Orientador: Prof. Dr. Jesuí Vergilio Visentainer

 Data da Defesa: 21/07/2017



INTRODUCTION Solanum americanum Mill. is a shrub endemic to the Americas, native to Brazil, popularly known as Erva Moura. Fruit can be called as berries (a generic term for small fruits, with small seeds, and can be consumed whole, rich in nutrients and antioxidants). It belongs to the family Solanaceae, one of the families with the greatest variety of plants and secondary metabolites and thus several biological activities, such as anti-inflammatory activity. The synthesis of secondary metabolites can begin after a specific stimulus such as ultraviolet radiation, temperature change, lack of nutrients, pathogen attack or being present in the plant from its embryonic stage, serving as an additional system of defense of the plant organism. Phenolic compounds, flavonoids and carotenoids are three major groups of substances resulting from secondary metabolism. Phenolic compounds are subdivided into groups of importance that vary according to the number of phenols (simple phenol or polyphenols). This study focused on anthocyanins (the main chromogenic substances found in red, blue and purple fruits) produced to protect plants against oxidative stress, acting as natural antioxidants. These so-called antioxidant compounds are substances that can inhibit free radical formation reactions, such as reactive oxygen species, and also inhibit carcinogenesis. Speciation of fruit composition has been used to identify compounds with anti-inflammatory and antioxidant potential. Fruits represent complex matrices, so the identification of the present compounds requires highly efficient, sensitive and reproducible separation and characterization techniques. AIMS The objective of this study was to develop and validate a rapid method for the determination of 3 different types of anthocyanins using ultra-fast liquid chromatography coupled to mass spectrometry (UFLC-MS/MS) and to evaluate the anti-inflammatory response of four different types of extracts obtained from ripe fruit of Solanum americanum Mill. in experimental models, and evaluate them qualitatively using UFLC-DAD/TOF-MS. MATERIAL AND METHODS Ripe fruits of Solanum americanum Mill. were collected in California, State of Paraná, Brazil. The exsicta was deposited herbarium of the Department of Botany, Federal University of Minas Gerais (BHCB: 179995). The ripe fruits were hygienized, homogenized, lyophilized and vacuum-packed and stored in a freezer at -22 °C until analysis. For the development and validation of a new method for determination of anthocyanin (cyanidin, pelargonidin and malvinidine), a conventional method was used, in which a 15 g-sample was diluted in acidified methanol (pH 3), and sonicated on ultrasonic bath for 20 minutes, filtered through a 0.22 μm membrane and compared to the proposed method. The fruits were macerated in a mortar with pestle, and the juice 10 extracted was filtered through a 0.22 μm membrane. Samples were immediately taken for UPLC-MS/MS injection. The validation parameters were determined according to the ICH Guide. The figures of merits analyzed on the same day were: linearity, precision, linear range, limit of detection and limit of quantification, estimated based on the signal-to-noise ratio of 3 and 10, respectively. For the biological analyses, four different types of extracts were prepared from the lyophilized sample (aqueous extract, chloroform extract, ethanol extract and methanol extract), all the solvents were evaporated in rotary evaporator with coupled vacuum, until complete evaporation; the extracts were tested in the biological assays (ear edema and paw edema) and analyzed by UFLC-DAD/TOF-MS. The experimental protocols were approved by the Animal Ethics Committee of the State University of Maringá (CEUA 2492160715/2015). Statistical analysis was performed using ANOVA followed by Tukey’s test (p <0.05) using the software GraphPad Prism 5.0 for biological assays. For the results of the Time-of-Flight - Mass Spectrometry analyses, Compass data analysis was used. RESULTS AND DISCUSSION A total of 34 compounds were identified in the four different extracts (aqueous: 13, ethanol: 10, methanol: 6 and chloroform: 4 compounds). During the extraction of the sample, the polarity of the solvents influenced the amount of compounds obtained, which were derived from different classes. The aqueous and ethanol extracts showed a higher number of compounds extracted in the analysis by UFLC-DAD-TOF/MS and the results obtained in the biological assays showed that, for the topical antiedematogenic activity (ear edema), all extracts were able to significantly inhibit the ear edema formation, whereas Chloroform and Methanol extracts showed a significant decrease in both concentrations (2.5 and 5.0 μg/ear). All extracts were able to reduce myeloperoxidase activity at the concentration of 5.0 μg/ear in the ear edema assay. Paw edema, induced by carrageenan, was used to evaluate the systemic effect of extracts by oral treatment. Oral treatment with aqueous and chloroform extract significantly inhibited the formation of edema within the fourth hour after application of the phlogistic agent. For the development of a new method, the chromatographic separation under optimal conditions of the equimolar mixture of the three anthocyanins of interest, namely cyanidin, pelargonidin and malvidinin were used. The results demonstrate an ultra-fast analysis technique for anthocyanin (1.30 min) when compared with the high performance liquid chromatography (HPLC) technique, one of the most used for this purpose, with analytical runs of approximately 20 to 60 min. The technique proposed by this study presents excellent reproducibility, since it presents a relative standard deviation of less than 5% for all investigated anthocyanins, high linearity (greater than 0.9) within the investigated range (1-1000 ng) and accuracy less than 10% and low limits of detection and quantification. Usually, the extraction of these compounds is carried out in acidified medium (HCl), but in order to make possible an extraction based on the principles of green chemistry, extraction was proposed only with maceration and the development of an analytical methodology that uses water as mobile phase, which further reduces the costs of this analytical technique. In order to check the efficiency of 11 the proposed methodology, samples of berries (Morus nigra L.; Prunus avium L.; Rubus idaeus L.; Solanum americanum Mill.; Vaccinium myrtillus L.; Vitis vinifera L.) were used for comparing the two methodologies of extraction and for verifying the concentrations of anthocyanins (proposed and conventional methodologies). Among the analyzed samples, better results were achieved for the proposed method in relation to the conventional method for Cyanidin and Delphinidin, whereas for Pelargonidin, only the conventional method was able to determine the concentration in fruit of Vaccinium myrtillus L. and Vitis vinifera L. This indicates that Pelargonidin is possibly bound to other compounds, such as sugars (xylose, arabinose, rhamnose) and acid hydrolysis increases its free concentration in the sample, and therefore it is quantified in a higher concentration in acid extraction, as observed in conventional methodology. CONCLUSIONS This study demonstrated an important anti-inflammatory activity of the extracts present in fruit of Solanum americanum Mill. The best results were obtained from aqueous and chloroform extracts in the topical and systemic evaluation, reducing the edema volume and the myeloperoxidase activity. The technique developed allows the extraction of the compounds of interest in analytical amounts, the detection and quantification of the same in scale of nanograms within only 1.30 minutes. This is the first time that concentrations of anthocyanins present in ripe fruit of Solanum americanum Mill. were reported; this study contributes to disseminate knowledge about another genus of the Family Solanaceae. Key words: Phenolic compounds; Anthocyanins; Chromatography; Acute Inflammation; Native Plants


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