Ability of great nothern and pinto beans to prevent high cholesterol caused by a diet rich saturated fat in a hamster model

ABILITY OF GREAT NORTHERN AND PINTO BEANS TO PREVENT HIGH CHOLESTEROL CAUSED BY A DIET RICH IN SATURATED FAT IN A HAMSTER MODEL by An Tien Nguyen A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirement For the Degree of Doctor of Philosophy Major: Food Science and Technology Under the Supervision of Professor Vicki Schlegel Lincoln, Nebraska July, 2018 ABILITY OF GREAT NORTHERN AND PINTO BEANS TO PREVENT HIGH CHOLESTEROL CAUSED BY A DIET RICH IN SATURATED FAT IN A HAMSTER MODEL An Tien Nguyen, Ph.D University of Nebraska, 2018 Advisor: Vicki Schlegel Great northern and pinto beans have been shown to possess a variety of bioactive components that potentially provide cholesterol lowering properties In this research project, three animal studies were carried out to investigate the ability and possible underlying mechanism of raw great northern and pinto beans as well as steamed pinto beans to mitigate the high cholesterol caused by a diet rich in saturated fat In each study, hamsters were randomly assigned into four different groups (n = 11), each received either a normal fat diet (NF), a diet rich in saturated fat (HSF) or HSF diets supplemented with % (w/w) whole beans or 0.5 % (w/w) bean hulls over a period of four weeks The results showed that the supplementation of the raw and steamed whole beans to the HSF diet significantly reduced plasma and liver cholesterol in experimental animals The decrease in plasma and / or hepatic cholesterol occurred concurrently with the increased excretion of cholesterol in the feces, indicating that the supplements promoted the elimination of cholesterol out of body The analyses of several important genes involved in cholesterol homeostasis revealed that the supplements primarily inhibited the absorption of cholesterol from the intestinal lumen Apart from acting on the cholesterol absorption pathway, the raw pinto beans were also able to inhibit hepatic cholesterol synthesis by suppressing the gene responsible for this activity In contrast, the cholesterol synthesis in the liver actually increased in great northern bean supplemented animals, which might help prevent the depletion of cholesterol due to high excretion The results also demonstrated that heat treatment during steaming negatively affected the efficacy of pinto beans in reducing cholesterol Among the hull supplements, only raw pinto bean hulls added at 0.5 % (w/w) was able to lower cholesterol, which might be attributed to its high level of phenolic compounds Nevertheless, the hulls played an important role in the overall capability of the beans to prevent high cholesterol caused by a diet rich in saturated fat, as demonstrated by their effects in molecular regulation of cholesterol homeostasis ACKNOWLEDGEMENT It would not have been possible for me to complete my Ph.D program as well as this research project without the help and supports from the kind people around me I am indebted to them and would like to take this chance to express my deepest gratitude Above all, I would like to sincerely thank my advisor, Dr Vicki Schlegel for her enthusiastic guidance throughout my whole academic life at the University of Nebraska – Lincoln She taught me how to plan the project and the lab work ahead of time, enabling me to complete my research in a timely manner She taught me to pay attention to the details and think critically to solve the problems She always helped me even when she had difficult times She inspired me a lot as a supportive advisor, critical thinker and professional writer Apart from academic activities, she also cared much about my personal life, for which I am very grateful I would like to deeply thank the other professors on my advisory committee, Dr Wehling, Dr Urrea and Dr Weller, who have given me invaluable suggestions and knowledge without which my research project would not have been completed successfully Thank you to all my committee members for their contributions to my program of study as well as my research plan, which helped me to be on the right track during my academic journey at UNL I was fortunate to have them on my committee Thank you to Dr Wehling for helping me develop critical thinking in food analysis, letting me use his instrument and reviewing my dissertation Thank you to Dr Urrea for generously providing me with the bean samples, helping me develop the knowledge in statistics and reviewing my dissertation Thank you to Dr Weller for his encouragement and caring about my progress and plans I would also like to thank Dr Carr for sharing invaluable knowledge of cholesterol metabolism, which is extremely critical to my research I would like to sincerely thank my lab partners, Sami Althwab and Haowen Qiu (Max) for their help with animal care and collecting samples Thank you to Sami for showing me every detail and instruction on animal experiments and his help with the analysis of cholesterol Thanks to you both for sharing your experience and knowledge It was a privilege experiencing academic life with them Thank you so much to the lab manager, Richard Zbasnik, for training me on how to use the analytical instruments, ordering chemicals, analyzing sugars and numerous other things that I cannot list here Thank you to all other lab members for always being supportive during my studying time at Dr Schlegel’s lab I am truly indebted to my wife and my son and other family members who have given me their unequivocal love and encouragement as I pursued my Ph.D Definitely my mere expression of thanks can never suffice I would like to acknowledge the Vietnamese government, UNL Department of Food Science and Technology and Dr Schlegel for providing financial support for my Ph.D study vi Table of Contents Literature Review and Project Objective / Significance: Heart health benefiting properties of dry edible beans (Phaseolus vulgaris L.), with an emphasis on great northern beans and pinto beans I Literature review A Introduction B Great northern beans and pinto beans C Bioactive composition of dry edible beans D Heart health promoting functionality of dry edible beans 10 E Conclusion 20 II Project Objective / Significance 20 III References 25 Chapter 1: Great northern beans prevent high cholesterol induced by a saturated fat diet by suppressing the expression of genes involved in small intestinal cholesterol absorption in hamsters 41 1.1 Abstract 41 1.2 Introduction 42 1.3 Materials and methods 43 1.4 Results 51 1.5 Discussion 60 1.6 References 64 vii Chapter 2: Raw pinto beans lower low density lipoprotein cholesterol in hamsters fed a diet rich in saturated fat by regulating genes involved in cholesterol homeostasis 73 2.1 Abstract 73 2.2 Introduction 74 2.3 Materials and methods 75 2.4 Results 83 2.5 Discussion 93 2.6 References 98 Chapter 3: Effect of thermal processing on ability of pinto beans to modulate cholesterol in hamsters fed a saturated fat diet 107 3.1 Abstract 107 3.2 Introduction 108 3.3 Materials and methods 110 3.4 Results 117 3.5 Discussion 125 3.6 References 130 Chapter 4: Summary 139 viii List of Figures I The evolution of dry edible beans represented by the seed II The production of dry edible beans in the United States in 2016 1.1: Plasma HDL and non-HDL cholesterol concentration of hamsters subjected to normal fat diet (NF), high saturated fat diet (HSF) or HSF diets supplemented with 5% whole beans (HSF+wGNB) or 0.5% hulls (HSF+hGNB) 53 1.2: Correlations between total cholesterol output and total liver cholesterol (A) and total plasma cholesterol (B) in hamsters subjected to a high saturated fat diet (HSF) and HSF diets supplemented with 5% whole beans (HSF+wGNB) and 0.5% hulls (HSF+hGNB) 57 1.3: mRNA levels of small intestinal MTP, ACAT2, NPC1L1, ABCG5 and ABCG8 (A) and hepatic SREBP2, HMGR, CYP51, CYP7A1 and LDLR (B) in hamsters subjected to a normal fat diet (NF), high saturated fat diet (HSF) or HSF diets supplemented with 5% whole beans (HSF+wGNB) or 0.5% hulls (HSF+hGNB) 58 2.1 Plasma HDL and non-HDL cholesterol concentration of hamsters subjected to a normal fat diet (NF), high saturated fat diet (HSF) or HSF diets supplemented with % pinto beans (HSF+wPB) or 0.5 of pinto bean hull (HSF+hPB) 87 2.2 Correlations between total cholesterol output and total liver cholesterol in hamsters subjected to a high saturated fat diet (HSF) and HSF diets supplemented with % pinto beans (HSF+wPB) and 0.5 % pinto bean hulls (HSF+hPB) 90 2.3 mRNA levels of hepatic SREBP2, HMGR, CYP51, CYP7A1 and LDLR (A) and small intestinal MTP, ACAT2, NPC1L1, ABCG5 and ABCG8 (B) in hamsters subjected to a normal fat diet (NF), high saturated fat diet (HSF) or HSF diets supplemented with % beans (HSF+wPB) or 0.5 of hulls (HSF+hPB) 91 ix 3.1 Plasma HDL, non-HDL cholesterol and triglyceride concentration of hamsters subjected to a normal fat diet (NF), high saturated fat diet (HSF) or HSF diets supplemented with % steamed pinto beans (HSF+wSP) or 0.5 % steamed pinto bean hulls (HSF+hSP) 120 3.2 Correlations between total cholesterol output and total plasma cholesterol in hamsters subjected to a high saturated fat diet (HSF) and HSF diets supplemented with % steamed pinto beans (HSF+wSP) and 0.5 % steamed pinto bean hulls (HSF+hSP) 123 3.3 mRNA levels of hepatic SREBP2, HMGR, CYP51, CYP7A1 and LDLR (A) and small intestinal MTP, ACAT2, NPC1L1, ABCG5 and ABCG8 (B) in hamsters subjected to a normal fat diet (NF), high saturated fat diet (HSF) or HSF diets supplemented with % steamed pinto beans (HSF+wSP) or 0.5 % steamed pinto bean hulls (HSF+hSP) 124 x List of Tables I Total phenolic content (TPC) and total flavonoid content (TFC) of pinto beans 1.1 Composition of treatment diets 47 1.2 Chemical compositions of great northern beans and their hulls 52 1.3 Hepatic lipid profiles of hamsters subjected to four diet treatments 55 1.4 Fecal lipid profiles of hamsters subjected to four diet treatments 55 1.5 Correlation of total plasma, hepatic and fecal cholesterols with hepatic and intestinal mRNA expression in hamsters subjected to HSF diets 59 2.1 Composition of treatment diets 80 2.2 Chemical composition of pinto beans and their hulls 85 2.3 Hepatic lipid profiles of hamsters subjected to four diet treatments 89 2.4 Fecal lipid profiles of hamsters subjected to four diet treatments 89 2.5 Correlation of total plasma, hepatic and fecal cholesterol levels with hepatic and intestinal mRNA expression in hamsters subjected to all HSF diets 92 3.1 Composition of treatment diets 114 3.2 Chemical composition of steamed pinto beans and their hulls 119 3.3 Hepatic lipid profiles of hamsters subjected to four diet treatments 122 3.4 Fecal lipid profiles of hamsters subjected to four diet treatments 122 3.5 Plasma, hepatic and fecal cholesterols in correlation with hepatic and intestinal mRNA expression in hamsters subjected to all HSF diets 126 130 supplemented diet in this study did not have a the similar effect It was noted that the amount of phenolic compound that each hamster consumed on average in this study was ~ 31.0 mg GAE while in previous study it was 59.2 mg GAE The degradation of phenolic compounds caused by steaming was the most likely reason for this difference as polyphenols are a potent HMGR suppressor (Liu, Wu, Guo, Meng, & Chang, 2018) In conclusion, although steaming caused the degradation of a marked quantity of bioactive components, wSP supplemented at % (w/w) into a HSF diet still effectively decreased the concentration of plasma cholesterol This reduction was achieved by the promotion of fecal cholesterol excretion in the form of neutral sterols Results of this study indicate that the underlying mechanism is the inhibition of intestinal NPC1L1 and MTP expression by the wSP which reduces cholesterol absorption and fecal cholesterol excretion, thereby reducing plasma cholesterol levels The wSP might provide additional protection against cardiovascular disease as the 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chemical composition of two market classes of dry edible beans, including great northern (GNB) and pinto beans (PB) in the raw state, and then to investigate the cholesterol lowering ability of these beans in vivo by using a hamster model The effect of heat during cooking was also taken into consideration for steamed PB, which was completed with another animal study to evaluate the effect of cooking on the ability of beans to modulate cholesterol For characterization of the beans, multiple chemical components, including macro constituents (crude protein, carbohydrate, lipid, ash and moisture content), soluble/insoluble fibers, oligosaccharides, total phenolic compounds, total flavonoids, total condensed tannins, total saponins, phenolic profiles and fatty acid composition, were analyzed to better understand the effect of the chemically diverse bean components on cholesterol homeostasis None of the beans differed significantly in their quantity of the macro nutrients, which were about 20 % protein, % lipid ad 65 % carbohydrates The total fiber was greater in GNB (~ 184 mg/g) than in PB (~ 159 mg/g) In contrast, total phenolic compounds were greater in PB (5.77 mg GAE/g) than in GNB (1.8 mg GAE / g) Similarly, PB contained more flavonoids and tannins (6.09 and 8.72 mg CE / g, respectively) compared to GNB (0.27 and 6.7 mg CE / g, respectively) The level of saponins was similar in the raw form of GNB and PB, which was approximately 28 mg Aescin / g Cooking (steaming) significantly reduced the quantity of bioactive compounds in PB, particularly total phenols, flavonoids, tannins and of saponins with 140 losses of approximately 2.2 mg GAE, 3.3 mg CE, 4.2 mg CE and mg Aescin equivalent, respectively, per gram of PB For the animal studies, the design included four experimental groups of hamsters, in which each received one of four different diets formulated based on a standard diet for rodents, including 1) diet containing a normal level of lipid (NF), 2) diet containing an additional 10 % of lipid rich in saturated fat (HSF), 3) HSF diet supplemented with % (w/w) beans and 4) HSF diet supplemented with 0.5 % (w/w) bean hulls The NF treatment was given as a negative control which represents a diet that is generally considered a healthy diet for heart health while the HSF diet represents a typical western diet with a high saturated fat component that is well known to elevate plasma cholesterol The other treatments including the supplementation of beans or bean hulls into the HSF diet were designed to evaluate the capability of beans or hulls to modulate cholesterol Bean hulls were used to better understand whether the effects solely come from bioactive components in the hulls or from more complex involvement of components in the whole beans After a period of feeding over four weeks, multiples parameters were analyzed with the most important markers being plasma, hepatic and fecal cholesterol levels The expressions of selected genes involved in cholesterol homeostasis were also analyzed using real-time RT-PCR to unfold any possible mechanisms underlying the cholesterol modulation exerted by the supplements In the first chapter, we showed that GNB are an effective cholesterol reducer by decreasing both plasma and liver cholesterol levels This reduction effect was achieved by the tremendous increase in cholesterol excretion in the feces The gene expression analysis showed that great northern beans suppressed NPC1L1 and ACAT2, thereby 141 promoting the excretion of fecal sterols Endogenous cholesterol synthesis was actually increased as shown by the upregulation of HMGR and CYP51 in bean fed hamsters, most likely to prevent the over depletion of cholesterol caused by secretion The hull played a role in the overall effect of the GNB as it affected the expression of several genes, such as ABCG5 and MTP This impact, however, was not expressed in biomarkers It must be noted that each hamster consumed ~ 10.4 g of GNB in a period of weeks, which translates into ~ 1.9 g fiber, ~ 18.6 mg total phenols, ~ 2.8 mg flavonoids, ~ 69 mg tannins and ~ 284 mg saponins In the second chapter, the cholesterol lowering capability of PB was clearly evident via the reduction of plasma and liver cholesterol in PB supplemented hamsters compared to HSF diet fed animals This reduction in cholesterol was associated with the elevation of cholesterol excretion The gene expression analysis showed that PB exerted cholesterol reducing ability by impacting both endogenous cholesterol synthesis as well as exogenous absorption In particular, expression of hepatic HMGR and SREPB2 as well as intestinal NPC1L1 and CAT2 was reduced in PB fed hamsters In this study, the PB hull also exhibited an ability to lower plasma non-HDL cholesterol and it clearly decreased the expression of HMGR and ACAT2 These results confirmed the crucial role of the hull, which contained a vast number of bioactive components, especially phenols and saponins, in the biological effect of the beans In this study, each hamster consumed 10.3 g PB over four weeks, which was equivalent to ~ 1.7 g fiber, ~ 60 mg total phenols, ~ 63 mg flavonoids, ~ 90.5 mg tannins and ~ 307 mg saponins The steaming significantly reduced the concentration of several bioactive components in PB In particular, the reduction of total phenols, flavonoids and condensed 142 tannins in steamed PB compared to the raw was 39, 55 and 48 %, respectively The third animal study showed that even after steaming, PB were able to lower both plasma nonHDL and liver cholesterol In addition, steamed PB promoted the excretion of cholesterol into feces The gene expression analysis showed that the underlying mechanism for the above effects of the cooked beans was the down regulation of intestinal MTP and NPC1L1 Regarding the bioactive components consumed, the average amount of steamed PB intake by each animal over four weeks was 8.8 g, which contained ~ 1.4 g fibers, 31 mg total phenols, ~ 24 mg flavonoids, ~ 40 mg tannins and ~ 208 mg saponins In general, GNB and PB shared some similarities in their ability to lower cholesterol They both reduced plasma and liver cholesterol and promoted the secretion of fecal sterols At the molecular level, both beans suppressed the expression of intestinal NPC1L1 and ACAT2 This effect might derive from the similarity in chemical composition of GNB and PB, such as protein, phenolic compounds and saponins However, several differences existed between GNB and PB with respect to their capability of cholesterol modulation For example, the magnitude of non-HDL cholesterol reduction caused by GNB was 51.5 %, which was higher compared to 31.9 % caused PB However, GNB also reduced the HDL levels in animals fed HSF diet whereas PB did not which is more beneficial In particular, the increase in de novo cholesterol synthesis in hamsters fed GNB was evidenced by the upregulation of HMGR and CYP51, while PB effectively reduced synthesis of cholesterol via the downregulation of HMGR and SREPB2 These differences might be the result of differences in the nature and quantity of bioactive compounds that hamsters consumed as mentioned above 143 It is evident from this work that thermal processing, steaming for instance, led to considerable losses of bioactive components in PB as described above Hence, the capability of PB to lower cholesterol was also compromised In particular, PB lost their ability to reduce endogenous cholesterol synthesis in the liver However, the bioactive compounds remaining after steaming PB were able to reduce both plasma HDL and nonHDL cholesterol In addition, the steamed PB also lowered plasma triglycerides to a level comparable to the normal fat diet, which was not exhibited by the raw beans From this result, it was very likely that heating not only caused a degradation of bioactive components but also modified the composition of bioactive compounds, or altered the interaction of between bioactive compounds regarding their action on the cholesterol homeostasis The additive or synergistic effect of multiple bioactive components on the cholesterol lowering ability of the beans was discussed in this research project but could not be fully elucidated because of certain limitations of this study However, the information obtained through these studies lay the foundation for future research For example, individual bioactive components can be isolated from beans and used as primary active ingredients in future studies, which might lead to the development of functional foods with bean-based bioactive substitutes or supplements containing the isolated ingredients In addition, animal studies can be conducted using bean cotyledon as the cholesterol modulator which, coupled with thorough characterization, will aid in understanding the synergistic and / or additive effect provided by beans Research can also expand to investigate the effect of bean consumption on the regulation of other important genes and proteins involved in cholesterol modulation, which will provide 144 greater insight into the potential of beans from multiple market classes to modulate cholesterol .. .ABILITY OF GREAT NORTHERN AND PINTO BEANS TO PREVENT HIGH CHOLESTEROL CAUSED BY A DIET RICH IN SATURATED FAT IN A HAMSTER MODEL An Tien Nguyen, Ph.D University of Nebraska, 2018 Advisor:... were carried out to investigate the ability and possible underlying mechanism of raw great northern and pinto beans as well as steamed pinto beans to mitigate the high cholesterol caused by a diet. .. Correlations between total cholesterol output and total plasma cholesterol in hamsters subjected to a high saturated fat diet (HSF) and HSF diets supplemented with % steamed pinto beans (HSF+wSP) and