br Methods br Results br Discussion
Discussion Literature showed SN was well studied for its antigastric ulcer, antiinflammatory, antihyperglycemic, and antihyperlipidemic properties. Although a recent publication reported that SN was found to exert its regulatory effect in animal cholesterol levels, the exact mechanism of action, particularly on the cholesterol Gemcitabine inhibition, is still unclear. Cholesterol homeostasis involves a balance in intestinal absorption, endogenous synthesis, bile acid activities, and fecal excretion. Intestinal cholesterol absorption involves several processes which include lipid digestion, micellar solubilization, cholesterol release from micelles into enterocytes, and secretion into lymph. The reduction of cholesterol absorption by reducing micellar cholesterol solubility is suggested for the treatment of hypercholesterolemia. Raederstorff and coworkers suggested that the uptake of cholesterol could interfere with the affinity of micelles into membranes and/or the affinity of cholesterol in micelles. Additionally, the binding of bile acids might act as a possible mechanism in regulating cholesterol levels. In detail, the bile acid sequestrants bind to bile acids in the intestine resulting in the formation of an insoluble complex which can be excreted in the feces. The activation of this process is known to play an important role in regulating the cholesterol levels in hypercholesterolemia condition.18, 20 In order to assess the inhibitory effect of SN extract on cholesterol absorption, Caco-2 cells were used as model of experimentation specifically for the cholesterol absorption study. Our results indicate that SN extract decreased cholesterol uptake in Caco-2 cells. In our attempt to address the possible involvement of the cholesterol absorption mechanism in observed activity, the effect of SN extract on cholesterol micelles size and solubility and its binding property to bile acids was studied. SN also demonstrated a dose-dependent decrease in solubility of cholesterol in micelles and a significant increase in size of micelles. In addition, SN revealed bile acids binding property (TC, GC, and TD). In fact, Fernandez suggested that soluble-fiber polysaccharides may play an important role in modifying the volume, bulk, and viscosity of the intestinal lumen. These effects were believed to interfere with the enterohepatic circulation and lipoprotein metabolism which may cause a resulting effect on lower cholesterol levels. Oat bran with native beta-glucans, polysaccharides, increased fecal bile acid excretion, as the β-glucans in oat bran entrap or encapsulate whole micelles in the small intestine. Besides, tea polyphenols (epigallocatechin gallate) are known to interact with TCs in micellar solutions, eventually inhibiting the solubilization of cholesterol. Meanwhile, the polyphenols in grape seed (gallic acid, catechin, and epicatechin) have high binding tendency to bile acids, which will cause a reduction in the solubilization of cholesterol in micelles. A similar activity was also found in black tea polyphenols as they decrease the in vitro micellar solubility of cholesterol and have been shown to interrupt intestinal cholesterol absorption in rats. In the present study, we found that SN contains polyphenolic compounds such as isoquercetin and catechin. Besides polyphenolic compounds, SN was also known to possess polysaccharides. Taken altogether, we postulated that the activities exhibited by SN extract could be possibly linked to the presence of polyphenols and polysaccharides. By contrast, intestinal cholesterol absorption can be specifically inhibited by cholesterol absorption inhibitors (ezetimibe). However, the treatment with ezetimibe showed no effect in NPC1L1 knockout mice, suggesting that NPC1L1plays a role in the uptake of cholesterol. Our results show for the first time that SN extract inhibited cholesterol uptake, and the observed activity possibly acted through an inhibitory effect on cholesterol transporters. Therefore, the experiment evaluated the combinatory effect of SN extract and ezetimibe. The results showed that the combination of SN extract and ezetimibe (0.04mg/mL) achieved a significantly greater reduction in cholesterol uptake than SN extract alone. However, the combinatory effect was still lower as compared to ezetimibe treatment alone. In the combination, additive effect refers to the sum of their effects, the synergistic effect refers to the effect which is greater than the sum of their effect, whereas an antagonistic effect is the opposite of synergistic effect. Our results suggest that SN and ezetimibe decreased cholesterol uptake and there was no synergetic effect with a combination of SN extracts and ezetimibe. Normally, ezetimibe does not completely inhibit cholesterol absorption, thus our results implied that SN may share the same mechanistic action sites with ezetimibe by inhibiting cholesterol transporter (NPC1L1) and/or effects on the internalization of transporter (NPC1L1) between intracellular compartments and membrane.