Conjugated and free sterols from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors and their effect on lipid metabolism and cholesterol transport in rat primary hepatocytes

Genes Nutr (2014) 9:367 DOI 10.1007/s12263-013-0367-1 RESEARCH PAPER Conjugated and free sterols from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors and their effect on lipid metabolism and cholesterol transport in rat primary hepatocytes Rocio A. Chávez-Santoscoy • Armando R. Tovar • Sergio O. Serna-Saldivar • Nimbe Torres • Janet A. Gutiérrez-Uribe Received: 20 August 2013 / Accepted: 8 November 2013 / Published online: 1 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013 Abstract Phytosterols have been widely studied for their cholesterol-lowering effect. Conjugated phytosterol forms have been found more active than free moieties. There are no reports about the sterol profile of black bean seed coats neither its effects on cholesterol metabolism. The aim of this research was to identify and quantify phytosterols from black bean seed coats and to determine their effects on cholesterol micellar solubility and on mRNA and key protein levels involved in lipid/cholesterol metabolism and cholesterol transport in primary rat hepatocytes. Free phytosterols, acylated steryl glycosides, and steryl glycosides were extracted from black bean seed coats. They were identified through HPLC–MS–TOF and quantified through HPLC equipped with UV–visible and evaporative light-scattering detectors. Free and conjugated phytosterols from the coats significantly increased the inhibitory effect of cholesterol micelle formation compared with stigmasterol, which was used as control (P \ 0.05). In addition, phytosterols of black bean seed coat decreased lipogenesis by the downregulation of lipogenic proteins such as sterol regulatory element-binding protein 1 and fatty acid synthesis (FAS) in primary rat hepatocytes. Regarding b-oxidation, phytosterols upregulated the expression of carnitine R. A. Chávez-Santoscoy  S. O. Serna-Saldivar  J. A. Gutiérrez-Uribe (&) Departamento de Biotecnologı́a e Ingenierı́a de Alimentos, Centro de Biotecnologı́a FEMSA, Tecnológico de MonterreyCampus Monterrey, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, NL, Mexico e-mail: A. R. Tovar  N. Torres Departamento de Fisiologı́a de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, C.P. 14000 Mexico, DF, Mexico palmitoyltransferase I and promoted the b-oxidation of long-chain fatty acids. Phytosterols inhibited cholesterol micellar solubility and reduced the activation of the liver X receptor, decreasing hepatic FAS and promoting hepatic b-oxidation of long-chain fatty acids. Keywords Black bean  Cholesterol lowering  Phytosterol composition  Lipid metabolism  Lipogenesis  b-Oxidation Abbreviations ABCG ATP-binding cassette subfamily G members ASG Acylated steryl glycosides CPT1 Carnitine palmitoyltransferase I ELSD Evaporative light-scattering detector FAS Fatty acid synthesis HMGCR 3-Hydroxy-3-methylglutaryl-CoA reductase LXR Liver X receptor Ph Phytosterols from black bean seed coat SG Steryl glycosides SREBP Sterol regulatory element-binding protein T T0901317 Introduction Phytosterols are structurally similar to cholesterol and act in the intestine lumen to lower cholesterol absorption via the higher excretion of fecal cholesterol (Ostlund 2004). The Food and Drug Administration (FDA) (2010) has considered these plant sterols as generally recognized as safe (GRAS) and established that the dietary intake necessary to achieve significant cholesterol plasma reductions 123 367 Page 2 of 9 (5–15 %) is 2 g per day. Similarly, the National Cholesterol Education Program (NCEP) (2001) recommends for adults the daily intake of 2 g phytosterols to reduce plasma LDL and the risk of cardiovascular diseases. However, a conventional Western diet provides only an average of 250 mg/day of phytosterols. Studies with the free forms of phytosterols related to their cholesterol-lowering effects have only been addressed toward the reduction in cholesterol absorption (Carr 2002; Guderian et al. 2007; Lee et al. 2012; Rasmussen et al. 2006; Trautwein and Duchateau 2003). One of the proposed mechanisms for the interference of cholesterol intestinal absorption is the inhibition of micellar solubility (Jesch and Carr 2006; Trautwein and Duchateau 2003). Remarkably, esterified phytosterols have been found more active in the cholesterol-lowering effect than free counterparts (Guderian et al. 2007; Rasmussen et al. 2006). Glycoside sterols (GS) and acylated steryl glycosides (ASG) are the major forms of phytosterols in many foods (Ostlund 2002), and according to Lin et al. (2009), these chemical forms have a similar reduction effect of cholesterol absorption compared with sterol esters. Further studies have demonstrated that the bioactive components responsible for reducing cholesterol absorption are ASG and SG, but not the free phytosterols (Lin et al. 2011). Lin et al. (2011) reported that ASG and SG present in the lumen and mucosa of mice reduced cholesterol absorption, and despite their low absorption, they significantly reduced both plasma and hepatic cholesterol. Other reports showed that ASG have also been used to enhance drug delivery because they are well recognized by some hepatic receptors, allowing the administrated drug to accumulate up to 80 % in the liver (Maitani et al. 2005). Despite the limited absorption of ASG, there is not knowledge whether small amounts of these compounds may exert biological effects in liver cells. There is limited information about the effect of specific free phytosterols (sitosterol and stigmasterol) on the expression of key genes of cholesterol metabolism. A significant decrease in the relative expression of hepatic sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and intestinal ATP-binding cassette subfamily G 5 (ABCG5) has been observed in hamsters fed with a highcholesterol diet and pure sitosterol or stigmasterol (Liang et al. 2011). SREBP is a family of transcription factors that regulate lipid metabolism and activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, as well as fatty acid synthesis (Horton 2002; Sato 2010). On the other hand, ABCG5 and 8 are transporters that belong to the family of reverse cholesterol 123 Genes Nutr (2014) 9:367 transport and are also responsible to limit intestinal absorption and promote biliary excretion of hepatic sterols (Graf et al. 2002). Beans are an important source of several phytosterols, but these phytochemicals have been quantified after the hydrolysis of the seed (Iriti et al. 2009; Nyström et al. 2012; Ryan et al. 2007). For instance, the most abundant phytosterol in kidney beans is sitosterol, containing 86.5 mg/100 g (Ryan et al. 2007). In addition, other authors reported that sitosterol is the main phytosterol found in common beans with a concentration of 27.2 mg/ 100 g (Iriti et al. 2009). For black beans, the most (...truncated)