles. Then, 20 mL of 30% NH3 2O, which is different from the 15 mL of 20% NH3 2O used in Ref. [30], were added to the solution. The color of bulk solution changed from orange to black immediately. The magnetite precipitates were washed twice with deionized water and once with 0.02 mol L-1 sodium chloride. The washed magnetite was stored in deionized water at a concentration of 40.0 g L-1.Then, the magnetite suspension prepared above (20 mL) was placed in a 250 mL round-bottom flask and allowed to settle. The supernatant was removed, and an aqueous solution of tetraethoxysilane [TEOS, 10% (v/v), 80 mL] was added, followed by glycerol (60 mL). The pH of the suspension was adjusted to 4.6 using glacial acetic acid, and the mixture was then stirred and heated at 90°C for 2 h under a nitrogen atmosphere. After cooling to room temperature, the suspension was washed sequentially with deionized water (3 × 500 mL), methanol (3 × 500 mL), and deionized water (5 × 500 mL). The silica magnetite composite was stored in deionized water at a concentration of 40.0 g L-1.
2.4. Preparation of silica-coated magnetite nanoparticles modified with salicylic acid
25 mL of silica-coated magnetite prepared as described above was washed with ethanol (2 × 100 mL) and then diluted to 150 mL with 3.3% SA solution and 16 mmol L-1 acetic acid solution (pH 4.5). The solution was transferred to a 500 mL 3- necked round-bottom flask and then stirred and heated at 60°C for 2 h under a nitrogen atmosphere. After that, the resulting nanospheres were washed with deionized water three times and twice with methanol, then dried into powders at room temperature under vacuum (Figure 1).
Figure 1. Schematic of the preparation of adsorbent (a) and solid phase extraction of the analytes (b).2.5. Recommended procedure for sorption and desorption of heavy metal ions
A series of sample solutions containing heavy metal ions were transferred into a 1 L beaker. The pH of the solution was adjusted to 6.0 using 0.01-0.1 mol L-1 HCl and/or NaOH solutions. After that, 0.11 g of the sorbent was added to the solution and the mixtures were dispersed by ultrasonication for 10 min at room temperature to attain equilibrium, and then magnetically separated (Figure 1). Then, the sorbent was washed with deionized water and afterwards, the metal ions retained on sorbent were eluted with the solution of the mixture of 6.0 mL of 1.0 mol L-1 HNO3. The analytes in the eluate were then determined by F-AAS.3. Results and discussion
3.1. Characteristics of modified magnetite nanoparticles
The surface and textural morphology of silica coated magnetite nanoparticles by SEM image is illustrated in Figure 2. As shown in Figure 2, the naked magnetite nanoparticles had a mean diameter of 29 nm. Using the ultrasonic vibrations caused the prepared nanoparticles were smaller and more homogenized particles [30]. After modification process, the modified nanoparticles prepared are in the range of 58-73 nm in diameter. This shows that the magnetite nanoparticles have been completely coated by the silica and SA. Also, this could be attributed to the reaction occurring only on the particle surface, and thus our attempt to prepare SA-silica coated magnetite nanoparticles in this work has been achieved.
Figure 2. SEM images of synthesized magnetite nanoparticles (left) and modified magnetite nanoparticles (right).The typical X-ray diffraction (XRD) profile of silica-coated magnetite nanoparticles is shown in Figure 3. The broad peak at around 2θ = 20° in the XRD pattern is due to the amorphous silica shell on the surface of the magnetite nanoparticles. The characteristic peaks of the magnetite nanoparticles were also clearly identified in the XRD pattern for a standard magnetite pattern (Joint Committee on Powder Diffraction Standards (JCPDS) file no. 19-0629) [30,31]. Also, with comparison of peaks of silica-coated magnetite nanoparticles and silica-coated magnetite nanoparticles modified with SA concluded although the magnetic particle surfaces were coated with SA, the very distinguishable FCC peaks of magnetite crystal were observed, which means that these particles have the phase stability. The different functional groups such as hydroxide and carboxylylic did not affect on crystallinity and morphology in this study [32].
Figure 3. X-ray diffraction pattern of silica coated magnetite nanoparticles.The specific surface area was determined using the Brunauer-Emmett-Teller (BET) equation applied to the adsorption data on nitrogen adsorption/desorption experiments. The results of the BET method showed that the average specific surface area of silica-coated magnetite nanoparticles modified with SA was 41.62 m2 g-1. It can be concluded from these values that this type adsorbent is nanoparticles with relatively large specific surface area.
The adsorbent was subsequently characterized by FT-IR spectral data. The FT-IR spectrum of silica-coated magnetite nanoparticles modified with SA has prominent bands at 1680 cm-1, 1486 cm-1, and 1387 cm-1 due to carboxylate, OH (bending) and phenolic group vibrations, respectively (Figure 4). This supports the immobilizing of SA onto silica-coated magnetite nanoparticles. The red shifts of the two peaks namely hydroxyl and carboxylic by 20-25 cm-1 for metal loaded adsorbent further suggest that chelation with salicylic acid functionality is responsible for the sorption of metal ions by adsorbent. Furthermore, the adsorbent shows good chemical stability with no less of capacity up to 4.0 mol L-1 of HCl/HNO3/H2SO4 used for stripping of metal ions. It can withstand alkaline medium up to 3.0 mol L-1 of NaOH.
Figure 4. FT-IR spectra (a) salicylic acid (b) Fe3O4 nanoparticles (c) SiO2 coated Fe3O4 nanoparticles (d) SiO2 coated Fe3O4 nanoparticles modified with salicylic acid.3.2. Variables Optimization of preconcentration process
3.2.1. Effect of pH for metal ions uptake
Solution acidity can show either of two different effects on metal adsorption: protonation of binding sites of the chelating molecules, and complexation or precipitation many metal ions by hydroxide ion. Therefore, since the solution pH is an important parameter to obtain quantitative recovery for heavy metal ions, this was the first parameter that was optimized. The influence of pH on the preconcentration of target metal ions over the pH range from 2.0 to 10.0 was studied (keeping the other parameters constant). As could be seen from Figure 5, quantitative recovery was obtained for Cr(III), Cu(II), Ni(II) and Cd(II) within the pH range 5.0-7.0. This may be attributed to the presence of free lone pair of electrons on oxygen atoms, which are suitable functional sites for coordination with the metal ions. The decrease in recovery at pH values lower than 5.0 may be due to the competition of proton with cations in binding to donor atoms. Considering these facts and in order to avoid an abrupt change in adsorption (which may occur due to minor changes in the pH), and also to preconcentrate of these ions simultaneously, pH 6.0 was selected as the optimal pH for all subsequent experiments.
Figure 5. Relation between pH and recoveries of analytes (N = 3).3.2.2. Effect of contact time
The efficiencies of the analytes deposition depend on the contact time of sample with the solid phase. It is necessary to require the preconcentrate of metal ions in short time. In this regard, replicate sets of analytes and adsorbents were prepared and investigating at different time intervals 2, 5, 8, 10, 15, 20, 30 min. Results showed that the rate of uptake of the analytes was quite high (Figure 6). Adsorption of Cd(II), Cu(II), Ni(II) and Cr(III) from the solution reached more than 95% at about 8 min. Therefore, ultrasonication time of 10 min was selected for further works.
Figure 6. Effect of contact time on recovery percentage of 20 μg L-1 heavy metal ions; pH 6.0; adsorbent 0.1 g.3.2.3. Effect of the adsorbent amount
Figure 7. Recovery percentage of metal ions at different adsorbent dosage.3.2.4. Effect of sediment time
Conventional SPE usually requires filtration or centrifugation to separate the adsorbent from aqueous solutions, which makes the method time-consuming. In this study, the adsorbent could be separated rapidly from the sample solution using an external magnetic field, due to the superparamagnetism of these nanoparticles. The effect of sediment time on the recovery of metal ions was investigated, and no significant effect was seen when the sedimentation time was greater than 60 s. A sediment time of 1 min was therefore selected in subsequent experiments.3.2.5. Effect of the type, concentration and volume of the eluent
The selection of suitable eluent was a difficult problem. As could be seen from Figure 4, the uptake of these metal ions was negligible at low pH; therefore, the acidic eluent is the best solution to obtain efficient extraction. The optimal eluent was a difficult problem because of the limitation of FAAS to tolerate organic solvents while the eluent should not destroy the solid phase. Various acids were used to identify the best eluent for the adsorbed metal ions on the adsorbent. The results are given in Table 1. Deionized water was found to be unsuitable for the purpose of elution as <0.5% recovery was achieved indicating that the metal ions were retained by the adsorbent by some strong bonding forces. Among of different eluents used, 1.0 mol L-1 of HNO3 provided higher recovery and reproducibility. Therefore, this solution was chosen as an eluent for the metal ions from nano-sized adsorbent.Table 1. Effect of type and concentration eluting solution (4 mL) on analytes recovery (%)
Subsequent experiments showed that even 2.0 mL of the eluent solution was enough for elution of metal ions, however, in all experiments metal ions were eluted by 4.0 mL of 1.0 mol L-1 of HNO3 solution because this volume was necessary for reading absorption signal of analytes by F-AAS.
3.2.6. Effect of the sample volume
Due to the low concentrations of trace metals in real samples, these analytes should be taken into smaller volumes for high preconcentration factor by using sample solutions with large volumes. Therefore the maximum applicable sample volume was determined by increasing the dilution of metal ion solution, while keeping the total amount of loaded metal ion fixed for analytes. Different feed volumes varied between 50 and 1000 mL. The recoveries were found to be stable until 800 mL and were chosen as the largest sample volume to work. In this study, the final solution volume to be measured by F-AAS was 4.0 mL; therefore the preconcentration factors were 200 for all metal ions. At volumes higher than 800 mL probably the analyte ions are not sorbed effectively because of low amount of adsorbent in those volumes. As stated previously, the final solution volume, after eluting the metal ions, was 4.0 mL, therefore the preconcentration factors of 200 was obtained for all analytes.3.3. Stability and reusability of adsorbent
The reusability and stability of the adsorbent was investigated. The capacity of the modified adsorbent was found to be apparently constant (less than 3%) after the repeated use of more than 9 cycles of sorption and desorption of the target analytes.3.4. Effect of potentially interfering ions
In view of the fact that flame atomic absorption spectrometry provides high selectivity, the only interference may be attributed to the preconcentration step. For application of recommended solid phase extraction to real samples, effects of some interfering species on the recovery of metal ions were investigated with the optimized procedure. Various species, which are inevitably associated with heavy metals, may interfere in the final determination through precipitate formation, redox reactions or competing complexation reactions. In order to assess the analytical applicability of the adsorbent to real samples, common chemical species such as sodium citrate, sodium tartrate, sodium oxalate, humic acid, fulvic acid, NO3-, CO32-, NH4+, SO42-, PO43-, Cl-, K+ and Na+ were checked for any interference in the sorption of these metals. The tolerance limit is defined here as the species concentration causing a relative error smaller than ± 5% related to the preconcentration and determination of the analytes. The tolerable limits of interfering ions are given in Table 2.Table 2. Effects of the matrix ions on the recoveries of the examined metal ions.
The allowable amount of Fe(III) ions as an interfering species was lower than the other investigated species in preconcentration of cadmium. The usage of masking agent such as NH4F for this interfering species in the present method resulted in suppressing the effect of Fe(III) interference and improving the selectivity. However, the tolerance ratio for Fe(III) ion could be raised to 700 times when 2 mL of 0.25 mol L-1 NH4F solution is also added when necessary.
3.5. Adsorption capacities
The capacity of the adsorbent is an important factor because it determines how much adsorbent is required to quantitatively remove a specific amount of metal ions from the solutions. The adsorption capacity was tested following the batch procedure. 110 mg of sorbent was equilibrated with 800 mL of various concentrations of Cu(II), Ni(II), Cd(II) and Cr(III) for 1 h. In order to reach the "saturation", the initial metal ions, concentrations were increased till the plateau values (adsorption capacity values) obtained. The results showed that adsorption capacity of various metal ions probably differ due to their size, degree of hydration and the value of their binding constant with the adsorbent. The maximum adsorption capacity has been found to be 39.9, 39.8, 27.8 and 17.3 mg g-1 for Cu(II), Cr(III), Cd(II) and Ni(II), respectively.3.6. Analytical precision and detection limits
Under the selected conditions, eight portions of standard solutions were enriched and analyzed simultaneously following the general procedure. Linearity was maintained between 0.73 μg L-1 to 0.40 mg L-1 for copper; 0.91 μg L-1 to 0.35 mg L-1 for nickel; 0.44 μg L-1 to 0.64 mg L-1 for chromium and 0.37 μg L-1 to 0.45 mg L-1 for cadmium, in initial solution. The detection limit (DL) of the present work was calculated under optimal conditions after the application of preconcentration and separation procedure of blank solutions analyzed by F-AAS. The DL was calculated as DL = kSb/m, where k is equal to 3 according to the desired confidence level (95%), Sb is the standard deviation of the blank signal and m is the slope of the analytical curve (n = 8). The detection limits were found to be 0.15, 0.22, 0.27 and 0.11 μg L-1 for Cr(III), Cu(II), Ni(II) and Cd(II), respectively. The relative standard deviation (RSD) of the eight replicate determinations was lower than 4.0% (Cr(III): 3.1%; Cu(II): 2.2%; Ni(II): 3.3%; Cd(II): 2.6%), which indicated that the method had good precision for the analysis of trace target ions in solution samples.3.7. Application to real samples
Certified reference materials were analyzed by developed method. The results are given in Table 3. The results show that the results are good agreement with the certified values for the investigated analyte ions. The proposed method was applied to a various water samples. The results were given Table 4. Proposed solid phase extraction method for determination of these metals in some water samples were applied successfully. Recovery values can be quantitatively except in all samples. Multivitamin capsule, IMS and hydrogenated oil samples were investigated as samples with complex matrices. These results indicate the applicability of the developed procedure; for selective preconcentration of target analytes; and that it is free of interference (Table 5). Also, the proposed procedure has been applied to the determination of copper, nickel, chromium and cadmium content; in tomato and spinach samples. The results are given in Table 6. As can be seen from the results in Table 6 the metal ions were quantitatively recovered from the food samples; by the proposed procedure. These results demonstrate, the applicability of the procedure for target ions determination in water samples.Table 3. Results for metal ions determination in certified reference samples obtained using the optimum conditions.
Table 4. Results for metal ions determination in various water samples obtained using the optimum conditions.
Table 5. Results for metal ions determination in various samples obtained using the optimum conditions.
Table 6. Results for metal ions determination in food samples obtained using the optimum conditions.
4. Conclusion
A simple, sensitive and selective method was developed for the preconcentration of cadmium, copper, nickel and chromium in various real samples. In summery, silica coated Fe3O4 nanoparticles modified with SA with well defined diameter prepared by such a simple, time-saving and low cost route using sol-gel method combined ultrasonic stirring. These nanoparticles have relatively high adsorption as compared to the similar materials because of their smaller size. The size of the produced modified maghemite nanoparticles was determined by X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM). The present method has following advantages over reported methods. Synthesized adsorbent is distinct in terms of sensitivity, selectivity towards investigated metal ions. Also, these magnetic nanoparticles carrying the target metals could be easily separated from the aqueous solution simply by applying an external magnetic field; no filtration or centrifugation was necessary. Furthermore, the proposed method gives an efficient and cost effective method with very low detection limits and good relative standard deviation and can be applied to the determination of traces of these ions in various real samples.Competing interests
The authors declare that they have no competing interests.Authors' contributions
HB carried out the synthesis of adsorbent and performed the some of experimental AA carried out the survey of prepared adsorbent, participated in the design of the study. sections. MRS performed the experimental sections, participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.Acknowledgements
The authors gratefully acknowledge the financial and technical support provided by the Research Council of Islamic Azad University, Yazd Branch.References
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THE END
ARTICLE-04
Amorphous calcium phosphate and its application in dentistry
1. Review
Amorphous calcium phosphate (ACP) is the initial solid phase that precipitates from a highly supersaturated calcium phosphate solution, and can convert readily to stable crystalline phases such as octacalcium phosphate or apatitic products. Its morphological form, structural model and X-ray diffraction patterns are typical for noncrystalline substances with short-range periodic regularity. ACP has been demonstrated to have better in vivo osteoconductivity than hydroxyapatite (HAP), better biodegradability than tricalcium phosphate, good bioactivity but no cytotoxicity [1]. These excellent biological properties make ACP widely used in dentistry, orthopaedics and medicine. This review provides an overview of the development, structure, composition and morphology characterization, phase transformation and biomedical application of ACP in dentistry.2. Development
Generally, it is believed that ACP was firstly described by Aaron S. Posner [1] in the mid 1960s. It was obtained as an amorphous precipitate by accident when mixing high concentrations (30 mM) of calcium chloride and sodium acid phosphate (20 mM) in buffer [2]. In X-ray diffraction, it was shown to have only two broad and diffuse peaks, with maximum at 25° 2θ. No other features were obvious and it was clearly not apatite. This pattern is typical for substances that lack long range periodic regularity. It was found that immediately after being mixed, the spontaneously formed precipitate was a non-crystalline, or amorphous, calcium phosphate with calcium to phosphorus molar ratio (Ca/P) of 1.50. After several hours, it could convert to poorly crystalline apatite on ageing. Afterwards, this solid converts slowly to crystalline apatite (Ca/P = 1.67) by an autocatalytic mechanism [3].In 1965, Eanes et al. identified ACP as a bone component [2]. ACP in bone, along with the apatite, might account for the broad diffraction pattern and variable composition of bone minerals. An age-dependent change in the ACP content of bone was also described, with the proportion of ACP decreasing with age [3,4]. In 1975, ACP was found in the mineralized cytoplasmic structure isolated from the blue crab hepatopancreas, with a very similar short-range atomic structure to synthetic amorphous calcium phosphate [5].
3. Structure
After the discovery of amorphous calcium phosphate, the early studies were focused on the structure of ACP. It was suggested that synthetic ACP particles, which appear as 300- 1000 Å spheres in the electron microscope, consist of a random assembly of ion clusters 9.5 Å in diameter, dimensions consistent with the chemical composition of Ca9(PO4)6 [5]. And the 15-20% of water found in synthetic amorphous calcium phosphate was shown to be mostly in the interstices between, and not within, the individual Ca9(PO4)6 clusters [6]. Aggregated ACP particles readily dissolve and crystallize to form apatite, a thermodynamically stable phase. The typical radial distribution of noncrystalline ACP cluster structures, calculated from the x-ray diffraction patterns, is only two broad and diffuse peaks showing the rapid drop-off of atomic periodicity. Short-range order exists in these amorphous structures but no long-range order such as that in crystalline hydroxyapatite [6]. Infrared analysis showed a similar lack of crystalline order about the PO4 anions in the ACP structure [7].It is now generally agreed that, both in vitro and in vivo, precipitation reactions at sufficiently high supersaturation and pH result in the initial formation of an amorphous calcium phosphate with a molar calcium/phosphate ratio of about 1.5, with a range of 1.34-1.50 in different pH and 1.50-1.67 when adding different amount of carbonates [8]. However, Wuthier et al reported that ACP, with Ca/PO4 molar ratio as low as 1.15, precipitated at more acidic preparative pHs, i.e.6.9 [9].
More importantly, it has been shown that ACP particles are nanometer particles. Primary particle sizes of ACP is about 40-100 nm. The morphology of ACP solids appears to be a curvilinear shape when viewed by TEM, rather than the faceted, angular shape of crystalline calcium phosphates. However, this curvilinear appearance has only been clearly established with dried ACP [10]. The initial flocculates collected immediately after precipitation of highly hydrated ACP have a low-contrast disk-shaped appearance. High-contrast spherical particles begin to appear as ACP suspensions age, and become the dominant shape with time [11].
The disordered structure makes ACP highly reactive with body fluid, resulting fast apatite reprecipitation. Accordingly, ACP has been evidenced to have better in vivo osteoconductivity than hydroxyapatite and better biodegradability than tricalcium phosphate [10]. The ACP precipitate, with little long-range order, is a highly unstable phase and hydrolyzes almost instantaneously to more stable phases. In the presence of other ions or under in vivo conditions, ACP may persist for appreciable periods due to kinetic stabilization [12]. Although the exact mechanism of stabilization of ACP is not understood, the presence of Mg2+, F-, carbonate, pyrophosphate, diphosphonates, or polyphosphorylated metabolites or nucleotides, in sufficient quantity will prevent the transformation of synthetic ACP to hydroxyapatite [13,14].
4. ACP in Biomineralization
It has been stated that ACP likely plays a special role as a precursor to bioapatite and as a transient phase in biomineralization [15]. In solutions, ACP is readily converted to stable crystalline phases such as octacalcium phosphate or apatitic products. One biomineralization strategy that has received significant attention in recent years is mineralization via transient precursor phases [16]. Transient amorphous mineral phases have been detected in biomineral systems in different phyla of the animal kingdom [17]. ACP has been previously reported in the otoliths of blue sharks and also shown to form as a precursor phase of carbonated hydroxyapatite in chiton teeth [18]. The presence of an abundant ACP phase has also been demonstrated in the newly formed zebrafish fin bony rays [19]. The disordered phase is a precursor of crystalline carbonated hydroxyapatite. It was found that the initially extracted amorphous mineral particles transformed into a crystalline mineral phase with time, and the proportion of crystalline mineral increased during bone maturation [19]. The transient ACP phase may conceivably be deposited directly inside the gap regions of collagen fibrils, but it may also be delivered as extrafibrillar particles [19]. This is consistent with the study that collagen mineralization via a transient ACP precursor phase in vitro to produce aligned intrafibrillar carbonated apatite crystals [20].Several studies in different systems in vivo also have reported the presence of transient precursor calcium phosphate phases in the deposition of carbonated hydroxyapatite. Beniash performed a comprehensive analysis of the mineral phases in the early secretory enamel of the mandibular mouse incisors using four physical characterization methods. It was suggested that the outer, younger, early secretory enamel contained a transient disordered ACP phase, which transformed with time into the final apatitic crystalline mineral [17].
A variety of proteins and ions have been proposed to be involved in the biomineralization of ACP to HAP [21,22]. Dentin matrix protein1 (DMP1) is one of such biomineralization proteins [23]. In the report of He, it has been shown that two peptide motifs identified in DMP1 [motif-A (ESQES) and motif-B (QESQSEQDS)] enhanced in vitro HAP formation when immobilized on a glass substrate. It was demonstrated in another study that the synthesized artificial protein composed of these peptide motifs of DMP1 facilitated reorganization of the internal structure of amorphous particles into ordered crystalline states, i.e., the direct transformation of ACP to HAP, thereby acting as a nucleus for precipitation of crystalline calcium phosphate [24].
5. Transformation to Octacalcium Phosphate and Apatite
Studies on the preparation of hydroxyapatite [Ca10(PO4)6-(OH)2], the synthetic prototype of bone mineral, showed that the precipitation of initial solid phase from a calcium phosphate solution depends on the degree of its supersaturation [8]. A noncrystalline ACP precursor, approximating Ca9(PO4)6 in composition forms under conditions of high supersaturation [1,15]. This precursor ACP, unless stabilized in some way, transforms to thermodynamically more stable calcium phosphate phases or will be taken place by an autocatalytic solution-mediate crystallization process. On the other hand, the first solid to form in low supersaturated solutions is hydroxyapatite with Ca/P ratio of 1.67 obtained without precursor phases. Therefore, ACP is considered as a "mandatory precursor to apatite", and apatite can be formed in dilute solutions without going through this precursor [15]. The pH value also affects the initial solid phase in the precipitation of calcium and phosphate ions. Octacalcium phosphate (OCP) is the crystalline phase that initially forms when the reaction pH is less than 9.25, whereas apatite preferentially forms at higher pHs [25]. It is known that ACP is often the first-formed deposit in vitro, at neutral pH and moderate supersaturation [26]. Transformation mechanism of ACP to apatite at physiological pH has been described as followings: firstly ACP dissolution, then a transient OCP solid phase reprecipitation through nucleation growth, and finally hydrolysis of the transient OCP phase into the thermodynamically more stable apatite by a topotactic reaction, which usually takes tens of hours [26].Based on the analysis of the measured precipitate induction time and the structure of the developing solid phase, Feenstra proposed that OCP might be an intermediate in the conversion of ACP to apatitic calcium phosphate [27]. Since OCP or apatite crystals are generally found in association with ACP spherules, it is possible that ACP acts as a template for the growth of these crystal phases. Their formation, however, appears to take place by consuming ions largely supplied from the surrounding solution, rather than from direct hydrolysis of the solid amorphous material. At pH 10, transformation of ACP to poorly crystalline HAP may proceed without changes in the local calcium environment, but with the development of longer range order in the structure.
However, in contrast to these results at pH = 10, under physiological conditions the picture is quite different. Tung used a titration method to study the conversion of high-concentration ACP slurry to an apatite. There was a typical conversion kinetics clearly indicating two processes: the first process consumes acid, with the conversion of ACP to an OCP-like intermediary and the second process consumes base with the conversion of the OCP-like intermediate to apatite or, possibly, direct conversion of ACP to apatite. It was proposed that a stoichiometric HAP could be formed when there is no OCP-like intermediate phase, and a nonstoichiometric apatite product could be formed when an OCP-like intermediate phase is involved [28].
6. Biomedical and Dental Applications
ACP has been widely applied in biomedical field due to its excellent bioactivity, high cell adhesion, adjustable biodegradation rate and good osteoconduction [29-32]. As discussed above, the first quantitative studies on synthetic ACP were done in the mid 1960s [1]. From then on, more and more attention has been attracted in the development and the application of ACP-containing products, especially in orthopedic and dental fields. It is also used as filler in ionomer cements to fill carious lesions or as a colloidal suspension in toothpastes, chewing gums or mouthwashes to promote demineralization of carious lesions and/or to prevent tooth demineralization.6.1 CPP-ACP
Casein phosphopeptides (CPP) contain the cluster sequence of -Ser (P)-Ser (P)-Ser (P)-Glu-Glu from casein [33,34]. Through these multiple phosphoseryl residues, CPP has a remarkable ability to stabilize clusters of ACP into CPP-ACP complexes, preventing their growth to the critical size required for nucleation, phase transformation and precipitation. In the United States, up to now, this product is primarily used for abrasive prophylaxis pastes and secondarily used for the treatment of tooth sensitivity especially after in-office bleaching procedures, ultrasonic scaling, hand scaling or root planing. However, its use for remineralizing dentin and enamel and preventing dental caries is an off-label application. Outside the United States, this product is marketed as GC Tooth Mousse [35,36].A clinical trial of a mouthwash containing CPP-ACP showed that the contents of calcium and inorganic phosphate in supragingival plaque increased after use of the mouthwash for a three-day period [37]. Rose measured the affinity of Streptococcus mutans to CPP-ACP. It was demonstrated that CPP-ACP bound with about twice the affinity to the bacterial cells [38]. Hence, CPP-ACP binds well to plaque, providing a large calcium reservoir within plaque and slowing diffusion of free calcium. Additional evidence reported also by Rose indicates that CPP-ACP would compete with calcium for plaque Ca binding sites. As a result, this will reduce the amount of calcium bridging between the pellicle and adhering bacterial cells and between bacterial cells themselves [39]. This is likely to restrict mineral loss during a cariogenic episode and provide a potential source of calcium for the inhibition of demineralization and assist in subsequent remineralization.
A human in situ caries model has been used by Reynolds to study the ability of 1.0% CPP, 60-mM CaCl2 and 36-mM sodium phosphate, pH 7.0, solution to prevent enamel demineralization [40]. Two exposures of CPP-ACP solution per day to one side of the enamel slabs produced 51 ± 19% reduction in enamel mineral loss compared to the control side. Plaque exposed to CPP-ACP had 2.5 times more Ca and phosphorus than control plaque [36]. Reynolds also used an in vitro model system to study the effects of CPP-ACP solutions on remineralization of artificial lesions in human third molars. After a ten-day remineralization period, all solutions deposited mineral into the bodies of the lesions, with 1.0% CPP-ACP (pH 7.0) solution replacing 63.9 ± 20.1% of mineral lost at an averaged rate of 3.9 ± 0.8 × 10-8 mol hydroxyapatite/m2/s. The remineralizing capacity was greater in the solutions with higher levels of CPP-stabilized free calcium and phosphate ions [41].
CPP-ACP and fluoride were shown to have additive effects in reducing caries experience [42]. Thus CPP-ACFP would add into the current fluoride-containing dentifrices as a toothpaste additive to improve the efficacy. Recent studies indicate that CPP-ACP can be incorporated into confectionery and drinks without adverse organoleptic effects [43]. CPP-ACP is a natural derivative of milk, therefore could have an important role as a food additive for the prevention of dental caries [44]. However, in 2008 Azarpazhooh systemically reviewed 98 articles on the clinical efficacy of casein derivatives and concluded that there was insufficient evidence (in quantity, quality or both) in existing clinical trials to make a recommendation regarding the long-term effectiveness of casein derivatives, specifically CPP-ACP, in preventing caries in vivo and treating dentin hypersensitivity or dry mouth [34].
6.2 ACP-filled polymeric composites
ACP has been evaluated as a filler phase in bioactive polymeric composites [45]. Skrtic has developed unique biologically active restorative materials containing ACP as filler encapsulated in a polymer binder, which may stimulate the repair of tooth structure because of releasing significant amounts of calcium and phosphate ions in a sustained manner [46-49]. In addition to excellent biocompatibility, the ACP-containing composites release calcium and phosphate ions into saliva milieus, especially in the oral environment caused by bacterial plaque or acidic foods. Then these ions can be deposited into tooth structures as apatitic mineral, which is similar to the hydroxyapatite found naturally in teeth and bone [50,51].However, it was reported that the orthodontic ACP-containing adhesive showed lower bond strength. Dunn conducted an in vitro study to compare ACP-containing vs. conventional resin-based orthodontic adhesives [52]. Foster also compared the shear bond strength of orthodontic brackets using ACP-containing adhesive with a conventional adhesive and a resin-modified glass ionomer. In both studies, ACP-containing adhesive was demonstrated with lower, but clinically satisfactory bond strength as an orthodontic adhesive [53]. When comparing four new ACP-containing bonding systems, including Aegis Ortho, with a conventional bracket bonding system (Transbond XT), it was found that the traditional bonding systems achieved greater bond strengths than the newer ACP-containing ones. According to the study, however, Aegis Ortho had bond strengths sufficient for orthodontic use at 24-hour post-cure time. But the bracket might drift because of low viscosity of the material during laboratory bonding. The authors also found that Aegis Ortho had lower flexural strength, which would explain for the material failure at the adhesive-bracket interface rather than the enamel adhesive interface [54].
Compared with more commonly used silanated glass or ceramic filler, more hydrophilic and biodegradable ACP-filled composites exhibited inferior mechanical properties, durability and water sorption characteristics [55]. The uncontrolled aggregation of ACP particulates along with poor interfacial interaction plays a key role in adversely affecting their mechanical properties [56]. Their clinical applicability may be compromised by relatively poor filler/matrix interfacial adhesion and also by excessive water sorption that occurs in both resin and filler phases of these composites [42,46].
However, it has been demonstrated that it is possible to improve the remineralizing potential of ACP composites by introducing Si or Zr elements during low-temperature synthesis of the filler. Si- and Zr- ACPs enhanced the duration of mineral ion release through their ability to slow down the intra-composite ACP to HAP conversion [57]. It was also possible that non-ionic and anionic surfactants and poly (ethylene oxide) (PEO) introduced during the preparation of ACP play a role on the particle size distribution and compositional properties of ACP fillers [58]. The hydrophilic PEO is widely used in water compatible polymer systems because of its proven ability to undergo multiple hydrogen bonding interactions and stabilize cations by multiple chelation. The incorporated PEO in ACP fillers would also be expected to affect ACP's tendency to form aggregates and the water content of the ACP-containing composites. These properties would eventually affect both ion release kinetics and mechanical stability of composites [59]. It was found that surfactants introduced during the precipitation of ACP stabilized the amorphous solid phase against the conversion to apatite. The particle size of ACP was moderately reduced because of the introduction of anionic surfactant. Addition of PEO resulted in more pronounced ACP agglomeration but no changes of ACP's water content. Both surfactants and PEO lead to no changes in dry biaxial flexure strength of composites compared to the control Zr-ACP composites. However, their strength was drastically reduced in contrast to the control after prolonged exposure to aqueous milieu.
6.3 ACP in bone repair materials
Various compounds from calcium phosphate family have been extensively investigated as hard tissue repair materials due to their excellent biocompatibility [60]. It has been shown that the rate of new bone formation coincides more closely with the resorption rate of poorly crystalline apatites and ACP [61]. Additionally, ACP showed better osteoconductivity in vivo than apatite and its biodegradability was higher than that of tricalcium phosphate [25].Clinically, it is widely accepted to use autograft and allograft materials to repair bone defects [29]. Recently, materials with ACP, hydroxyapatite and other calcium phosphate family members have been extensively investigated for alternative bone repair due to the limitations of traditional materials such as potential immunogenicity, insufficient supply and so on [62,63]. ACP and ACP/biopolymer composites have emerged as a new class of bone tissue engineering scaffold materials. Their excellent biocompatibility and osteoconductibility make them great materials for bone substitution and repair.
It has been shown that bone-like apatite materials have optimal surface characteristics for osteoblast cells to adhere, proliferate and differentiate, as a result, to favor bone formation and regeneration. An amorphous carbonated calcium phosphate ceramic was encapsulated within bioresorbable PLAGA microspheres and sintered to form a bioresorbable, highly porous, 3-dimensional scaffold. These noncrystalline and carbonated materials may be ideal for tissue ingrowth and potentially suitable for bone repair applications [64].
ACP was also incorporated into porous poly (L-lactic acid) (PLLA) to create a desired pore wall surface within bone tissue engineering scaffolds [65]. After being soaked in PBS, ACP aggregates in the composite experienced a fast and in situ transformation into bone-like apatite. The cell culture results also demonstrated that ACP/PLLA composite had an enhancement in cytocompatibility [65]. It has been demonstrated that ACP/PLLA material, which can experience morphological variations in the microstructure is also supposed to be a suitable candidate as scaffold for cartilage tissue engineering [63,65].
7. Conclusions
ACP is usually formed as a metastable phase when calcium and phosphate ions in aqueous solution react to precipitate. The x-ray diffraction pattern, structure, morphology and infrared analysis results of ACP solids show typical noncrystalline characters within short-range order, instead of long-range periodic regularity. ACP act as an important intermediate product for in vitro and in vivo apatite formation. A variety of proteins and ions can increase the stability of ACP. ACP becomes increasingly significant in orthopedics and dentistry because of their excellent biocompatibility and mechanical properties. It is believed that ACP will be used even more extensively in the future due to due to the fast development of tissue engineering techniques and applied material science.Competing interests
The authors declare that they have no competing interestsAuthors' contributions
JZ, YL, WS and HZ have all been involved in drafting this review and have given final approval of the version to be published.Acknowledgements and Funding
WS would like to thank Worldwide Universities Network Development Fund (No.201001168) and The Natural Scientific Fund of Jiangsu (No. BK2010118)References
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THE END
ARTICLE-05
Simple isatin derivatives as free radical scavengers: Synthesis, biological evaluation and structure-activity relationship
Isatin is an endogenous indole present in mammalian tissues and fluids [11]. The substance was initially discovered as a component of endogenous monoamine oxidase (MAO) inhibitory activity, tribulin, and subsequently identified as a selective inhibitor of MAO B [12]. Further investigations have shown that isatin acts as an antagonist of both atrial natriuretic peptidestimulated and nitric oxide-stimulated guanylate cyclase activity [13-15]. Isatin has a distinct and discontinuous distribution in rat brain and other tissues; the highest concentrations in the brain are found in the hippocampus and cerebellum [10]. Many Isatin derivatives, such as isatin hydrazono, isatin Mannich bases, isatin based spiroazetidinones and 3-(methylene)indolin-2-ones, have also been reported to possess neuroprotection activity [16-19].
To develop more potent small molecules with enhanced free radical scavenger properties, a series of N-substituted isatin derivatives was synthesized by substitution reactions (as shown in Scheme 1), and the cytoprotective effect on the apoptosis of PC12 cells induced by H2O2 was screened.
Scheme 1
Synthesis of N-substituted isatin derivatives.
Results and Discussion
Chemistry
The N-substituted isatin derivatives were synthesized by reactions of substitution reaction. The reaction between isatin and halohydrocarbon has been reported being carried out in the presence of NaOEt using EtOH as solvent or in the presence of NaH using DMF as solvent [16]. The reactants and the solvents involved in the reactions must be anhydrous. To develop a simple method to synthesize N-substituted isatin derivatives, we firstly screened the effect of the base and solvent on the yield of the reaction of isatin and bromoethane (C2H5Br), and the results was shown in Table 1.Table 1. The substitution reaction between isatin and bromoethane
In this reaction, the protons transfers from N-H (a Brösted acid) to a Brösted or Lewis base via the hydrogen-bonded covalent and ionic complexes [20], producing the isatin anion which is the nucleophilic reactant to the halohydrocarbon. Higher solvent polarity can promote the proton-transfer equilibrium and leads to the higher yield [20]. From this table, it can be found that K2CO3-DMF system was an effective promotion for this reaction and other base-solvent systems were not effective with the yield no more than 60%. The possible reason might be that weak base can not help the proton transfer at the beginning effectively, but the too strong bases will lead to the substitution reaction between bromoethane and OH-. DMF exhibits the highest yield of 89% with K2CO3 for its highest solvent polarity, so the K2CO3-DMF was selected as the reactant reaction system in the following synthesis, and the results were shown in Table 2.
Table 2. Synthesis of N-substituted isatin derivatives
Bioactivity
The chemical modification of lead compound 1, focusing on the N-substituent, was carried out to further improve the free scavenging ability. A series of new N-substituted isatin derivatives (compounds 2-12) was synthesized. The free radical scavenging properties of these derivatives were evaluated to elucidate structure-activity relationships. The protective effect on the apoptosis of PC12 cells induced by H2O2 by free radical scavenging of these compounds against H2O2 were evaluated by cell survival assay in PC12 cells using a reported method [21]. The results were given in Table 3.Table 3. Inhibitory and protective effects of N-substituted isatin derivatives
From the table, we can find almost all of the compounds showed potent activity at the condensation of 2 μg/ml, which were more effective than VE ((±) α-Tocophreol with the percentage of 22.5%). There is a noteworthy phenomenon that the activities of all compounds at the condensation of 2 μg/ml are more potent than that at the condensation of 20 μg/ml, and the mechanism will be interesting for the further investigation. Compound 3 and 8 exhibited the most potent activity with the protective effect of 69.8% and 69.5% at the condensation of 2 μg/ml respectively, which are more potent than that at the condensation of 20 μg/ml.
Almost all of these compounds were weakly cytotoxic to PC12 cells at the concentrations of 2-20 μg/ml except compound 11 and 12. Almost all compounds are cytotoxic to PC12 cells at the concentrations of 200 μg/ml, the PC12 cells inhibitory effects are more than 40%. Based on the factors, we can conclude the addition of halogenous atom in the substituents (compound 11 and 12) enhance the cytotoxicity at the concentrations of 2-20 μg/ml.
The substitution reaction between isatin and halohydrocarbon (C1 to C6) gave compounds 2-7, which provided the appropriate material for the structure-activity relationship analyses. The cytoprotective activities of N-substituted isatin derivatives with the alkyl group containing one to six carbon atoms were shown in Figure 1. The activity approximately declines as the increase of the chain of the alkyl group. With a further analysis, it was found that there was a clear odd-even effect in these activities. The activities of N-substituted isatin derivatives with odd carbon atoms alkyl group (one, three and five carbon atoms, corresponding compound 2, 4 and 6, marked with solid pillars in Figure 1) decline as the chain of the alkyl group increases, and the same regulation can be found in the activities of the N-substituted isatin derivatives with even carbon atoms alkyl group (two, four and six carbon atoms, corresponding compound 3, 5 and 7, marked with virtual pillars in Figure 1). This regulation exhibits both under the condensation of 2 μg/ml and 20 μg/ml, and the activities of N-substituted isatin derivatives with even carbon atoms alkyl group are more potent than the that of N-substituted isatin derivatives with parallel odd carbon atoms alkyl group. Besides, by the structure-activity relationship analyses, it was found that the unsaturated bond of the substituent (compound 8-10) can improve the activity compared with the other substituents with similar carbon atoms.
Figure 1. The cytoprotective activities of N-substituted isatin derivatives with the alkyl group containing 1-6 carbon atoms (The corresponding compounds are compounds 2-7.).Experimental
All starting materials and solvents (A.R. grade) were commercially available and were used without further purification. NMR spectra were recorded using a Bruker Drx-400 spectrometer operating at 400 MHz for 1H. Mass spectra were recorded on a Micromass Platform spectrometer using a direct-inlet system operating in the electron impact (EI) mode at 75 eV. Elemental analyses were obtained using a Carlo Erba 1106 elemental analyzer.General synthesis of N-alkyl substituted isatin derivatives
Isatin (1 mmol) and halohydrocarbon (1.2 mmol) were dissolved in DMF (20 ml), and 3 mmol anhydrous K2CO3 was added. The mixture was stirred under room temperature until the disappearance of isatin, as evidenced by thin-layer chromatography. The solvent was removed in vacuo and the residue was separated by column chromatography (silica gel, petroleum ether/ethyl acetate = 20:1), giving N-alkyl substituted isatin compound (compound 2-12).1-Methylindoline-2,3-dione (Compound 2)
1H-NMR (D6-DMSO, 400 MHz): 7.66 (1 H, td, J = 1.2, 7.6 Hz), 7.52 (1 H, d, J = 7.6 Hz), 7.12 (2 H, t, J = 7.6 Hz), 3.12 (3 H, s); MS (EI) m/z: 161 (M+); Anal. Found: C, 67.01; H, 4.40; N, 8.66 (%). Calc. for (C9H7NO2): C, 67.07; H, 4.38; N, 8.69 (%).1-Ethylindoline-2,3-dione (Compound 3)
1H-NMR (CDCl3, 400 MHz): 7.57 (2 H, m), 7.09 (1 H, t, J = 7.6 Hz), 6.89 (1 H, d, J = 7.6 Hz), 3.76 (2 H, q, J = 7.6 Hz), 1.29 (3 H, t, J = 7.6 Hz); MS (EI) m/z: 175 (M+); Anal. Found: C, 68.59; H, 5.22; N, 8.01 (%). Calc. for (C10H9NO2): C, 68.56; H, 5.18; N, 8.00 (%).1-Propylindoline-2,3-dione (Compound 4)
1H-NMR (D6-DMSO, 400 MHz): 7.58 (1 H, d, J = 6.8 Hz), 7.55 (1 H, t, J = 7.6 Hz), 7.09 (1 H, t, J = 7.6 Hz), 6.88 (1 H, d, J = 8 Hz), 3.67 (2 H, t, J = 7.2 Hz), 1.72 (2 H, m, J = 7.2-7.6 Hz), 0.98 (3 H, t, J = 7.6 Hz); MS (EI) m/z: 189 (M+); Anal. Found: C, 69.88; H, 5.89; N, 7.35 (%). Calc. for (C11H11NO2): C, 69.83; H, 5.86; N, 7.40 (%).1-Butylindoline-2,3-dione (Compound 5)
1H-NMR (CDCl3, 400 MHz) δ: 7.60 (2 H, m), 7.12 (1 H, t, J = 7.6 Hz), 6.91 (1 H, d, J = 8.4 Hz), 3.73 (2 H, t, J = 7.6 Hz), 1.69 (2 H, m), 1.42 (2 H, m), 0.98 (3 H, t, J = 7.2 Hz); MS (EI) m/z: 203 (M+); Anal. Found: C, 70.90; H, 6.59; N, 6.90 (%). Calc. for (C12H13NO2): C, 70.92; H, 6.54; N, 6.89 (%).1-Pentylindoline-2,3-dione (Compound 6)
1H-NMR (CDCl3, 400 MHz) δ: 7.60 (2 H, m), 7.12 (1 H, t, J = 7.6 Hz), 6.91 (1 H, d, J = 8.0 Hz), 3.72 (2 H, t, J = 7.6 Hz), 1.71 (2 H, m), 1.37 (4 H, m), 0.91 (3 H, t, J = 6.8 Hz); MS (EI) m/z: 217 (M+); Anal. Found: C, 71.88; H, 7.00; N, 6.44 (%). Calc. for (C13H15NO2): C, 71.87; H, 6.96; N, 6.45 (%).1-Hexylindoline-2,3-dione (Compound 7)
1H-NMR (CDCl3, 400 MHz) δ: 7.60 (2 H, m), 7.11 (1 H, t, J = 7.6 Hz), 6.90 (1 H, d, J = 7.6 Hz), 3.72 (2 H, t, J = 7.6 Hz), 1.70 (2 H, m), 1.31-1.38 (6 H, m), 0.89 (3 H, t, J = 6.4 Hz); MS (EI) m/z: 231 (M+); Anal. Found: C, 72.72; H, 7.40; N, 6.01 (%). Calc. for (C14H17NO2): C, 72.70; H, 7.41; N, 6.06 (%).1-Allylindoline-2,3-dione (Compound 8)
1H-NMR (D6-DMSO, 400 MHz): 7. 63 (1 H, t, J = 7.6 Hz), 7.55 (1 H, d, J = 7.2 Hz), 7.12 (1 H, t, J = 7.6 Hz), 7.04 (1 H, d, J = 7.6 Hz), 5.84 (1 H, m, J = 5.2-5.6 Hz), 5.32 (1 H, d, J = 17.2 Hz), 5.18 (1 H, d, J = 10.4 Hz), 4.30 (2 H, d, J = 4.8 Hz); MS (EI) m/z: 187 (M+); Anal. Found: C, 70.60; H, 4.84; N, 7.49 (%). Calc. for (C11H9NO2): C, 70.58; H, 4.85; N, 7.48 (%).1-Benzylindoline-2,3-dione (Compound 9)
1H-NMR (D6-DMSO, 400 MHz) δ: 7.56 (2 H, m), 7.42 (2 H, d, J = 7.6 Hz), 7.30 (2 H, t, J = 7.6 Hz), 7.27 (1 H, m), 7.10 (1 H, t, J = 7.6 Hz), 6.96 (1 H, m), 4.90 (2 H, s); MS (EI) m/z: 233 (M+); Anal. Found: C, 75.99; H, 4.65; N, 5.92 (%). Calc. for (C15H11NO2): C, 75.94; H, 4.67; N, 5.90 (%).Ethyl 2-(2,3-dioxoindolin-1-yl)acetate (Compound 10)
1H-NMR (CDCl3, 400 MHz) δ: 7.62 (1 H, d, J = 7.6 Hz), 7.57 (1 H, t, J = 7.6 Hz), 7.14 (1 H, t, J = 7.6 Hz), 6.77 (1 H, d, J = 7.6 Hz), 4.47 (2 H, s), 4.22 (2 H, q, J = 7.2 Hz), 1.26 (3 H, t, J = 7.2 Hz); MS (EI) m/z: 233 (M+); Anal. Found: C, 61.84; H, 4.72; N, 6.00 (%). Calc. for (C12H11NO4): C, 61.80; H, 4.75; N, 6.01 (%).1-(2-Chloroethyl)indoline-2,3-dione (Compound 11)
1H-NMR (D6-DMSO, 400 MHz) δ: 7.67 (1 H, td, J = 8, 1.2 Hz), 7.56 (1 H, dd, J = 7.6, 1.2 Hz), 7.29 (1 H, d, J = 8.0 Hz), 7.14 (1 H, dd, J = 7.6, 0.8 Hz), 4.10 (2 H, t, J = 6.4 Hz), 3.70 (2 H, t, J = 6.4 Hz); MS (EI) m/z: 211 (M+); Anal. Found: C, 58.86; H, 3.99; N, 13.70 (%). Calc. for (C10H8ClNO2): C, 58.82; H, 3.95; N, 13.72 (%).1-(2-Bromoethyl)indoline-2,3-dione (Compound 12)
1H-NMR (D6-DMSO, 400 MHz) δ: 7.67 (1 H, td, J = 8, 1.2 Hz), 7.57 (1 H, dd, J = 7.6, 1.2 Hz), 7.29 (1 H, d, J = 8.0 Hz), 7.14 (1 H, dd, J = 7.6, 0.8 Hz), 4.11 (2 H, t, J = 6.4 Hz), 3.71 (2 H, t, J = 6.4 Hz); MS (EI) m/z: 254 (M+); Anal. Found: C, 47.31; H, 3.19; N, 5.50 (%). Calc. for (C10H8BrNO2): C, 47.27; H, 3.17; N, 5.51 (%).Competing interests
The authors declare that they have no competing interests.Authors' contributions
GC has formulated the research idea and prepared the manuscript draft version, YW prepared the manuscript for submission and coordinated further formalities, SM and QS carried out the chemical and biological studies, XH conceived of the study, participated in its design and coordination. All authors have read and approved the final manuscript.Acknowledgements
This work was financially supported by the grant from Scientific Research Program Funded by Shaanxi Provincial Education Department (Program No.11JK0560).References
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