Background
Hyperlipidemia is a group of metabolic disorders characterized by hypercholesterol and/or hypertriglyceride in blood circulation. The prevalence of hyperlipidemia has dramatically increased worldwide due to a sedentary lifestyle and consumption of high fat diets [
1,
2]. Long-term hyperlipidemia is an important risk factor in contributing to the development of cardiovascular diseases (CVD). The risk of developing cardiovascular diseases in subjects with hyperlipidemia was twice as high as in subjects with normal lipid levels [
3]. Currently, the inhibition of dietary fat digestion and absorption from the small intestine is an attractive target for the management of hyperlipidemia [
4,
5]. For example, ezetimibe is a cholesterol absorption inhibitor that can be used as monotherapy or a combination therapy with a first-line drug such as a statin. Evidence has demonstrated that a combination of ezetimibe and a statin caused the lowering of low-density lipoprotein (LDL) cholesterol and improved cardiovascular outcomes in patients after acute coronary syndromes [
6,
7]. However, the number of patients with elevated liver enzyme levels showed a significant increase with ezetimibe-statin combination therapy [
8]. Therefore, current studies attempt to search for effective phytochemical compounds from dietary fruits, vegetables and herbal medicines as lipid-lowering agents [
9,
10].
Phytochemical compounds in fruits and vegetables have been targeted to promote beneficial health effects, especially the prevention of pathophysiological conditions such as dyslipidemia, diabetes, hypertension and cancer [
11,
12]. Anthocyanins are one of the largest groups of natural pigments responsible for red, purple and blue colors in fruits and vegetables. They have been proven to possess favorable antioxidant, anti-inflammatory and anti-diabetic properties under both in vitro and in vivo study [
13]. Interestingly, a meta-analysis revealed that supplementation of anthocyanins reduced serum lipid profiles in dyslipidemia patients [
14]. Cyanidin-3-rutinoside (C3R), a naturally occurring anthocyanin, is widely distributed in a high number of dietary sources, such as blackberry, mulberry and black raspberry [
15,
16]. This compound has demonstrated promising benefits for reduction of postprandial glucose through inhibition of pancreatic α-amylase and intestinal α-glucosidase [
17,
18]. Another study suggests C3R could inhibit the glucose uptake in Caco-2 cells [
19]. In vitro data supported the underlying mechanism and indicated that C3R might modulate postprandial glycemia by inhibiting carbohydrate digestive enzymes and decreasing glucose transport in the small intestine. C3R also regulated glucose uptake and increased GLUT4 expression in 3T3-L1 adipocytes through activation of the PI3K/Akt pathways [
20]. Our studies found that C3R inhibited monosaccharide- and methylglyoxal-induced protein glycation in bovine serum albumin [
21,
22]. A previous study has shown the vascular relaxing activity of C3R and its protection against methylglyoxal-induced vascular dysfunction in rats [
23]. However, available data on C3R mostly focused on its anti-diabetic and anti-glycation activity. Existing researches have never established the potential effectiveness of C3R in relation to lipid-lowering activity. Therefore, the objective of the study was to determine whether C3R could inhibit pancreatic lipase, cholesterol esterase, and the binding of bile acid as well as the reduction of cholesterol micellization. Since the blood cholesterol level is also influenced by absorption of cholesterol in the small intestine, the effect of C3R on cholesterol uptake in the enterocytes was also determined.
Discussion
Clinical evidence revealed that supplementation of anthocyanin reduced total cholesterol, triglyceride and the level of low-density lipoprotein cholesterol (LDL-C) and increased the level of high-density lipoprotein cholesterol (HDL-C) in patients with dyslipidemia [
28,
29]. The previous study reported that consumption of purified mulberry anthocyanins containing cyanidin-3-rutinoside (C3R) effectively decreased serum lipid levels in high-fat fed mice [
30]. However, the specific mechanisms of action by which C3R decreases serum lipid level are still unknown. We present the first report on lipid-lowering mechanisms of C3R, including inhibition of lipid digestive enzymes and absorptive processes.
Inhibition of fat digestive enzymes results in delaying the process of hydrolyzing dietary fats. Pancreatic lipase is the primary digestive enzyme that converts triglyceride substrates to monoglycerides and free fatty acids, which may form micelles that serve as necessary intermediates for absorption into enterocytes [
4]. The inhibition of pancreatic lipase activity is the most widely studied mechanism for the identification of potential anti-obesity agents [
4]. The results from the pancreatic lipase inhibitory activity of C3R are in agreement with previous reports suggesting that pure anthocyanins, cyanidin-3-glucoside (C3G) and peonidin-3-glucoside, and anthocyanins extracted from dietary sources demonstrated inhibitory action against pancreatic lipase [
31,
32]. Since understanding the mechanism of enzyme inhibition has become the basis of development of pharmaceutical agents, the study of the structure-enzyme activity relationship provides helpful information for drug design [
33]. According to the results, C3R displayed a mixed-type competitive inhibitor against pancreatic lipase. The binding mode of C3R was assumed to be one inhibitor that can bind either to the active site of a free enzyme or to the enzyme-substrate complex. When
Ki and
Ki’ dissociation constants were compared, it was found that the
Ki value of C3R was 1.9 times higher than the
Ki’ value, suggesting that binding of C3R to a free form of enzyme was stronger than the binding of C3R to enzyme-substrate complex. The results indicated that the pancreatic lipase inhibitory activity of C3R was non-competitive predominant over competitive. However, the type of inhibition of C3R differs from earlier reports that cyanidin and its glycosides, including C3G and cyanidin-3,5-diglycoside, were identified as a competitive inhibitor [
31,
34]. It has been reported that cyanidin and C3G had three potential binding sites for porcine lipase-colipase complex within and near the active site, and cyanidin bound more effectively than C3G within (− 9.8 vs. -7.0 kcal/mol) and near (− 8.8 vs. -8.0 kcal/mol) the active site [
35]. Therefore, the different type of enzyme inhibition might be due to their glycosides at the 3-
O-position of cyanidin, which may affect the binding site and binding affinity of the anthocyanins to the enzyme [
36]. Therefore, further study is needed to evaluate the interaction between pancreatic lipase and C3R containing rutinose sugar at the 3-
O-position using the molecular docking study.
Pancreatic cholesterol esterase plays an important role in the hydrolyzing of dietary cholesterol ester (10–15% of total cholesterol in food) into non-esterified cholesterol which can be incorporated into mixed micelles and absorbed by enterocytes [
37]. It has been reported that the inhibition of pancreatic cholesterol esterase caused a reduction in cholesterol absorption in hamsters fed a high cholesterol diet [
38]. The current findings firstly revealed that C3R inhibited pancreatic cholesterol esterase activity, which is consistent with previous reports describing the potency of plant and fruit extracts against cholesterol esterase [
25,
39]. It has been stated that the structure of flavonoids is similar to that of cholesterol ester, which could irreversibly bind to the active site of the enzyme, resulting in inhibition of enzyme activity [
40]. According to the basic structural feature of flavonoid compounds, C3R may act in the same manner.
Generally, the principle steps in absorption of dietary cholesterol are emulsification of dietary fat, micellization of cholesterol, and absorption of mixed micelles in the proximal jejunum [
41]. One of the cholesterol-lowering mechanisms of flavonoids (tea catechins) is the interruption of cholesterol incorporation into micelles [
42]. The present study indicated that micellization of cholesterol was inhibited by C3R. In support of this, the recent study revealed that cyanidin-3-glucoside (C3G) added to micellar solution precipitated with cholesterol and formed an insoluble complex [
31]. Therefore, it could be hypothesized that C3R may prevent the formation of micelles due to having the same core chemical structure as C3G with a different type of sugar at the β-glycosidic linkage.
In addition to cholesterol, bile acids are also essential components in the formation of mixed micelles [
41]. C3R exhibited the binding ability of bile acids, including taurocholic acid, taurodeoxycholic acid and glycodeoxycholic acid. C3R showed the strongest binding capacity to taurocholic acid, a primary bile acid directly synthetized by the liver, suggesting that C3R may disrupt the endogenous bile acid pool, causing the stimulation of bile acid synthesis from cholesterol and leading to the reduction of blood cholesterol [
43]. Moreover, the binding of bile acids causes the formation of an insoluble complex and increases fecal bile excretion as well as disrupts the formation of micelles [
43]. Taken together, these findings support the notion that C3R may act as the bile acid sequestrant, resulting in increased cholesterol metabolism and decreased cholesterol absorption into blood circulation.
Intestinal Caco-2 cells are a widely used human intestinal in vitro model that obtain results that usually correlate with human figures [
44]. Since intestinal cholesterol absorption affects the levels of cholesterol in blood circulation, the effect of C3R on cholesterol uptake was determined in Caco-2 cells [
44]. Our results demonstrated that C3R inhibited cholesterol uptake in both free cholesterol (containing only bile salt taurocholate) and mixed micelles (mimicking the mixed micelles found in dietary conditions). These results indicated that the action of C3R was independent of cholesterol micelle formation, and the reduction of cholesterol uptake was not due to a toxic effect of C3R on cell viability (data not shown). The results are consistent with previous reports that anthocyanins in black rice extract are a potent agent to reduce cholesterol uptake in enterocytes [
31]. Interestingly, C3R showed sustained inhibitory effects similar to ezetimibe. This agent has a distinct mechanism as a selective cholesterol uptake inhibitor through binding of NPC1L1 at the brush border of the small intestine [
45]. Our results found that anthocyanin C3R markedly decreased mRNA expression of NPC1L1 in Caco-2 cells. Nevertheless, the short-term mechanism by which C3R inhibits cholesterol uptake does not appear to relate to the suppression of NPC1L1 mRNA expression in Caco-2 cells. This conclusion is drawn from the fact that a significant decrease in NPC1L1 mRNA expression was observed only at 24 h incubation with C3R, whereas there was no significant alteration of NPC1L1 mRNA expression at 2 h and 6 h. We suggest that C3R may interact with NPC1L1 protein to inhibit the cholesterol uptake after a short exposure period [
45]. In addition, the suppression of NPC1L1 mRNA expression by C3R appears to be the long-term mechanism for reducing cholesterol uptake into the enterocytes.
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