Anti-atherosclerotic potential of gossypetin via inhibiting LDL oxidation and foam cell formation

https://doi.org/10.1016/j.taap.2013.06.027Get rights and content

Highlights

  • The anti-atherosclerotic effect of gossypetin in vitro was examined.

  • Gossypetin inhibited LDL oxidation.

  • Gossypetin showed potential in reducing on the formation of foam cells.

  • Gossypetin functions against ox-LDL through PPARa activation and PPARγ depression.

Abstract

Gossypetin, a flavone originally isolated from Hibiscus species, has been shown to possess antioxidant, antimicrobial, and antimutagenic activities. Here, we investigated the mechanism(s) underlying the anti-atherosclerotic potential of gossypetin. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay showed that the addition of > 50 μM of gossypetin could scavenge over 50% of DPPH radicals. The inhibitory effects of gossypetin on the lipid and protein oxidation of LDL were defined by thiobarbituric acid reactive substance (TBARS) assay, the relative electrophoretic mobility (REM) of oxidized LDL (ox-LDL), and fragmentation of apoB in the Cu2 +-induced oxidation of LDL. Gossypetin showed potential in reducing ox-LDL-induced foam cell formation and intracellular lipid accumulation, and uptake ability of macrophages under non-cytotoxic concentrations. Molecular data showed that these influences of gossypetin might be mediated via peroxisome proliferator-activated receptor α (PPARα)/liver-X receptor α (LXRα)/ATP-binding cassette transporter A1 (ABCA1) and PPARγ/scavenger receptor CD36 pathways, as demonstrated by the transfection of PPARα siRNA or PPARγ expression vector. Our data implied that gossypetin regulated the PPAR signals, which in turn led to stimulation of cholesterol removal from macrophages and delay atherosclerosis. These results suggested that gossypetin potentially could be developed as an anti-atherosclerotic agent.

Introduction

Flavonoids are polyphenolic compounds found in fruits and vegetables and plant-derived products like red wine and tea, and can be divided into different subclasses such as anthocyanidins, flavonols, and flavones. The intake of dietary flavonoids has been related to a reduced risk for several diseases, such as cardiovascular and chronic inflammatory diseases (Geraets et al., 2007, Nijveldt et al., 2001). These positive health effects associated with the intake of flavonoids have been ascribed to their well-known antioxidant properties and to inhibiting effects on a wide range of enzymes (Middleton and Kandaswami, 1993, Rice-Evans et al., 1996). The presence of 3-hydroxyl group of flavonoids is important to their radical-scavenging capabilities higher than ascorbic acid (Wang et al., 2005). Gossypetin (3,5,7,8,3′,4′-hexahydroxy flavone) originally isolated from the flowers of Hibiscus species, has been shown to be quite effective in suppressing N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG) mutagenicity, exhibiting as much as 70% inhibition (Francis et al., 1989). Previous studies also showed that gossypetin possessed antimicrobial activity (Jeong et al., 2009, Miceli et al., 2009) and prevented allergic reaction (Yoshikawa et al., 1995, Yoshikawa et al., 1996). Hibiscus sabdariffa is rich in two important flavonoids, quercetin and gossypetin, as well as anthocyanins. Consuming foods such as H. sabdariffa has been shown to help neutralize cancer-causing agents, decrease oxidative stress and atherosclerosis (Lin et al., 2011a).

Atherosclerosis is a complicated vascular disorder, and low-density lipoprotein (LDL) is implicated as a major risk factor for the progression of this disease (Ross, 1999). Oxidized LDL (ox-LDL) is also well-known as a key factor in the development of atherosclerosis stimulating macrophage foam cell formation in the aorta. It is associated with the accumulation of cholesterol in macrophages by imbalance of cholesterol influx and efflux (Ross, 1999). Recent data support the importance of macrophage surface proteins CD36 and class A scavenge receptors (SR-As), which can specially bind ox-LDL in the atherosclerosis (Yang et al., 2011). Furthermore, CD36 expression participates in cholesterol efflux and is controlled by regulating the transcription factor regulator peroxisome proliferator-activated receptors (PPARs), liver X receptor (LXR), and ATP-binding cassette transporter A1 (ABCA1) (Chawla et al., 2001, Chinetti et al., 2001). It has been demonstrated that SR-A and CD36 are the target genes for PPARγ (Tontonoz et al., 1998). All of these seemed to suggest that controlling the balance of cholesterol transport is strongly associated with regulation of atherosclerosis. To date, synthetic compounds such as PPAR agonists and LXR agonists have been demonstrated to have the effect of delaying atherosclerosis by stimulating cholesterol removal from macrophages (Larrede et al., 2009). Thus, seeking natural compounds for modulation of macrophage functions presents an attractive strategy for the prevention and treatment of atherosclerosis.

Many studies have demonstrated that flavonoids in medical and edible plants have various pharmacological activities, such as antioxidant, anti-inflammatory, anti-carcinogenic, and anti-atherogenic activities (de Whalley et al., 1990, Gaziano et al., 1992). Here, we aimed to investigate the putative effect of gossypetin, a hexahydroxylated flavonoid, on atherosclerosis. Whether the compound could influence LDL oxidation, foam cell formation, and protein expression of the cholesterol regulatory-related signals still needs to be clarified.

Section snippets

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity

The effect of gossypetin (purity > 99.0%) (ChromaDex Inc., St. Santa Ana, CA, USA) on the DPPH radical was estimated according to the method of Yang et al. (2011). A reaction mixture containing methanol (3 mL), DPPH (1 mM) and gossypetin (1–1000 μM) was allowed to stand at room temperature for 30 min before mixing with redistilled water (1 mL) and toluene (3 mL). The solution was then centrifuged, and the absorbance of the upper phase was measured at 517 nm. The DPPH mixture without sample solution was

Gossypetin inhibited the protein oxidation and lipid peroxidation of LDL

In order to explore the capacity of free radical scavenging of gossypetin, the spectrophotometric measurement of DPPH consumption in the presence of gossypetin was performed. As can be observed in Fig. 1A, gossypetin at concentrations of > 50 μM scavenged over 50% of free radicals. Therefore, gossypetin may exhibit the antioxidant ability on the DPPH blocking in a dose-dependent manner.

Next, the effects of various concentrations of gossypetin on Cu2 +-mediated oxidation of LDL were determined by

Discussion

In recent years, attention has been focused on the antioxidant properties of pure components for an application in chemoprevention of disease. Previous studies have shown that consumption of flavonoid compounds mitigates the risk of cardiovascular diseases including atherosclerosis (Nicholson et al., 2008). The average daily human intake of these compounds in the UK and USA has been estimated to be 1 g or more (Pierpoint, 1986). Flavonoids have many properties including scavenging free radicals (

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the grant from the National Science Council (NSC99-2632-B-040-001-MY3), Taiwan.

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