molecules Article Rutin and Quercetin Decrease Cholesterol in HepG2 Cells but Not Plasma Cholesterol in Hamsters by Oral Administration Ning Liang 1,2,3, Yuk-Man Li 1, Zouyan He 1, Wangjun Hao 1, Yimin Zhao 1 , Jianhui Liu 1, Hanyue Zhu 1, Erika Kwek 1, Ka-Ying Ma 1, Wen-Sen He 4 and Zhen-Yu Chen 1,* 1 School of Life Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong 999077, China; [email protected] (N.L.); [email protected] (Y.-M.L.); [email protected] (Z.H.); [email protected] (W.H.); [email protected] (Y.Z.); [email protected] (J.L.); [email protected] (H.Z.); [email protected] (E.K.); [email protected] (K.-Y.M.) 2 College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China 3 Health Science Center, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs Innovation Platform for Natural Small Molecule Drugs, Department of Pharmacy, School of Medicine, Shenzhen University, Shenzhen 518060, China 4 School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China; [email protected] * Correspondence: [email protected]; Tel.: +852-3943-6382 Abstract: Rutin (R) and quercetin (Q) are two widespread dietary flavonoids. Previous studies regarding the plasma cholesterol-lowering activity of R and Q generated inconsistent results. The present study was therefore carried out to investigate the effects of R and Q on cholesterol metabolism Citation: Liang, N.; Li, Y.-M.; He, Z.; in both HepG2 cells and hypercholesterolemia hamsters. Results from HepG2 cell experiments Hao, W.; Zhao, Y.; Liu, J.; Zhu, H.; Kwek, E.; Ma, K.-Y.; He, W.-S.; et al. demonstrate that both R and Q decreased cholesterol at doses of 5 and 10 µM. R and Q up-regulated Rutin and Quercetin Decrease both the mRNA and protein expression of sterol regulatory element binding protein 2 (SREBP2), low- Cholesterol in HepG2 Cells but Not density lipoprotein receptor (LDLR), and liver X receptor alpha (LXRα). The immunofluorescence Plasma Cholesterol in Hamsters by study revealed that R and Q increased the LDLR expression, while only Q improved LDL-C uptake Oral Administration. Molecules 2021, in HepG2 cells. Results from hypercholesterolemia hamsters fed diets containing R (5.5 g/kg diet) 26, 3766. https://doi.org/10.3390/ and Q (2.5 g/kg diet) for 8 weeks demonstrate that both R and Q had no effect on plasma total molecules26123766 cholesterol. In the liver, only Q reduced cholesterol significantly. The discrepancy between the in vitro and in vivo studies was probably due to a poor bioavailability of flavonoids in the intestine. Academic Editors: It was therefore concluded that R and Q were effective in reducing cholesterol in HepG2 cells in vitro, Ryszard Amarowicz and Adriano whereas in vivo, the oral administration of the two flavonoids had little effect on plasma cholesterol Costa de Camargo in hamsters. Received: 18 May 2021 Accepted: 16 June 2021 Keywords: rutin; quercetin; cholesterol; hamsters; LXR; SREBP2; LDLR Published: 21 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Hypercholesterolemia is a major cause of cardiovascular diseases. Healthy diets and an iations. active lifestyle can improve the plasma lipid profile by reducing blood total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) while elevating high-density lipoprotein cholesterol (HDL-C) [1,2]. Cholesterol homeostasis is regulated by its absorption, synthesis, conversion, and excretion. In the lumen of the small intestine, cholesterol is transported into Copyright: © 2021 by the authors. enterocytes, where it is esterified and packed into chylomicrons followed by entering the Licensee MDPI, Basel, Switzerland. lymphatic system [3,4]. Synthesis of cholesterol is mainly carried out in the liver, starting This article is an open access article with acetyl CoA, mediated by regulation of the activity of 3-hydroxy-3-methylglutaryl- distributed under the terms and CoA reductase (HMG-CoA R). The LDL receptor (LDLR) is responsible for the removal conditions of the Creative Commons of cholesterol from the circulation and lowering plasma LDL-C [5,6]. Sterol regulatory Attribution (CC BY) license (https:// element binding protein 2 (SREBP2) governs the expression of both HMG-CoA R and creativecommons.org/licenses/by/ LDLR. Cholesterol 7α-hydroxylase (CYP7A1) is a key enzyme for converting excessive 4.0/). Molecules 2021, 26, 3766. https://doi.org/10.3390/molecules26123766 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 12 Molecules 2021, 26, 3766 2 of 12 HMG-CoA R and LDLR. Cholesterol 7α-hydroxylase (CYP7A1) is a key enzyme for con- verting excessive cholesterol to bile acids in the liver [7]. Another regulator is liver X re- ceptorcholesterol α (LXRα), which to bile regulates acids in thethe liverencoding [7]. Anotherof CYP7A1 regulator [8]. is liver X receptor α (LXRα), Rutinwhich (R) regulates is a natural the encodingflavonoid of present CYP7A1 in [many8]. foods [9–11] (Figure 1). R has been reported toRutin possess (R) several is a natural biological flavonoid functions, present including in many foodsanti-oxidative, [9–11] (Figure anti-diabetic,1). R has been anti-adipogenic,reported to anti-inflammatory, possess several biological and anti-cancer functions, activities including [9,12–14]. anti-oxidative, R is structurally anti-diabetic, composedanti-adipogenic, of quercetin anti-inflammatory,(Q) as its aglycone and and rutinose anti-cancer as its activities sugar moiety [9,12– [15]14]. (Figure R is structurally 1). As withcomposed R, Q is also of quercetin widely present (Q) as itsin many aglycone foods, and such rutinose as buckwheat, as its sugar apple, moiety onion, [15]( redFigure 1 ). wine, andAs with cranberry R, Q is [16–18]. also widely Q has present also been in de manymonstrated foods, such to possess as buckwheat, anti-oxidative, apple, anti- onion, red viral, anti-atherosclerotic,wine, and cranberry and [16 anti-inflammatory–18]. Q has also been activities demonstrated [19–23]. Various to possess studies anti-oxidative, have been carriedanti-viral, out anti-atherosclerotic,to investigate the effects and anti-inflammatory of R and Q on cholesterol activities [metabolism19–23]. Various using studies differenthave models, been carriedincluding out vascular to investigate endothelial the effects cells, ofintestinal R and Qepithelial on cholesterol cells, macro- metabolism phages,using hepatic different cells, male models, Swiss including ablino mice, vascular male Wistar endothelial rats, male cells, Sprague intestinal Dawley epithelial rats, cells, macrophages, hepatic cells, male Swiss ablino mice, male Wistar rats, male Sprague Dawley male New Zealand white rabbits, male C576J/BL mice, and male golden Syrian hamsters rats, male New Zealand white rabbits, male C576J/BL mice, and male golden Syrian [24–27]. However, these studies generated inconsistent results regarding the cholesterol- hamsters [24–27]. However, these studies generated inconsistent results regarding the lowering activity of R and Q. We had previously shown that consumption of rutin-rich cholesterol-lowering activity of R and Q. We had previously shown that consumption of buckwheat was effective in reducing plasma cholesterol in hypercholesterolemia ham- rutin-rich buckwheat was effective in reducing plasma cholesterol in hypercholesterolemia sters; however, R and Q appeared not to be the active ingredients [16]. This arouses our hamsters; however, R and Q appeared not to be the active ingredients [16]. This arouses interest to further investigate if R and Q act differently in vitro and in vivo in affecting our interest to further investigate if R and Q act differently in vitro and in vivo in affecting cholesterol metabolism. cholesterol metabolism. Figure 1. Chemical structures of rutin and quercetin. Figure 1. Chemical structures of rutin and quercetin. The objectives of the present study were to (i) study the effect of R and Q on cholesterol Themetabolism objectives using of the HepG2 present cells study as anwerein vitroto (i) model;study the (ii) effect examine of R the and effect Q on of choles- R and Q on terol metabolismblood cholesterol using HepG2 using hamsters cells as an as anin vitroin vivo model;model; (ii) and examine (iii) investigate the effect of the R interaction and Q on bloodof R and cholesterol Q with using the expression hamsters ofas genesan in vivo involved model; in and cholesterol (iii) investigate metabolism. the inter- We chose action hamstersof R and asQ anwith in vivothe expression model because of genes hamsters involved are relativelyin cholesterol hyper-responsive metabolism. toWe dietary chose cholesterol,hamsters as excrete an in vivo bile acids,model and because synthesize hamsters cholesterol are relatively and bile hyper-responsive acids in a manner to similar dietaryto cholesterol, that in humans excrete [28 ,bile29]. acids, and synthesize cholesterol and bile acids in a man- ner similar to that in humans [28,29]. 2. Results 2. Results2.1. Effects on Cell Viability 2.1. Effects onTo Cell estimate Viability the cytotoxicity of R and Q, HepG2 cells were incubated with various doses Toof estimate R and Q the for 24cytotoxicity h. The results of R indicateand Q, thatHepG2 R and cells Q were exhibited incubated a different with cytotoxicityvarious in µ doses HepG2of R and cells. Q for R 24 did h. not The affect results cell indica viabilityte that at concentrations R and Q exhibited ranging a different from 3 to cytotox- 100 M, while µ icity inQ HepG2 had a significant cells. R did toxicity not affect in HepG2 cell viability cells from at concentrations 25 M onwards ranging (Figure from2A). Therefore,3 to 100 the effects of R and Q on HepG2 cells were evaluated at 5 and 10 µM. µM, while Q had a significant toxicity in HepG2 cells from 25 µM onwards (Figure 2A).