Colorimetric determination of amino acids using genipin from Gardenia jasminoides

Genipin, a hydrolysate of geniposide from gardenia fruits, produces blue pigments on reaction with amino acids. The colorimetric detection of amino acids using this genipin reaction was evaluated and compared with the well-known ninhydrin reaction. The molar absorptivities of the blue pigments, the reaction products of genipin with various amino acids, were greater than those of the respective ninhydrin reaction products. When asparagine was reacted with genipin, the molar absorptivity was about 14 times higher than with ninhydrin. The absorbance of the genipin–amino acids increased linearly with increase of amino acid concentration, indicating that genipin could be a useful reagent for quantitation of amino acids. Thin layer chromatographic analysis showed that the genipin reaction produces clear and stable colored spots. The blue ninhydrin reaction spots were usually bleached in 24 h at room temperature, while the genipin reaction spots remained unchanged for several months. The addition of 0.1 mM Cu²⁺ and Fe³⁺ decreased the absorbance of Gly–ninhydrin pigment by 50% and 98%, respectively, but those metal ions did not affect the absorbance of the Gly–genipin pigment.     

Characterization of ring‐opening polymerization of genipin and pH‐dependent cross‐linking reactions between chitosan and genipin

In this study, a novel chitosan‐based polymeric network was synthesized by crosslinking with a naturally occurring crosslinking agent—genipin. The results showed that the crosslinking reactions were pH‐dependent. Under basic conditions, genipin underwent a ring‐opening polymerization prior to crosslinking with chitosan. The crosslink bridges consisted of polymerized genipin macromers or oligomers (7 ～ 88 monomer units). This ring‐opening polymerization of genipin was initiated by extracting proton from the hydroxyl groups at C‐1 of deoxyloganin aglycone, followed by opening the dihydropyran ring to conduct an aldol condensation. At neutral and acidic conditions, genipin reacted with primary amino groups on chitosan to form heterocyclic amines. The heterocyclic amines were further associated to form crosslinked networks with short chains of dimmer, trimer, and tetramer bridges. An accompanied reaction of nucleophilic substitution of the ester group on genipin by the primary amine group on chitosan would occur in the presence of an acid catalysis. The extent in which chitosan gels crosslinked with genipin was significantly dependent on the crosslinking pH values: 39.9 ± 3.8% at pH 5.0, 96.0 ± 1.9% at pH 7.4, 45.4 ± 1.8% at pH 9.0, and 1.4 ± 1.0% at pH 13.6 (n = 5, p < 0.05). Owing to the different crosslinking extents and different chain lengths of crosslink bridges, the genipin‐crosslinked chitosan gels showed significant difference in their swelling capability and their resistance against enzymatic hydrolysis, depending on the pH conditions for crosslinking. These results indicated a direct relationship between the mode of crosslinking reaction, and the swelling and enzymatic hydrolysis properties of the genipin‐crosslinked chitosan gels. The ring‐opening polymerization of genipin and the pH‐dependent crosslinking reactions may provide a novel way for the preparation and exploitation of chitosan‐based gels for biomedical applications. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1985–2000, 2005     

Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin

The reaction mechanism of chitosan, bovine serum albumin (BSA), and gelatin with genipin (a natural crosslinking reagent) was examined with infrared, ultraviolet–visible, and ¹³C NMR spectroscopies; protein‐transfer reaction mass spectrometry; photon correlation spectroscopy; and dynamic oscillatory rheometry. Two reactions that proceeded at different rates led to the formation of crosslinks between primary amine groups. The fastest reaction to occur was a nucleophilic attack on genipin by a primary amine group that led to the formation of a heterocyclic compound of genipin linked to the glucosamine residue in chitosan and the basic residues in BSA and gelatin. The second, slower, reaction was the nucleophilic substitution of the ester group possessed by genipin to form a secondary amide link with chitosan, BSA, or gelatin. A decreased crosslinking rate in the presence of deuterium oxide rather than water suggested that acid catalysis was necessary for one or both of the reactions to proceed. The behavior of the gel time with polymer concentration was consistent with second‐order gelation kinetics resulting from an irreversible crosslinking process, but was complicated by the oxygen radical‐induced polymerization of genipin that caused the gels to assume a blue color in the presence of air. The lower elastic modulus attained after a given time during crosslinking of the globular protein BSA as compared to the coiled protein gelatin, despite possessing more crosslinkable basic residues, demonstrated the importance of protein secondary and tertiary structures in determining the availability of sites for crosslinking with genipin in protein systems. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3941–3953, 2003     

Synthesis and characterization of a novel chitosan‐based network prepared using naturally occurring crosslinker

Novel chitosan‐based polymeric networks were synthesized and characterized by FTIR, UV and C¹³ NMR. A naturally occurring crosslinking reagent—genipin, which has been used in herbal medicine and in the fabrication of food dyes, was used to prepare crosslinked chitosan hydrogel. The heterocyclic compound of genipin crosslinked chitosan was formed by a nucleophilic attack by amino group on the olefinic carbon atom at C‐3 of deoxyloganin aglycone, followed by opening of the dihydropyran ring and attack by the secondary amine group on the intermediate aldehyde group. Additional, secondary amide linkages could be established in the genipin crosslinked chitosan network by the reaction of ester group of genipin with amino group of chitosan. This bifunctional linkages of genipin with amino group of chitosan leads to form crosslinked networks. Genipin reacted with nucleophilic reagent such as chitosan could further go through a polymerization to form oligomer‐bridge in the crosslinked network. The finally formed chitosan networks are blue color, elastic and exhibits pH‐dependent swelling characteristics. The swelling ratio of the chitosan hydrogel increased at pH lower than 3 and higher than 11 due to the hydrolysis of amide linkage in the genipin crosslinked chitosan network by acid or alkaline, followed by the protonation of amine group or ionization of carboxyl acid group in the network. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2804–2814, 2000     

哈茨木霉CGMCC 2979生物转化栀子中的京尼平苷制备京尼平

采用微生物直接转化药材的方法,将栀子中的京尼平苷转化为京尼平,无需糖苷酶和京尼平苷的制备. 在培养温度为30 ℃,pH 6.1以及栀子载量为80 g/L的条件下,48 h京尼平苷的转化率为97.8%. 转化后的京尼平通过XAD-16N大孔树脂偶联硅胶层析的方法,制备得到纯度大于95%的京尼平,收率为62.3%. 在催化、转化机制研究中,从哈茨木霉CGMCC2979的发酵液中分离得到了分子量为74.4 kDa的京尼平苷β-葡萄糖苷酶,该酶最优催化条件为50 ℃和pH 4.0-5.0. K_m和V_max分别为3.6 mmol/L和775 μmol/h/mg蛋白. 本文提供了一种简便、高效制备京尼平的新方法.     

Kinetic analysis of genipin degradation in aqueous solution

Degradation of genipin (GP), a low toxicity natural protein crosslinking agent, in aqueous solution was monitored by HPLC at various pH levels. Degradation of GP was consistent with a mechanism consisting of a first order reaction with a reversible first step. Formation of the intermediate was slowest at more neutral pHs while formation of the irreversible product was correlated to increasing alkalinity. Degradation at all pHs was enhanced by the presence of phosphate ions. Degradation of GP most likely proceeds via the reversible opening of the dihydropyran ring by water followed by irreversible polymerization of the intermediate. Degraded solutions containing no detectable GP or intermediate, however, are still capable of crosslinking proteins.     

A validated HPLC‐MS/MS method for determination of genipin‐1‐o‐glucuronic acid in rat plasma after administration of genipin and its application to a pharmacokinetic study

A sensitive and specific method was developed and validated for the quantitation of one major metabolite of genipin in rats plasma. The major metabolite was isolated from rat bile via semi‐preparative HPLC technology and its chemical structure was identified as genipin‐1‐o‐glucuronic acid (GNP‐GLU), which was for the first time used as a standard compound for quantitative analysis in rat plasma after administration of genipin. The application of high‐performance liquid chromatography–tandem mass spectrometry in negative mode in multiple reaction monitoring mode was investigated. Chromatographic separation was achieved on an Eclipse XDB‐C₁₈ column using a mobile phase consisting of water with 0.1% formic acid (A)–acetonitrile (B). The limit of detecation was 0.214 ng/mL and the lower limit of quantification was 0.706 ng/mL. The calibration curve was linear from 1.27 to 3810 ng/mL for plasma samples, with a correlation coefficient of 0.9924. The intra‐ and inter‐day precisions and accuracy were all within 15%. The recoveries of GNP‐GLU and puerarin were above 90.0 and 76.2%, respectively. The highly sensitive method was successfully applied to estimate pharmacokinetic parameters of GNP‐GLU following oral and intravenous administration of genipin to rats. Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC-MS/MS method to determine genipin in rat plasma after hydrolysis with sulfatase and its application to a pharmacokinetic study

A sensitive high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of genipin in rat plasma after hydrolysis with sulfatase. Genipin could not be detected directly as it could be transformed into other forms such as conjugated‐genipin immediately after administration. The conjugated genipin could be hydrolyzed by sulfatase to genipin. The conditions of hydrolysis were investigated. Genipin and the internal standard, peoniflorin (IS), were separated on a reversed‐phase column by gradient elution and detected using an electrospray ion source on a 4000 QTrap triple‐quadrupole mass spectrometer. The quantification was performed using multiple reaction monitoring with selected precursor‐product ion pairs of the transitions m/z 225.0 → 122.7 and m/z 479.1 → 449.1 for genipin and peoniflorin. The assay was linear over the concentration range of 1.368–1368 ng/mL, with correlation coefficients of 0.9989. Intra‐ and inter‐day precisions and accuracy were all within 15%. The lower limit of quantification was 1.368 ng/mL. The recoveries of genipin and peoniflorin were more than 53.3 and 51.2%. The highly sensitive method was successfully applied to estimated pharmacokinetic parameters of genipin following oral and intravenous administration to rats. The absolute bioavailability of genipin was 80.2% in rat, which is the first report. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of geniposide and its metabolites genipin and genipinine in culture of Aspergillus niger by HPLC

When cultivated with Aspergillus niger, geniposide, an important drug, is transformed into genipin and genipinine. A simple and rapid HPLC method for simultaneous determination of geniposide and its two metabolites in broth of A. niger is described. The chromatographic separation was achieved on a C18 ODS column (250 x 4.6 mm) by gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the gradient mixtures. The flow rate was 1 mL/min, the detection wavelength was 238 nm and the column temperature was kept at 28 degrees C. The retention times of geniposide, genipin and genipinine were 10.9, 13.8 and 21.5 min, respectively. The mean absolute recoveries of three analysts were over 98%. Quantification limits were 0.01 microg/mL for geniposide and 0.02 microg/mL for the two metabolites. The method was applied for the quantification of geniposide, genipin and genipinine during fermentation and the evaluation of the bioavailabilities of these three compounds in Caco-2 monolayer.     

Soybean and Lupine Addition in Hen Nutrition—Influence on Egg Immunoreactivity

Modifying hen fodder is a common way of changing eggs composition today. However, there is no information on the effect of the source of protein in the fodder replacement on egg allergenicity. This research aimed to detect potential differences in the immunoreactivity and protein composition of eggs from hens fed with fodder containing legume. The aim of the first step of the study was to select the proper solvent for extracting allergenic proteins from hen eggs. Two of them (containing Tween 20 and Triton 100) were selected, based on protein profile and concentration analysis. Egg-white- and egg-yolk-proteins extracts prepared with them were checked for potential differences, using SDS-PAGE electrophoresis, and then the Western-blot method, using sera from children allergic to eggs and soy. Preliminary studies on the influence of fodder composition on the composition of egg proteins suggest that the addition of soy and lupine to fodder modifies the expression of egg proteins. The observed differences in the immunoreactivity of proteins contained in hen egg-white samples do not seem to be as significant as the appearance of protein with a molecular weight of ~13 kDa in the yolk of eggs obtained from soybean-fed hens. This protein may increase the immunoreactivity of eggs for children allergic solely to soy.     

Transformation of geniposide into genipin by immobilized β-glucosidase in a two-phase aqueous-organic system

Genipin is the bioactive compound of geniposide and a natural cross-linking agent. In order to improve the preparation process of genipin, the hydrolysis of geniposide to genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was studied. β-glucosidase was immobilized by the crosslinking-embedding method using sodium alginate as the carrier. The optimum reaction temperature, pH value and time were 55 °C, 4.5 and 2.5 h, respectively. To reduce genipin hydrolysis and byproduct production the reaction was carried out in an aqueous-organic two-phase system comprising ethyl acetate and sodium acetate buffer. The product was analyzed by HPLC, UV, IR, and NMR. The yield of genipin was 47.81% and its purity was over 98% (HPLC).     

Kinetic study of chitosane/genipin system using DSC

Knowledge of the the kinetic study of chitosan/genipin allow to know the different effects that time and temperature have on the cure reaction of the material. The total enthalpy of reaction, the glass transition temperature and the partial enthalpies have been determined using DSC in dynamic mode. Two models, one based on chemical kinetics and the other accounting for diffusion were used. The incorporation of the diffusion factor in the second model allowed for the cure kinetics to be predicted the whole range of conversion.     

Elucidation of Metal‐Ion Accumulation Induced by Hydrogen Bonds on Protein Surfaces by Using Porous Lysozyme Crystals Containing RhIII Ions as the Model Surfaces

Metal‐ion accumulation on protein surfaces is a crucial step in the initiation of small‐metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh^III ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino‐acid residues to capture Rh^III ions. ii) Water molecules pre‐organized by hydrogen bonds can stabilize Rh^III coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino‐acid residues can be replaced by Rh^III ions to form polynuclear structures with the residues. iv) Rh^III aqua complexes are retained on amino‐acid residues through stabilizing hydrogen bonds even at low pH (≈2). These metal–protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.     

Homogeneous immunoassay for human IgG using oriented hen egg IgY immobilized on gold sol nanoparticles

Homogeneous immunoassays using (red) gold nanoparticles represent an attractive detection scheme because of the option of photometric readout. We have applied oriented immobilization of hen egg immunoglobulin Y (IgY) on gold nanoparticles when developing a homogeneous immunoassay for human IgG. In oriented immobilization, as opposed to random immobilization, the antigen binding capabilities of the antibodies are retained. It is shown that such immunoassay has significantly better sensitivity in comparison with methods based on conventional immobilization of affinity-purified antibodies. It is also shown that hen egg IgY is better suited than rabbit antibodies, because much more antibody can be immobilized on gold nanoparticles without any destabilization, probably because of the more acidic nature of these antibodies. In addition, hen egg IgY can be supplied in higher quantity and can be prepared more easily than IgG from rabbits. Bleeding and slaughtering of animals is not needed. The assay presented here has a wide detection range (30–500?ng?.mL^?1) and a limit of detection as low as 30?ng.mL^?1 of human IgG.

Synthesis of monoterpene alkaloid derivatives from the iridoid glucoside geniposide

Nine novel monoterpene alkaloid derivatives (3(a)-(c), 4(a)-(c), 5(a)-(c)) were prepared as a reaction of genipin 2 with L-amino acid methyl hydrochlorides utilized the reaction of reductive amination. Genipin was obtained by beta-glucosidase, catalyzed hydrolysis of the iridoid glucoside geniposide 1. The chemical structures were confirmed by 1D-, 2D-NMR and MS.     

A new method for microcapsule characterization: use of fluorogenic genipin to characterize polymeric microcapsule membranes

Numerous microcapsule systems have been developed for a wide range of applications, including the sustained release of drugs, cell transplantation for therapy, cell immobilization, and other biotechnological applications. Despite the fact that microcapsule membrane is a dominant factor governing overall microcapsule performance, its characterization is challenging. We report a new method for characterizing microcapsule membranes, using the most common alginate-poly-L-lysine-alginate (APA) microcapsule as an example. Our data demonstrate that genipin, a naturally derived reagent extracted from gardenia fruits, interacts with poly-L-lysine (PLL) and generates fluorescence. This fluorescence allows clear visualization and easy analysis of the PLL membrane in the APA microcapsules using confocal laser scanning microscopy. The results also show that PLL binding correlates to the reaction variables during PLL coating such as PLL concentration and coating time. In addition, five other different microcapsule formulations consisting of PLL and/or chitosan membranes were examined, and the results imply that this method can be extended to characterize a variety of microcapsule membranes. These findings suggest that genipin can serve as a fluorogenic marker for rapid characterization of microcapsule membranes, a superior method that would have important implications for microcapsule research and potential in many other applications.     

Animal glues in mixtures of natural binding media used in artistic and historic objects: identification by capillary zone electrophoresis

Animal glues were often used in historic and artistic objects, e.g. as paint ground, as binders for pigments, or as adhesives. The sources were egg, casein, or different collagens. For restoration and conservation purposes it is important to know which kind of animal glue a museum object contains. Capillary electrophoresis can deliver such information, because it enables differentiation among the three proteinaceous glue classes according to their different amino acid patterns after hydrolysis. This work deals with the most relevant problem in practice, whether this identification is obstructed by the presence of other binders, with which they are mixed in many real samples; in particular, interference from plant gums and drying oils was investigated. Capillary electrophoresis of the hydrolysates (after reaction with 6 mol L^–1 HCl) was performed with an acidic background electrolyte consisting of chloroacetic acid (51.9 mmol L^–1) adjusted with LiOH to pH 2.26. The underivatised analytes were detected with a contactless conductivity detector. It was found that the constituents of the plant gums (monosaccharides) or drying oils (long-chain fatty acids and short-chain dicarboxylic acids) never interfered with identification of the animal glues, as shown for artificial mixtures of the different binders even at tenfold excess over the animal glue, and for egg tempera samples. The method was used to identify the filling material from a statue from the eighteenth century.     

Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT-isooctane-water reverse micelles

The kinetics of 1-naphthyl phosphate and phenyl phosphate hydrolysis, catalyzed by human prostatic acid phosphatase (PAP) entrapped in AOT-isooctane-water reverse micelles, has been studied over surfactant hydration degree (w0) range 5 to 35. Continuous spectrophotometric acid phosphatase assays, previously prepared, were employed. PAP was catalytically active over the whole w0 studied range. In order to determine steady-state reaction constants the experimental data were fitted to Hill rate equation. Positive cooperativity in substrate binding was observed, as it was earlier found in aqueous solutions. The extent of cooperativity (expressed as the value of the Hill cooperation coefficient h) increased from 1 to 4, when the micellar water-pool size was growing, at fixed enzyme concentration. In the plots of catalytic activity (kcat) versus w0, the maxima have been found at w0=10 (pH 5.6) and 23 (pH 3.8). It is suggested that catalytically active monomeric and dimeric PAP forms are entrapped in reverse micelles of w0=10 and 23, respectively.     

GC-MS identification of proteins in wall painting samples: A fast clean-up procedure to remove copper-based pigment interferences

A new approach was explored to purify proteins in a multi-step procedure for the characterisation of proteinaceous materials (casein, animal glue, and egg) in artwork samples by gas chromatography-mass spectrometry. High concentrations of inorganic salts, such as azurite, have been found to impair the determination of protein via amino acid analysis. The effect of varying concentrations of copper-based pigments on the quantification of amino acids was evaluated through the analysis of replica paintings prepared with the three types of proteinaceous materials. Glycine, aspartic and glutamic acids are the amino acids most affected by the presence of copper salts. In the case of high concentration of salts, this interference hampers the correct identification of the proteins. To eliminate the inorganic salts, a C18 pipette tip was used to clean-up the ammonia extracts before the acidic hydrolysis step. The clean-up procedure allows us to prevent the influence of the inorganic salts and thus allows correct protein identification, though the quantitative recovery of proteinaceous material is quite low. The effectiveness of the optimised procedure was evaluated by analysing samples from two Italian wall paintings from the 13th and the 14th centuries. Without the clean-up it would not have been possible to detect the presence of a mixture of egg and animal glue in one case, and that of egg in the other one.     

Classification of protein binders in artist's paints by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: an evaluation of principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA)

Proteomics techniques are increasingly applied for the identification of protein binders in historical paints. The complex nature of paint samples, with different kinds of pigments mixed into, and degradation by long term exposure to light, humidity and temperature variations, requires solid analysis and interpretation methods. In this study matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra of tryptic-digested paint replicas are subjected to principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) in order to distinguish proteinaceous binders based on animal glues, egg white, egg yolk and milk casein from each other. The most meaningful peptide peaks for a given protein class will be determined, and if possible, annotated with their corresponding amino acid sequence. The methodology was subsequently applied on egg temperas, as well as on animal glues from different species. In the latter small differences in the MALDI-TOF mass spectra can allow the determination of a mammal or sturgeon origin of the glue. Finally, paint samples from the 16(th) century altarpiece of St Margaret of Antioch (Mlynica, Slovakia) were analysed. Several expected peaks are either present in lower abundance or completely missing in these natural aged paints, due to degradation of the paints. In spite of this mammalian glue was identified in the St Margaret samples.