Serotonin (5-hydroxytryptamine), a neurotransmitter in the central nervous system (CNS), is synthesised from the essential amino acid L-tryptophan by a short metabolic pathway with oxitriptan (5-hydroxytryptophan) and tryptamine as intermediate compounds. Serotonin has an enormous influence over many brain functions i.e. performing an essential role in the modulation of anger, mood, body temperature, and appetite. However, the exogenous serotonin scarcely accesses the CNS via serotonergic pathways from the bloodstream, while its precursor, oxitriptan, grants the access. Therefore, oxitriptan is recognized as dietary supplement source and has been used in clinical treatment since decades for migraine. Oxitriptan has been produced commercially by extraction from plants.
In this research, a new ultrasound-assisted extraction (UAE) of serotonin and its precursor (tryptophan, tryptamine and oxitriptan) has been optimized and validated. Box-Behnken design in conjunction with response surface methodology was used based on six factors and three levels to evaluate the significances of studied factors prior to optimize the UAE condition. The effects of solvent composition (0−50% ethanol in water), extraction temperature (10−70 oC), solvent-solid ratio (10:1−20:1), solvent pH (3−7), amplitude (30−70 %) and cycle (0.2−0.7 s-1) on the extraction yields were taken into account. Subsequently, extraction kinetic (6−18 min) was studied to reach high recovery of serotonin and its precursors extracted from the matrix. The levels of serotonin and its precursors in the UAE extracts were quantified simultaneously by Ultrahigh Performance Liquid Chromatography coupled with fluoresce detector (UPLC-FD) within 3 min of analysis run time.
The significant (p<0.05) response surface models with high coefficients of determination values of 0.78 were fitted for the experimental data. Solvent composition was found to have a greatly significant effect on the response value (p<0.0001) and the extraction temperature also have a significant effect (p <0.05). The optimal UAE condition was as follows: extraction time 6 min, ultrasound amplitude 30%, cycle 0.7 s-1, extraction temperature 40oC, water as the extraction solvent at pH 5 and solvent-solid ratio 20:1. Complete validation of the method was performed. The developed method presented recoveries between 90% and 106% for the target analytes and showed high precision in terms of CV lower than 3.5% for intermediate precision (except serotonin with 10.5%).
The suitability of the method was checked by application to Vitis vinifera L plants grown under controlled conditions, supplied with different doses of nitrogen and collected at different times during the plants growth. Hence, this particular ultrasound extraction method was demonstrated to be an excellent alternative for the extraction of serotonin and its precursors from plant sample.