RESUMO
The current study explored the hepatotoxicity among closed-ring genipin, open-ring tautomer of genipin and gardenia blue that generated from genipin and amino acid reaction using HepaRG cells to identify the material basis of genipin-induced hepatotoxicity in vitro. The effects of temperature, pH value and different kinds of amino acids on the chemical structure tautomerism between closed-ring and open-ring tautomer of genipin and the production of gardenia blue were investigated firstly, which aimed to explicit the conditions that could distinguish the closed-ring genipin and its open-ring tautomer, and the conditions generating gardenia blue, which were applied to prepare different kinds of gardenia blue; the CCK-8 kit was employed to analyze the hepatotoxicity of closed-ring genipin, open-ring tautomer of genipin and gardenia blue. From the results, it was found that, the structure transformation from close-ring to open-ring of genipin could be inhibited under the condition with acid environment; being essential groups to generate gardenia blue, the primary amino group and the open-ring tautomer of genipin reacting to generate the dihydropyridine ring was probably the key structure of gardenia blue; the structure characteristics existed apparent distinction at the reactive temperature of 37 ℃ and 80 ℃; compared to the culture condition with pH = 7.4, the concentration of genipin with close-ring in culture medium was significantly increased at pH = 5, but the cell viability did not decreased; the cell toxicity of gardenia blue was apparently lower than open-ring tautomer of genipin, and even some kinds of gardenia blue showed growth promoting effect on HepaRG cells. Here, it was suggested potentially that open-ring tautomer of genipin be the important material basis to induce hepatotoxicity, which could provide a cue and lay a foundation for the elucidation of the underlying mechanism of genipin-induced hepatotoxicity.
RESUMO
ObjectiveTo study the chemical structure of gardenia blue pigment and its inhibitory activity against monoamine oxidase B （MAO-B）， in order to seek a potential feasible way for rational utilization and value enhancement of iridoids in Gardeniae Fructus. MethodIridoid glycosides in Gardeniae Fructus were hydrolyzed by cellulase to obtain their aglycones and reacted with amino acids. Then， the products were purified by column chromatography packed with D101 macroporous resin and preparative liquid chromatography to obtain gardenia blue pigments， and the gardenia blue pigments were identified by nuclear magnetic resonance （NMR） and mass spectrometry （MS）. Benzylamine was used as the reaction substrate of MAO-B and in vitro incubated with gardenia blue pigment monomers， high performance liquid chromatography （HPLC） was employed to determine the production of benzaldehyde for evaluating the inhibitory effect of gardenia blue pigments on MAO-B， the mobile phase was methanol （A） -50 mmol·L-1 potassium phosphate buffer （B， pH 3.2） （2∶3）， and the detection wavelength was 245 nm. ResultEight compounds of gardenia blue pigment A-H were synthesized and identified. In MAO-B inhibition test， compared with geniposide， the inhibitory activity of gardenia blue pigment D and E was significantly enhanced （P<0.05）. Compared with the 6β-hydroxygeniposide， the inhibitory activity of gardenia blue pigment G and H was significantly enhanced （P<0.05， P<0.01）. All the four gardenia blue pigments showed better MAO-B inhibitory activity than the prototype compounds. ConclusionGardenia blue pigment is a simple compound formed by one molecule of amino acid and one molecule of iridoid. Some gardenia blue pigments have better MAO-B inhibitory activity than the prototype compounds. The activity of gardenia blue pigment produced by different substrates is different， and the high-value gardenia blue pigment can be prepared based on experimental optimization， which can expand the application range of gardenia blue pigment and enrich the comprehensive utilization of iridoids from Gardeniae Fructus.