In vitro study of cholesterol micellar solubility of Cymbopogon citratus and its phenolic fractions - Optimization of methodology and application

Dissertação de Mestrado em Química Farmacêutica Industrial apresentada à Faculdade de FarmáciaO colesterol é um composto de natureza lipídica, essencial ao funcionamento de todas as células do organismo humano, podendo ser sintetizada a nível endógeno. Cerca de 30 a 40% é sintetizado pelo organismo, sendo o restante obtido pela alimentação, em particular por ingestão de produtos de origem animal. As doenças cardiovasculares são a principal causa de morte e hospitalização nos países desenvolvidos e tem vindo a aumentar nos países em via de desenvolvimento. São diversos os fatores de risco, como é o caso das dislipidemias, isto é, elevados níveis de lipídios (colesterol, triglicerídeos ou ambos) transportados pelas lipoproteínas no sangue. Um dos principais problemas associados à dislipidemia é a formação de placas de aterosclerose que podem acumular-se e depositar-se nas artérias. Isto leva a que possa ocorrer uma diminuição da quantidade de sangue que circula nas artérias, podendo levar mesmo à total oclusão da artéria, dando origem ao acidente vascular cerebral (AVC) e ao enfarte do miocárdio, entre outras situações graves. O aumento da concentração de colesterol no sangue (hipercolesterolémia) associado ao consumo de dieta rica em lípidos, o consumo de grandes quantidades de álcool, a falta de exercício físico, e ainda, a diabetes e hipertensão têm sido apontados como as principais causas das doenças cardiovasculares. Atualmente encontra-se disponível uma grande variedade de terapêuticas medicamentosas para o controlo da hipercolesterolémia. Porém os efeitos adversos destas terapias provocam muitas vezes uma fraca adesão e mesmo o abandono da terapêutica levando muitas pessoas a recorrer aos produtos naturais.O recurso a estudos in vitro permite avaliar, de forma rápida e pouco onerosa, o potencial de plantas medicinais no controlo do colesterol e, consequentemente, selecionar as mais promissoras para a validação subsequente através de ensaios in vivo e clínicos. Uma das vias de redução do colesterol exógeno é a inibição da formação de micelas no intestino, essencial à absorção do colesterol. Pretendeu-se com este trabalho otimizar um método que permita estudar in vitro a solubilidade micelar do colesterol na presença de extratos de folhas do Cymbopogon citratus (infuso e infuso deslipidificado) e comprovar com esta metodologia a potencial atividade desta planta, descrita como tendo atividade hipocolesterolémica e atividade hipolipemiante. Adicionalmente, avaliou-se a contribuição dos seus compostos fenólicos (ácidos fenólicos, flavonóides e taninos) para essa mesma atividade. Os resultados obtidos permitiram concluir que ambos extratos, assim como a fração de ácidos fenólicos, flavonóides e taninos apresentam capacidade para a destruição de micelas, particularmente as duas últimas classes de fenólicos, para as quais se obtiveram percentagens de destruição micelar superiores a 90% para concentração exíguas de flavonóides e taninos (1 µg/mL e 25 µg/mL respetivamente). Estes resultados sugerem que este é um dos mecanismos possíveis pelo qual se observa o efeito hipocolesterolémico desta planta e que os compostos fenólicos, maioritariamente os flavonóides e taninos contribuem para essa atividade. Em conclusão, foi otimizado um teste rápido para o estudo in vitro da solubilidade micelar do colesterol na presença de extratos e frações fenólicas do Cymbopogon citratus, o qual permite evidenciar um dos mecanismos conducentes à atividade hipolipemiante e hipocolesterolémia. Este trabalho permitiu ainda aquilatar o potencial hipolipemiante do Cymbopogon citratus, assim como inferir que essa atividade, pelo menos em parte, se relaciona com os compostos fenólicos, nomeadamente flavonóides e taninos.Cholesterol is a compound of a lipid nature, essential to the functioning of all Human bodily cells, which can be synthetized at an endogenous level. Between 30 and 40% are synthetized within the organism, with the remaining acquired through the diet, in particular through animal products. Cardiovascular diseases are the leading cause of death and hospital admission in the developed countries and is currently increasing in the developing countries. There several risk factors, such as dyslipidaemia, that is, the high amount of lipids (cholesterol, triglycerides, etc.) transported by the lipoproteins of the blood. One of the major problems associated with dyslipidaemia is the formation of arteriosclerosis plaques that can deposit and accumulate in the arteries. This can lead to a diminishing amount of blood circulating in the arteries, up to the full blocking of the artery, which leads to strokes, myocardial infarction, among other serious illnesses. The increase in cholesterol concentration of the blood (hypercholesterolemia), associated to the consumption of a diet rich in lipids, the consumption of great amounts of alcohol, the lack of exercise, hypertension and diabetes have been sown to be the primary causes of heart diseases. Currently there is a great variety of available therapies through medication to control hypercholesterolemia. Nonetheless, the adverse effects of those therapies can often cause a weak acceptance and high abandonment of the therapies, with many people searching for natural products for the same effect. The in vitro study allows the assessment, quickly and cheaply, of the potential of medicinal plants in the control of cholesterol and, sequentially, the selection of the most promising for subsequent validation through in vivo and clinical trials. One of the ways to reduce exogenous cholesterol is the inhibition of the intestinal micelle formation, essential to the absorption of cholesterol. With this essay, the intention was to optimize a method to allow the in vitro study of the micelle solubility of cholesterol in the presence of leaves extracts of the plant Cymbopogon citratus (infusion and non-lipid infusion) and prove, with this methodology, the potential activity of this plant, already described as having hypercholesterolemic activity and lipid-lowering activity in literature. Additionally, the contribution of its phenolic compounds (phenolic acids, flavonoids and tannins) for such activity was evaluated. The results obtained allow to conclude that both extracts, as well as the phenolic acid, flavonoids and tannins show a micelle destruction capacity, particularly the two last classes of phenolic compounds to which percentages over 90% of micelle destruction were obtained for similar flavonoids and tannins concentrations (1 µg/mL and 25 µg/mL respectively). These results suggest that this is one of the possible mechanisms by which the cholesterol-lowering and fat-lowering effects are observed for this plant and mostly flavonoids and tannins are the phenolic compounds contributing to this activity. In conclusion, a rapid-test was optimized for the in vitro study of micelle solubility of cholesterol in the presence of extracts and phenolic fractions of Cymbopogon citratus, which allows for a conducting mechanism to the fat-lowering and cholesterol-lowering activities. This essay allowed also to evaluate the fat-lowering potential of Cymbopogon citratus as well as inferring that this activity is, at the least partly, related to the phenolic compounds, namely flavonoids and tannins

Screening of antioxidant activity in microalgae

Due to the toxicity of synthetic antioxidants, the demand for alternative sources of antioxidants over the years has increased. The interest in such compounds is related to their importance in human health and food quality. Microalgae, a group of organisms with high morphological diversity and able to produce a wide variety of biochemical compounds are considered a promising, non expensive source of antioxidant compounds (Guedes et al., 2013a). This work reports the screening of several different species of microalgae for their antioxidant behavior using the 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid radical cation (ABTS•+) spectrophotometric assay for antioxidant activity (Guedes et al., 2013b) to 50 microalgae from the Coimbra Collection of Algae (ACOI). This method is based on the discoloration of a radical solution expressed as a radical inhibition activity. Recorded values obtained varied between 0.19 and 12.31 mg.L1 equivalent ascorbic acid concentration. In order to confirm the results, the initial screening was followed by the application of the ABTS•+ assay and another common method for antioxidant activity namely that using 2,2-diphenyl-1-picrylhydrazyl (DPPH•) to 8 selected strains from the first 50 and belonging to different taxonomic groups. The DPPH• method consists on the decrease of absorbance of a solution containing the radical in the presence of antioxidants (Brand-Williams et al., 1994). The results obtained for these 8 strains ranged from 2 to 30 mg.L-1 equivalent ascorbic acid concentration for the ABTS•+ assay and from 7 to 13% inhibition of DPPH•. Both sets of results indicate an interesting antioxidant potential in microalgae belonging to the groups Eustigmatophyceae and Chlorophyceae. Tested species of these groups showed ABTS•+ values comparable to grape and raspberry ethanolic extracts, confirmed also by the DPPH• method

Effect of Phenolic Compounds from Cymbopogon citratus (DC) Stapf. Leaves on Micellar Solubility of Cholesterol

Dyslipidemias are one of the risk factors for cardiovascular diseases, the leading cause of death and hospitalization worldwide. One way to control cholesterol levels is to control the exogenous cholesterol intake in the body. Natural polyphenolic compounds, namely theaflavins from plant extracts such as black tea, showed the ability to inhibit the formation of the micellar structure, essential for the absorption of cholesterol in the intestine. There are several methodologies to determine this effect, many of which are expensive and time-consuming. Due to these facts, the main purposes of this work were to optimize an inexpensive colorimetric method to study, in vitro, the micellar solubility of cholesterol and applied it to plant extracts. In this work, Cymbopogon citratus leaf extracts, its phenolic fractions, and flavonoids were evaluated. The non-delipidified infusion (CcI) obtained a maximum percentage of micelle destruction of 59.22% for a concentration of 50 μg/mL and the delipidified infusion (CcdI) obtained a maximum percentage of micelle destruction of 58.01% for a concentration of 200 μg/mL. In the case of the fraction of phenolic acids (CcPAs), 23.85% of maximum micellar destruction was recorded for the concentration of 100 μg/mL, while for the fraction of flavonoids (CcF), the micellar destruction was 92.74% at 1 μg/mL, and for the tannin fraction (CcT) of 99.45% at 25 μg/mL. Luteolin presented a percentage of micelle destruction of 94.83% in the concentration of 1 ng/mL, followed by luteolin-7-O-glucoside with 93.71% and luteo-lin-6-C-glucoside with 91.26% at the concentrations of 25 ng/mL and 50 ng/mL, respectively. These results suggest the capability of polyphenols from Cymbopogon citratus to prevent the cholesterol absorption in the gut by micellar destruction, and its contribution for cholesterol-lowering activity

Re-Thinking Table Salt Reduction in Bread with Halophyte Plant Solutions

Sodium intake higher than it is physiologically necessary has been associated with some non-communicable diseases such as hypertension, cardiovascular disease, and stroke. Bread is commonly consumed and is a major source of sodium in the human diet. Among the interventions to reduce the salt content in bread, the incorporation of salty taste halophyte powder could be a promising strategy. In the present work, Sarcocornia perennis was incorporated as a food ingredient to substitute the salt (sodium) of white wheat bread (1.2% NaCl/0.47% sodium, flour basis). Powdered dried S. perennis was incorporated into bread by replacing the same amount of sodium (0.47%, flour basis) and half of the sodium concentration (0.235%, flour basis), respectively, B100 and B50 bread samples. The bread samples were analyzed to evaluate the impact of the sodium chloride replacement by S. perennis powder on total baking loss, specific volume, crumb color, textural properties, microbial activity, nutritional and mineral composition and sensory evaluation. The incorporation of S. perennis increased the specific volume but had no relevant impact on the textural properties of bread. Furthermore, the substitution of sodium chloride by S. perennis powder allowed a more colored (greenish and yellowish) and dark crumb leading to a lower whiteness index. Compared with control bread, the addition of S. perennis powder promoted a significant increase of all bread’s nutrients and minerals, namely calcium, phosphor, iron, and manganese. Besides the improvement of bread quality, B100, and B50 bread samples were both sensorily well accepted and with similar scores to all the evaluated sensorial attributes. Moreover, the reduction of sodium to half (0.235% sodium (flour basis) in bread (B50) did not affect the acceptability of tasters, as compared with B100 (0.47% sodium (flour basis)). Both new bread formulation has microbiological quality as ready-to-eat product. However, taking into account greater stability over time for microbial spoilage, mainly caused by fungi and yeasts, B50 bread is more promising. The B50 bread sample is also a potential strategy to obtain a sodium reduction of 50% in bread, which could be essential to reduce the overall sodium daily intake and bring important economic and public health benefits

Crepis vesicaria L. subsp. taraxacifolia Leaves: Nutritional Profile, Phenolic Composition and Biological Properties

Crepis vesicaria subsp. taraxacifolia (Cv) of Asteraceae family is used as food and in traditional medicine. However there are no studies on its nutritional value, phenolic composition and biological activities. In the present work, a nutritional analysis of Cv leaves was performed and its phenolic content and biological properties evaluated. The nutritional profile was achieved by gas chromatography (GC). A 70% ethanolic extract was prepared and characterized by HLPC-PDA-ESI/MSn. The quantification of chicoric acid was determined by HPLC-PDA. Subsequently, it was evaluated its antioxidant activity by DPPH, ABTS and FRAP methods. The anti-inflammatory activity and cellular viability was assessed in Raw 264.7 macrophages. On wet weight basis, carbohydrates were the most abundant macronutrients (9.99%), followed by minerals (2.74%) (mainly K, Ca and Na), protein (1.04%) and lipids (0.69%), with a low energetic contribution (175.19 KJ/100 g). The Cv extract is constituted essentially by phenolic acids as caffeic, ferulic and quinic acid derivatives being the major phenolic constituent chicoric acid (130.5 mg/g extract). The extract exhibited antioxidant activity in DPPH, ABTS and FRAP assays and inhibited the nitric oxide (NO) production induced by LPS (IC50 = 0.428 ± 0.007 mg/mL) without cytotoxicity at all concentrations tested. Conclusions: Given the nutritional and phenolic profile and antioxidant and anti-inflammatory properties, Cv could be a promising useful source of functional food ingredients

Crepis vesicaria L. subsp. taraxacifolia Leaves: Nutritional Profile, Phenolic Composition and Biological Properties

Crepis vesicaria subsp. taraxacifolia (Cv) of Asteraceae family is used as food and in traditional medicine. However there are no studies on its nutritional value, phenolic composition and biological activities. In the present work, a nutritional analysis of Cv leaves was performed and its phenolic content and biological properties evaluated. The nutritional profile was achieved by gas chromatography (GC). A 70% ethanolic extract was prepared and characterized by HLPC-PDA-ESI/MSn. The quantification of chicoric acid was determined by HPLC-PDA. Subsequently, it was evaluated its antioxidant activity by DPPH, ABTS and FRAP methods. The anti-inflammatory activity and cellular viability was assessed in Raw 264.7 macrophages. On wet weight basis, carbohydrates were the most abundant macronutrients (9.99%), followed by minerals (2.74%) (mainly K, Ca and Na), protein (1.04%) and lipids (0.69%), with a low energetic contribution (175.19 KJ/100 g). The Cv extract is constituted essentially by phenolic acids as caffeic, ferulic and quinic acid derivatives being the major phenolic constituent chicoric acid (130.5 mg/g extract). The extract exhibited antioxidant activity in DPPH, ABTS and FRAP assays and inhibited the nitric oxide (NO) production induced by LPS (IC50 = 0.428 ± 0.007 mg/mL) without cytotoxicity at all concentrations tested. Conclusions: Given the nutritional and phenolic profile and antioxidant and anti-inflammatory properties, Cv could be a promising useful source of functional food ingredients

Sea Purslane as an Emerging Food Crop: Nutritional and Biological Studies

alophyte plants are highly adapted to salt marsh ecosystems due to their physiological and ecological characteristics. Halimione portulacoides (L.) Aellen is one abundant halophyte shrub that belongs to a Chenopodiaceae family and Caryophyllales order and is found on sandy or muddy coastlines and salt marshes. In this study, the leaves of sea purslane (H. portulacoides) grown in Figueira da Foz (Portugal) were characterized at nutritional and mineral concentration. Moreover, different methanolic extracts were obtained from the leaves, and the antioxidant activity was assessed by several methods. From a nutritional point of view, this halophyte plant may be considered a good source of dietary fiber, protein, natural minerals such as calcium, magnesium, manganese, copper, and potassium. The primary sugar found in leaves of sea purslane is maltose, followed by sucrose, glucose, and fructose. Finally, leaves showed a high content of phenolic compounds and considerable antioxidant activity. The novel products butter and pasta enriched with powder dried leaves of H. portulacoides revealed the plant’s potential to be used as a salt substitute and a good alternative to enhance the sensory characteristics of products, with additional health benefits. The nutritional characteristics and the phytochemical value highlight H. portulacoides as a potential candidate crop in saline agriculture and to be used as a new vegetable, especially as a premium food in the novel “salty veggies” market or as a kitchen salt substitute. View Full-Textinfo:eu-repo/semantics/publishedVersio