Influence of dietary tender cluster beans (Cyamopsis tetragonoloba) on biliary proteins, bile acid synthesis and cholesterol crystal growth in rat bile
Graphical abstract
Introduction
Cholesterol gallstone (CGS) disease is a gastroenterological disorder wherein various factors are involved in the CGS formation in the gallbladder. Even if the same degree of super saturation of cholesterol is observed in the bile of a normal individual and a CGS afflicted individual, appearance of cholesterol crystallization will be dissimilar in the bile of the CGS patient when compared to that of the normal individual [1]. Cholesterol gallstone formation involves procrystalizing and anticrystalizing factors viz., calcium, bilirubin, high molecular weight (HMW) proteins, low molecular weight (LMW) proteins and increased cholesterol saturation index, cholesterol: phospholipid ratio and expression of genes involved in bile acid synthesis. Cholesterol-7α-hydroxylase and cholesterol-27-hydroxylase are major rate-limiting enzymes in bile acid synthesis. Bile acid synthesis pathway is important to remove excess of cholesterol from liver. Recently, we have reported that tender cluster beans (Cyamopsis tetragonoloba) and garlic (Allium sativum) have the potential to reduce the formation of cholesterol gallstone in experimental mice [2]. Tender cluster beans are considered as soluble dietary fiber-rich vegetable which obstruct the cholesterol absorption in the intestine and also produce short chain fatty acids (SCFA) by large intestinal microbial flora. These SCFA have an impact of reducing endogenous cholesterol synthesis, fatty acid and very low density lipoproteins [3]. Garlic acts through its active principle diallyl disulfide, inactivating enzymes and substrates containing thiol groups in an exchange reaction; increases hydrolysis of triacylglycerols through induction of lipase activity; and reduces the biosynthesis of triacylglycerols as NADPH is made unavailable for the process. The hypocholesterolemic effect of garlic stems in part from decreased hepatic cholesterogenesis [4].
Besides these, cluster beans and garlic may also have an effect on biliary proteins and thus on cholesterol nucleation time in bile. Hence the present study investigates biliary proteins in the bile of experimental rats maintained on these dietary interventions and also cholesterol nucleation time in a model bile system. HMW proteins and LMW proteins were separated on gel permeation chromatography and by electrophoresis. These proteins were appropriately inserted and checked for the nucleation time and appearance of cholesterol crystals in rat bile of different dietary groups and in a model bile system. This particular investigation was undertaken to observe the impact of these two food ingredients on proteins present in the bile that are likely to influence cholesterol crystallization in a supersaturated bile, cholesterol crystal growth in model bile system and also expression of CYP7A and CYP27 genes.
Section snippets
Chemicals
Cholesterol, bile salts, dipalmitoyl phosphatidylcholine, triolein, 3α-hydroxysteroid dehydrogenase, standard bile acids, 7β-hydroxycholesterol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium azide, acrylamide, bis-acrylamide, tetramethyl ethylenediamine (TEMED), ammonium persulphate, protein markers, DNA ladder, TRI-reagent, diethyl pyrocarbonate (DEPC), RNA-later, urethane and alpha cellulose were purchased from Sigma–Aldrich Chemicals (St. Louis, MO, USA). Intramedic
Biliary lipid profile
As shown in Table 1, in HCD fed animals the bile acid content was increased by about 1.5-fold compared to control group. On the other hand, CB, garlic and combination of both along with HCD increased the bile acid content compared to control as well as lithogenic diet. Cholesterol level in the bile of HCD fed animals was increased by 3.0-fold compared to control. In the case of CB and garlic incorporated with control diet there was no significant alteration compared to control. In animals fed
Discussion
Dietary tender CB was evidenced to possess anti-cholelithogenic potential in mice both in terms of preventing the experimental induction of CGS and also regressing the pre-established CGS [2]. The anti-cholelithogenic influence of tender cluster beans is attributable to their cholesterol-lowering effect in blood and liver, and the ability to lower cholesterol saturation index by altering the biliary lipid composition, and in other words biliary cholesterol homeostasis. Formation of cholesterol
Acknowledgment
The first author (CKR) is thankful to the Indian Council of Medical Research, New Delhi for awarding Senior Research Fellowship.
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