# Polyphenol-Rich Black Elderberry Extract Stimulates Transintestinal Cholesterol Excretion
## 4 Discussion  
Hypercholesterolememia is one of the primary risk factors for the prevalence of CVD [39]. Cholesterol homeostasis is tightly regulated by the interplay of the liver and in the intestine [4, 6]. Therefore, the hypolipidemic effects of drugs or dietary components target either  
Figure 5: The effect of BEE on fatty-acid metabolism in Caco-2 cells. Cells were treated with 50 or 100 ug/mL BEE for 24 h. The expression of genes involved in fatty-acid metabolism was measured by qRT-PCR. Data are expressed as relative expressions of control. Bars with different letters are significantly different (\(p<0.05\)). Values are means \(\pm\) SEM; \(n=6\). Figure 6: The effect of BEE on the expression of sirtuins (SIRTs) in Caco-2 cells. Cells were treated with 100 ug/mL BEE for 24 h. The expression of SIRT isoforms was measured by qRT-PCR. Data are expressed as relative expressions of control. \(p\) values of significant differences are shown as above bars. Values are means \(\pm\) SEM; \(n=6\). blocking cholesterol synthesis in the liver or inhibiting intestinal cholesterol absorption. Classically, hepatobiliary cholesterol excretion mediated by reverse cholesterol transport has been considered the only way to remove cholesterol from the body [12]. Recently, several publications reported the direct contribution of TICE to fecal neutral sterol in mice models and humans [40; 41; 42; 43; 44; 45; 46]. In this non-biliary cholesterol excretion pathway, the plasma lipoprotein-derived cholesterol is transported from the basolateral to the apical side of enterocytes for direct excretion [47]. The underlying mechanisms regulating TICE are still unclear, but the significant contribution of TICE to cholesterol excretion gives an attractive strategy for preventing CVD. The protective effects of polyphenols against hypercholesterolemia are well known. Black elderberry (_Sambucus nigra_) is well known for its cardioprotective effects by exerting hypolipidemia and improving lipid profiles, oxidative stress, and inflammation [22; 23; 24]. Black elderberry alleviated oxidative stress, insulin resistance, and inflammation in 3T3-L1 cells [29]. In particular, black elderberry improved cholesterol profiles and HDL functions in animal studies [23]. High contents of polyphenol in the black elderberry are claimed to be responsible for these effects. Natural products high in polyphenols altered the genes involved in the TICE pathway [37; 38]. In the present study, to gain insight into the mechanism of BEE on intestinal cholesterol metabolism, we investigated if BEE could alter the genes involved in the biosynthesis and flux of cholesterol in Caco-2 cells. The genes for intestinal cholesterol metabolism were markedly induced by BEE treatment. HMGR is a rate-limiting enzyme for cholesterol biosynthesis, and its inhibitor, statin, is widely prescribed to lower plasma cholesterol [48; 49]. SREBPP2, a master of regulation for cholesterol metabolism, is primarily responsible for the transcription of HMRG and LDLR [50]. There was a significant reduction in both mRNA and protein levels of HMGR and SREBPP2 by BEE. Intestinal free cholesterol absorption is mediated by several transporters and enzymes such as NPC1L1, SR-B1, ABCA1, ACAT2, and MTTP [10; 51]. We observed that BEE significantly decreased mRNA and protein levels of NPCIL1 in Caco-2 cells. NPC1L1, a sterol transporter, present in the apical membrane of enterocytes, mediates cholesterol absorption in the intestine [52; 53]. Ezetimibe, an NPC1L1 inhibitor, has been used for lowering cholesterol by inhibiting cholesterol absorption [54; 55]. Therefore, the reduction in NPC1L1 by BEE treatment also supports the hypocholesterolem effective of black elderberry. ABCA1 is responsible for basolateral cholesterol efflux [56]. We observed significant decreases in both mRNA and protein levels of ABCA1 in Caco-2 cells. Cholesterol absorption in the intestine is closely involved with chloramphenicol assembly and secretion.