New analytical approaches to assess the phenolic composition of guava (psidium guajava l.) leaves with anti-diabetic related risk factors bioactivity

The present doctoral dissertation encompasses new analytical approaches to assess the phenolic composition of Psidium guajava L. leaves as a possible source as nutraceutical against diabetes mellitus (DM), compiling the identification, quantification, and also the evaluation of in vivo bioactivity of the phenolic composition of the leaves towards DM and some of its risk factors. It is divided into two main parts; the first one is the INTRODUCTION, which encompasses a brief explanation of DM disease, and an overview of the description and importance of guava tree, the nutritional value of the fruit, and the composition and the methods most used for the analysis of the phenolic compounds of the leaves, useful to know the analytical research that had been previously done with the leaves and to properly focus on further studies. The second part includes the “EXPERIMENTAL PART. RESULTS AND DISCUSSION” carried out in the current PhD thesis and is sub-divided into two sections. Section I provides an extend characterization of the phenolic composition of guava leaves by using several advanced analytical techniques. Chapter 1 concerns the optimization of the most common ancient remedy, the infusion, and it is compared with the bath-ultrasound assisted extraction, by one-variable-at-a-time method. It is a first attempt to characterize extracts rich in phenolic compounds from P. guajava L. leaves by means of identification and quantification of the compounds by high-performance liquid chromatography coupled to diode array detection and quadrupole mas spectrometer (HPLC-DAD-ESI-Q-MS), as well as the evaluation of their antioxidant capacity by FRAP and ABTS assays. Chapter 2 deals with the optimization of the extraction of phenolic compounds by bath-ultrasound assisted extraction and one-variable-at-a-time method, as well as the accurate identification of several compounds (some of them for the first time) by high-performance liquid chromatography coupled to diode array detection and quadrupole time of flight spectrometry (HPLC-DAD-ESI-QTOF-MS). Besides, it includes the comparison between the main varieties of guava leaves (pyrifera and pomifera vars.) according to the phenolic composition and the antioxidant capacity by FRAP and ABTS assays. Chapter 3 studies the influence that the different oxidative states have on the phenolic composition of guava leaves var pyrifera by high-performance liquid chromatography coupled to diode array detection and quadrupole time of flight spectrometry (HPLC-DAD-ESI-QTOF-MS), and on antioxidant capacity (FRAP and ABTS), following the extraction method optimized in the previous chapter. Moreover, cyaniding-3-O-glucoside has been identified for the first time. Chapters 4 and 5 were prepared in collaboration with the Inter-Departmental Centre for Agri-Food Industrial Research (CIRI Agrifood) and the Department of Agro-Food Sciences and Technologies from the University of Bologna (Italy) during a pre-doctoral stay founded by CEI-Biotic mobility fellowship (3 months). In Chapter 4, the optimization, by means of response surface methodology (Box-Behnken design (BBD)), of proanthocyanidins extraction from guava leaves var. pyrifera is carried out assisted by bath-ultrasound. The influence of the factors (time, temperature, solvent ratio and acid ratio) is evaluated in the response of the sum of the target compounds. Identification and quantification of this family of bioactive compounds is done by normal phase high-performance liquid chromatography coupled to fluorescence detection and quadrupole mass spectrometer (NP-HPLC-FLD-ESI-Q-MS). Also, the composition of guava leaves at different oxidative states is evaluated under the optimal conditions. In Chapter 5, the sonotrode-ultrasound assisted extraction efficiency is evaluated at laboratory scale. Based on response surface methodology (BBD), the response of the factors (time, solvent ratio, and ultrasound power) is studied in the antioxidant capacity (DPPH and TEAC) and the main families of bioactive compounds present in guava leaves. Identification and quantification is carried out by high-performance liquid chromatography coupled to triple quadrupole mass spectrometer (HPLC-ESI-QqQ-MS). Besides, the best process conditions are applied to pyrifera and pomifera vars. The Section II focuses on the bioactivity of guava leaves. Chapter 6 provides a view over the last decade of the health benefits, in vitro and in vivo, as well as some food applications reported with guava leaves. Chapter 7 was prepared in collaboration with the Center for Biomedical Research (CIBM) and the Department of Pharmacology (CIBER-EHD and CIBER-Enfermedades Cardiovasculares, respectively) from the University of Granada. Following the same analytical scheme as chapter 2, here, is assessed the bioactivity of guava leaves var. pyrifera in mice with diet-induced obesity towards the DM related risk factors. Lastly, the annex section was prepared in collaboration with “Advanced NMR methods and metal-based catalysts” research group from the University of Almeria. This appendix includes the fingerprint of guava leaf var. pyrifera by nuclear magnetic resonance (NMR) spectroscopy. The structure of this work has not the same appearance as the chapters, since it has not been concluded yet. Nevertheless, it has been included because it has been achieved during this period.