New analytical method for the quantification of cholesterol-lowering phytosterols in 22 legume samples

Pulses represent one of the traditionally most important dietary components worldwide, supplying proteins, dietary fibres, minerals, and vitamins. Legumes represent a very important source of nutrients such as proteins, mainly complex carbohydrates, as well as dietary fibre and minerals, including magnesium and phosphorus. They also have a low-fat content, mainly of the unsaturated type [1,2]. They also contain numerous bioactive molecules such as polyphenols (flavonols, anthocyanins, phenolic acids, flavanols, etc), and soyasaponins with hypocholesterolemic action. Moreover, legumes are a rich source of phytosterols, a group of chemical compounds of plant origin able to reduce blood levels of cholesterol and in particular of Low-Density Lipoprotein or "LDL", and the consequent risk of atherosclerosis [3,4]. In this work, an HPLC-DAD method was developed for the quantification of cholesterol and 7 main PSs (β-sitosterol, campestanol, campesterol, fucosterol, sitostanol, stigmasterol, and stigmastanol) in legumes samples. The method was validated, and good results were obtained in terms of linearity, repeatability, LOD, and LOQ. Then, a process of extraction for phytosterols from legumes was optimized by carefully modifying a standard method reported in the literature. The developed new procedure through ultrasound-assisted extraction (U.A.E) allowed a reduction of extraction time by half while maintaining a high extraction efficiency. Then, the developed HPLC-DAD method, after being validated, was applied to 22 legume samples. Six phytosterols were observed in legumes and the highest total phytosterols content was found in Black Chickpeas (699.9 mg/kg) followed by Brown Lentil, Whole Fava Beans, and Corona Beans. These results confirm that legumes should be included in the diet as great sources of cholesterol-lowering agents [5].