METABOLOMIC ASSESSMENT WITH LC-QTOF-MS OF THE NUTRACEUTICAL EFFECT OF ROSMARINUS OFFICINALIS EXTRACTS ON PLASMA SAMPLES OF DIABETIC RATS

Diabetes mellitus is one of the major worldwide health problems. The peculiarity of this disease is to have high levels of oxidative stress, which plays a crucial role in the pathogenesis of other complications, such as nephropathy, neuropathy and retinopathy. Rosmarinus officinalis L. extracts, processed by supercritical fluid extraction (SFE), have long been recognized as having antioxidant properties. To investigate the potential nutraceutical properties of these extracts on type 1 diabetes, a metabolomic fingerprinting study was carried out using LC-QTOF-MS. Metabolomic fingerprinting is a technique which study as many analytes as possible to get a complete view of a disease and of the different levels of metabolites involved, and finally to identify possible biomarkers.In this work, Sprague-Dawley male rats (12 ± 2 weeks of age), treated with streptozotocin, were used in controlled conditions in which they received, by intragastric administration, five doses of rosemary extract containing 10% folic acid (used to improve the endothelial progenitor cell function) dispersed in 1 mL vehicle or only 1 mL of vehicle. There were also healthy rats that received the same treatment, thus generating four groups of samples: treated or non-treated (only vehicle) controls, treated and non-treated diabetics.Plasma fingerprints of control and diabetic rats, focused on the most polar constituents, were obtained by LC-QTOF-MS. Retention of polar compounds from plasma in hydrophilic interaction liquid chromatography (HILIC) mode and use of solvents readily compatible with mass spectrometry is a new approach that was initially explored by testing different stationary phases. The HILIC columns used in this study included an Ascentis Si (15 cm × 2.1 mm, 5 µm, Supelco), a TSK gel Amide-80 (5 cm × 2.0 mm, 3 µm, Tosoh), a Luna HILIC (10 cm × 2.0 mm, 5 µm, Phenomenex) and a ZIC-HILIC (10 cm × 2.1 mm, 3.5 µm, Merck). Unfortunately, the results were not as good as it was expected and this was mainly due to the lack of precision. It was therefore decided to analyze the plasma samples under reversed-phase (RP) conditions, using a Discovery HS C18 column (15 cm × 2.1 mm, 3 µm, Supelco), with a mobile phase composed by water (with 0.1% of formic acid) and ACN (with 0.1% of formic acid), under gradient elution. The injection volume was 10 µL; the flow rate was 0.6 mL/min and the column temperature was set at 30 °C. Data were collected in the positive ion mode with a QTOF mass analyzer, operated in the full scan mode from 50 to 1000 m/z. Experimental data from RP-HPLC were further processed by multivariate analysis to find compounds with statistically significant changes. Then, MS/MS experiments were performed under the same experimental conditions and identifications were carried out by studying the fragmentation pattern of the target analytes or using reference standards. Finally, the biochemical interpretation of potential biomarkers associated with the disease was performed. The compounds that were found to be affected by the nutraceutical treatment include:•Lysophosphatidylcholines (LPCs), represent the first group of identified compounds. LPCs are generated from ox-LDL or from inflammatory cells, and are very important intermediates of different metabolic pathways. These compounds were found to increase in diabetic rats after the treatment.•Pipecolic acid, the major metabolite of lysine degradation. This compound was found to increase in diabetic rats after the treatment.•Lipoic acid, an antioxidant synthesized in mitochondria. It is a necessary co-factor for mitochondrial α-ketoacid dehydrogenases, and thus plays a critical role in mitochondrial energy metabolism. This metabolite was found to increase in diabetic rats after the treatment.•Carnitine and derivates, which is important to transport fatty acids into the mitochondria. Also these compounds were found to be affected by the treatment.In conclusion, the LC-QTOF-MS analysis allowed us to study a large number of compounds, very useful in metabolomic fingerprinting, and, with the help of chemometric analysis, to select masses with statistically significant changes. The levels of LPCs, pipecolic acid, lipoic acid, carnitine and derivates were found to be improved after the nutraceutical treatment, that can be considered useful to counteract some of the deleterious effects of type 1 diabetes.