Article pubs.acs.org/JAFC
Plasma Cholesterol-Lowering Activity of Gingerol- and ShogaolEnriched Extract Is Mediated by Increasing Sterol Excretion Lin Lei,† Yuwei Liu,† Xiaobo Wang,† Rui Jiao,‡ Ka Ying Ma,† Yuk Man Li,† Lijun Wang,† Sun Wa Man,† Shengmin Sang,§ Yu Huang,# and Zhen-Yu Chen*,† †
Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China ‡ College of Science and Engineering, Jinan University, Guangzhou, China § Center for Excellence in Post-Harvest Technologies, North Carolina A&T State University, North Carolina Research Campus, Kannapolis, North Carolina 28081, United States # School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China ABSTRACT: The present study investigated the cholesterol-lowering activity of gingerol- and shogaol-enriched ginger extract (GSE). Thirty hamsters were divided into three groups and fed the control diet or one of the two experimental diets containing 0.5 and 1.0% GSE. Plasma total cholesterol, liver cholesterol, and aorta atherosclerotic plaque were dose-dependently decreased with increasing amounts of GSE added into diets. The fecal sterol analysis showed dietary GSE increased the excretion of both neutral and acidic sterols in a dose-dependent manner. GSE down-regulated the mRNA levels of intestinal Niemann−Pick C1like 1 protein (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP binding cassette transporter 5 (ABCG5), whereas it up-regulated hepatic cholesterol-7α-hydroxylase (CYP7A1). It was concluded that beneficial modification of the lipoprotein profile by dietary GSE was mediated by enhancing excretion of fecal cholesterol and bile acids via up-regulation of hepatic CYP7A1 and down-regulation of mRNA of intestinal NPC1L1, ACAT2, and MTP. KEYWORDS: ginger, ABCG5/8, ACAT2, cholesterol, CYP7A1, MTP, NPC1L1
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susceptibility of LDL to oxidation.10 In humans, patients with cardiac disease given ginger extract decreased their plasma TC, TG, and LDL-C as well as very low density lipoprotein cholesterol (VLDL-C) compared with the placebo group.11 Cholesterol-lowering nutraceuticals and functional foods have been classified into two major types, namely, inhibitors of cholesterol absorption and modulators of cholesterol homeostasis.12,13 Cholesterol absorption in the intestine is a very complex process that requires several proteins, transporters, and enzymes mainly including Niemann−Pick C1-like protein 1 (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP binding cassette transporters 5 and 8 (ABCG5/8).12 Cholesterol homeostasis is maintained by several nuclear receptors, proteins, and enzymes mainly including sterol regulatory element binding protein 2 (SREBP2), liver X receptor (LXRα), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA-R), LDL receptor (LDL-R), and cholesterol-7αhydroxylase (CYP7A1).12 However, no study to date has examined how dietary ginger or its extract affects the cholesterol absorption pathway and homeostasis. The present study was designed to investigate the underlying mechanism by which gingerol- and shogaol-riched ginger
INTRODUCTION Most coronary heart diseases (CHD) involve atherosclerosis, which refers to the accumulation of cholesterol and other lipids along the inside wall of coronary arteries. Elevated plasma total cholesterol (TC) and low-density lipoprotein (LDL-C) are the major risk factors for atherosclerosis, whereas a high concentration of plasma high-density lipoprotein (HDL-C) is protective against atherosclerosis. In recent years, many phytochemicals from functional foods have attracted much interest due to their potential as functional nutraceuticals to treat hypercholesterolemia, especially for patients whose plasma TC concentration is marginally high but do not warrant the prescription of cholesterol-lowering medications. Ginger is among these nutraceuticals and functional foods. It has been shown that ginger possesses anti-inflammatory, antioxidant, antitumor, and antithrombotic activity.1,2 The pungent components including gingerols, shogaols, and their derivatives are claimed to be the active compounds. It has been shown that gingerols and shogaols are the most abundant in fresh ginger.3,4 Ginger has been shown to possess a cholesterol-lowering activity in various animal models. In rats fed a high-cholesterol diet, ginger extract decreased plasma TC and LDL-C.5−7 In rabbits fed a cholesterol-enriched diet, ginger could significantly reduce plasma TC, triacylglycerols (TG), and LDL-C and also prevent the progression of atherosclerosis.8,9 In apolipoprotein E-deficient mice, ginger extract similarly attenuated atherosclerotic lesions in a dose-dependent manner accompanied by reducing plasma TC, TG, and LDL-C as well as decreasing © XXXX American Chemical Society
Received: September 9, 2014 Revised: October 5, 2014 Accepted: October 7, 2014
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dx.doi.org/10.1021/jf5043344 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
extract (GSE) could reduce plasma TC, with focus on the interaction of dietary GSE with the gene expressions of hepatic SREBP2, LXRα, HMG-CoA-R, LDL-R, and CYP7A1 as well as intestinal NPC1L1, ACAT2, MTP, and ABCG5/8.
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MATERIALS AND METHODS
Diet. Three diets were prepared as previously described with minor modifications.14 The control diet (CTL) was prepared by mixing the following powdered ingredients (g/kg): cornstarch, 507; casein, 242; lard, 50; sucrose, 119; mineral mix, 40; vitamin mix, 20; gelatin, 20; DLmethionine, 1; and cholesterol, 1. Two GSE diets were similarly prepared by adding 0.5% GSE (L-GSE) or 1.0% GSE (H-GSE) into the control diet. All three diets contained 0.1% cholesterol by weight and had 68.2, 26.4, and 5.4% energy from carbohydrate, protein, and fat, respectively. A 0.1% cholesterol diet in this study could induce plasma cholesterol concentration to about 240 mg/dL or 6.2 mM, which is borderline for the diagnosis of human hypercholesterolemia. The rationale for adding 0.5 and 1.0% GSE into diet was that the recommended dose of dietary supplements, for example, phytosterols, is 2−3 g per day to decrease plasma LDL-cholesterol, translating to a dose of 0.8−1.2 g plant sterol esters/1000 kcal provided that humans daily consume about 2500 kcal of energy. In the present study, GSE was added in diets at two doses of 0.5 and 1.0%, which were equivalent to 0.8 and 1.6 g per 1000 kcal for humans, respectively. Hamsters. Thirty male Golden Syrian hamsters (aged 2.5 months) were randomly divided into three groups (n = 10 each) and fed one of the three diets for 6 weeks. Hamsters were housed in an animal room at 23 °C with a 12/12 h light−dark cycle. Food consumption was measured daily, and the fecal outputs from each hamster were collected weekly. Blood samples were collected from the retro-orbital sinus under light anesthesia using a mixture of ketamine, xylazine, and saline (v/v/v; 4:1:5) at weeks 0, 3, and 6 after overnight fasting. At the end of week 6, hamsters were sacrificed under carbon dioxide anesthesia. Organs were removed, washed with saline, weighed, and frozen in liquid nitrogen. The first 5 cm of duodenum was discarded, and the remaining 30 cm of the small intestine was kept. All samples were stored in a −80 °C freezer before analysis. The whole experimental protocol was approved and performed under the guidelines of the Animal Experimental Ethical Committee, The Chinese University of Hong Kong. HPLC Analysis of Gingerols and Shogaols. GSE was obtained from Sabinsa Corp. (Piscataway, NJ, USA). Individual gingerols and shogaols were separated and quantified using an HPLC-ECD system (ESA, Chelmsford, MA, USA), which consists of an ESA model 584 HPLC pump, an ESA model 542 autosampler, an ESA organizer, and an ESA electrochemical detector (ECD) coupled with two ESA model 6210 four sensor cells.15,16 A Gemini C18 column (150 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) was used for chromatographic analysis at a flow rate of 1.0 mL/min. The mobile phases consisted of solvent A (30 mM sodium phosphate buffer containing 1.75% acetonitrile and 0.125% tetrahydrofuran, pH 3.35) and solvent B (15 mM sodium phosphate buffer containing 58.5% acetonitrile and 12.5% tetrahydrofuran, pH 3.45). The gradient elution had the following profile: 20% B from 0 to 3 min; 20−55% B from 3 to 11 min; 55−60% B from 11 to 12 min; 60−65% B from 12 to 13 min; 65−100% B from 13 to 40 min; 100% B from 40 to 45 min; and then 20% B from 45.1 to 50 min. The cells were then cleaned at a potential of 1000 mV for 1 min. The injection volume of the sample was 10 μL. The eluent was monitored by the Coulochem electrode array system (CEAS) with potential settings at −100, 0, 100, 200, 300, 400, and 500 mV. Data for Figure 1 were from the channel set at 300 mV of the CEAS. Individual gingerols and shogaols were identified by comparing the retention times of authentic standards. HPLC analysis found that GSE mainly consisted of 29.4% 6-gingerol, 4.5% 8-gingerol, 5.3% 10gingerol, 4.3% 6-shogaol, 0.5% 8-shogaol, and 1.8% 10-shogaol (Figure 1). Analysis of Plasma Lipids. Commercial enzymatic kits from Infinity (Waltham, MA, USA.) and Stanbio Laboratories (Boerne, TX, USA) were used to determine the plasma TC, HDL-C, and TG,
Figure 1. HPLC chromatogram of gingerol- and shogaol-enriched ginger extract (GSE). Peaks: 1, 6-gingerol; 2, 8-gingerol; 3, 6-shogaol; 4, 10-gingerol; 5, 8-shogaol; 6, 10-shogaol. respectively. Non-HDL-C was calculated by deducting HDL-C from TC. Analysis of Hepatic Cholesterol. Total cholesterol in the liver was determined using a gas chromatograph (GC) method as we previously described.14 In brief, total lipids were extracted from the liver into a solvent mixture of chloroform/methanol (2:1, v/v) with the addition of 5α-cholestane (1.0 mg) as an internal standard. The total lipids were saponified, followed by the conversion of cholesterol to its trimethylsilyl ether (TMS) derivative. Cholesterol-TMS derivative was determined using a Shimadzu GC-14B gas chromatograph equipped with a fused silica capillary column (SAC-5, 30 m × 0.25 mm, i.d.; Supelco, Inc., Bellefonte, PA, USA) and a flame ionization detector. Analysis of Atherosclerotic Plaque. The severity of atherosclerosis in the endothelial layer of the aorta was measured as previously described.17 In brief, aortas were cut open vertically, stained with saturated oil red for 30 min, and washed with 2-propanol three times and then distilled water twice before scanning with a table scanner (Epson 1220 perfection, Epson Co., Tokyo, Japan). The area of atherosclerotic plaque was analyzed by a computer images analyzing program (Sigma Scan Pro 5.0, SPSS, Inc., Chicago, IL, USA). Analysis of Hepatic and Fecal Lipids. In brief, total lipids from 100 mg of liver or 300 mg of feces were extracted with the addition of heptadecanoic acid (0.9 mg) as an internal standard by chloroform/ methanol (2:1, v/v). The solvents were evaporated under a gentle stream of nitrogen, and the lipids were then methylated with 14% BF3 in methanol. The resultant total fatty acid methyl esters (FAME) were analyzed on a Shimadzu GC 2010 equipped with a flame ionization detector and a HP Innowax capillary column (30 m × 0.5 μm, i.d. = 0.32 mm). The temperature was increased from 150 to 200 °C at a rate of 15 °C/min, further increased to 25 °C at a rate of 2 °C/min, and finally kept at 250 °C for 15 min. Injector and detector temperatures were set at 250 and 270 °C, respectively. Helium was used as a carrier gas at a rate of 1.5 mL/min.18 Analysis of Fecal Sterols. Total fecal outputs from each hamster at weeks 1 and 6 were freeze-dried, ground, and thoroughly mixed before analysis. Both fecal neutral and acidic sterols were measured according to the previous method.19 In brief, 300 mg fecal samples were weighed with the addition of 5α-cholestane (0.5 mg) as an internal standard for neutral sterols and hyodeoxycholic acid (0.3 mg) as an internal standard for acidic sterols. The fecal samples were first saponified, followed by extraction of the total neutral sterols with cyclohexane. The fecal neutral sterols were then converted to their TMS derivatives before GC analysis. Similarly, the acidic sterols in the aqueous phase were extracted, converted to their corresponding TMS derivatives, and subjected to GC analysis. Real-Time PCR. mRNA of intestinal NPC1L1, ACAT2, MTP, and ABCG5/8 and hepatic SREBP2, HMG-CoA-R, LDL-R, LXRα, and B
dx.doi.org/10.1021/jf5043344 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Table 1. Changes in Plasma Total Cholesterol (TC), Triacylglycerols (TG), HDL-Cholesterol (HDL-C), Non-HDL-Cholesterol (Non-HDL-C), Liver Cholesterol, and Atherosclerotic Plaque in Hamsters Fed the Control (CTL) and the Two Experimental Diets Containing 0.5% GSE (L-GSE) and 1.0% GSE (H-GSE) for 0, 3, and 6 Weeksa CTL
L-GSE
H-GSE
regression (R)
P value
week 0 TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) non-HDL-C (mg/dL) non-HDL-C/HDL-C HDL-C/TC
125.7 ± 11.1 69.6 ± 15.0 92.8 ± 16.9 33.9 ± 14.7 0.39 ± 0.24 0.74 ± 0.11
126.2 ± 16.2 62.2 ± 10.0 90.6 ± 11.7 35.6 ± 20.5 0.41 ± 0.27 0.73 ± 0.13
125.8 ± 11.7 63.6 ± 8.5 89.9 ± 10.9 35.9 ± 13.4 0.41 ± 0.17 0.72 ± 0.09
−0.009 −0.329 −0.046 0.029 −0.002 −0.018
0.961 0.082 0.812 0.880 0.994 0.926
TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) non-HDL-C (mg/dL) non-HDL-C/HDL-C HDL-C/TC
268.9 ± 31.7a 156.3 ± 83.7.9 147.4 ± 11.5a 121.5 ± 34.8a 0.84 ± 0.30 0.55 ± 0.07
230.3 ± 46.5b 116.8 ± 41.3 136.7 ± 21.1a 93.6 ± 34.3ab 0.69 ± 0.24 0.60 ± 0.08
195.5 ± 20.4c 107.5 ± 23.7 118.4 ± 16.5b 77.1 ± 22.4b 0.67 ± 0.24 0.61 ± 0.09
−0.670 −0.289 −0.588 −0.520 −0.255 0.258
0.001 0.128 0.001 0.004 0.183 0.176
TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) non-HDL-C (mg/dL) non-HDL-C/HDL-C HDL-C/TC liver cholesterol (mg/g) atherosclerotic plaque (%)
259.4 ± 32.5a 140.5 ± 48.1 132.8 ± 22.5a 126.6 ± 34.2 0.99 ± 0.35 0.51 ± 0.09 48.2 ± 8.3a 17.1 ± 10.8a
240.6 ± 18.3a 144.7 ± 42.2 117.6 ± 18.9ab 122.9 ± 21.8 1.08 ± 0.31 0.49 ± 0.07 38.8 ± 7.9b 7.4 ± 1.6b
209.1 ± 24.3b 124.5 ± 41.4 102.7 ± 14.8b 106.4 ± 27.8 1.07 ± 0.33 0.50 ± 0.09 38.7 ± 5.8b 7.2 ± 3.9b
−0.645 −0.074 −0.564 −0.295 0.074 −0.093 −0.472 −0.524
0.001 0.703 0.001 0.120 0.635 0.630 0.023 0.010
week 3
week 6
a Data are expressed as the mean ± SD. Mean values in a row with different letters differ significantly (p < 0.05). R is the coefficient of correlation for the regression analysis on the dose-dependent effect of GSE on target lipoproteins.
Table 2. Changes in Major Liver Fatty Acids and Daily Excretion of Fecal Fatty Acids in Hamsters Fed the Control (CTL) and the Two Experimental Diets Containing 0.5% GSE (L-GSE) and 1.0% (H-GSE) for 6 Weeksa CTL
L-GSE
H-GSE
regression (R)
P value
7.71 ± 1.25a 5.72 ± 1.24 14.18 ± 2.56a 28.85 ± 5.99a 1.31 ± 0.24a 33.72 ± 6.76a 3.65 ± 0.80 2.03 ± 0.45 1.10 ± 0.22 7.51 ± 1.58 55.41 ± 10.64a
6.49 ± 0.31b 4.61 ± 0.48 11.74 ± 0.72b 24.83 ± 2.31b 1.18 ± 0.10ab 29.30 ± 2.61b 3.19 ± 0.36 1.62 ± 0.19 0.84 ± 0.11 6.25 ± 0.65 47.29 ± 3.41b
6.57 ± 0.80b 4.95 ± 0.68 12.17 ± 1.33b 24.56 ± 1.66b 1.10 ± 0.11b 28.63 ± 2.01b 3.21 ± 0.39 1.73 ± 0.30 0.95 ± 0.20 6.53 ± 0.91 47.33 ± 3.20b
−0.461 −0.326 −0.417 −0.426 −0.492 −0.445 −0.315 −0.337 −0.286 −0.327 −0.444
0.012 0.084 0.024 0.021 0.007 0.016 0.096 0.074 0.133 0.084 0.016
0.615 0.515 0.586 0.605 0.472 0.579 0.697 0.705 0.603
