Abstract
The untargeted metabolomics analysis was used to evaluate the metabolites produced after drink genipap juice and identify biomarkers of exposure. The identification of a biomarker of food or diet intake becomes a new tool in the dietary assessment. Genipap (Genipa americana L) is a native fruit from Amazonia largely consumed and well-known in folk medicine as diuretic, digestive, laxative, antiseptic, healing and to treat anemia, uterine cancer, and measles. The biological effect on a living organism is assigned to compounds presents in this fruit with a wide range of bioactivity. To identify metabolites produced after genipap juice intake that can be biomarkers of genipap consumption, we analyzed the urine of sixteen healthy free-living men before and after genipap juice intake. The experimental design was divided into two different expositions, one acute and other medium-term in an open-label crossover clinical trial. In the acute intervention, the volunteers consumed a shot of 500 mL of genipap juice. In the medium-term exposition, the volunteers drank 250 mL of genipap juice two times a day for 19 days, and in the 20th day they drunk 500 mL of water with sugar (control drink). The urine samples were collected for 24 h in 3 different bottles corresponding 0 – 6 h, 6 – 12 h and 12 – 24 h after drinking. The samples from acute and medium-term exposition were compared with control samples collected before genipap juice intake in the same conditions of the juice intake. The metabolites from urine samples were extracted using Milli-Q water and analyzed on ultra-high performance liquid chromatography coupled to mass spectrometer Orbitrap. To perform the T-test analysis with a false discovery rate < 0.05 in log-transformed and Pareto-scaled data, the software Metaboanalyst 3.5 was used. The principal components analysis and partial least squares discriminant analysis were also performed to highlight experimental differences between groups. The value of the area under the curve of the receiver operator characteristic curve validated the identified biomarkers. 33 metabolites were putatively annotated in acute study and 26 in the medium-term. 22 of these metabolites were common in the two expositions. 15 compounds from iridoid class were putatively identified using the basic chemical rules and the software Qual Browser. These structures correspond to genipic acid, its derivatives forms and derivative forms of genipinic acid. A group with nine endogenous and exogenous metabolites was validated as biomarkers of genipap acute juice intake with high confidence. The biochemical analysis of blood samples showed a low and significant reduction of enzymes glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase after a medium-term of genipap juice consumption. The physical alterations related to juice intake were: 7 of the volunteers related an increase in the disposition, 6 had an increase in the sleep, 7 had a decrease in body weight, 3 said that the feces became pastier and others 3 had diarrhea. Thus, we conclude that iridoids present in genipap fruit are bioavailable, it was the first time that this bioavailability was reported. These iridoids have different kinetics of absorption by the human body. Some metabolites are more rapidly excreted than others, but all continue to be excreted even after 24 h after the exposition. Thus in this study genipap juice intake showed safety and tolerability and no toxic effect was observed. Furthermore, more investigation is necessary to identify the possible therapeutic activity of this fruit.