Article pubs.acs.org/JAFC
Cholesterol-Lowering Activity of Tartary Buckwheat Protein Chengnan Zhang,† Rui Zhang,‡ Yuk Man Li,† Ning Liang,† Yimin Zhao,† Hanyue Zhu,† Zouyan He,† Jianhui Liu, Wangjun Hao,† Rui Jiao,§ Ka Ying Ma,† and Zhen-Yu Chen*,† †
Food & Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China ‡ Institute of Special Animal and Plant Science, China Academy of Agricultural Sciences, Changchun, China § College of Science and Engineering, Jinan University, Guangzhou 510630, China S Supporting Information *
ABSTRACT: Previous research has shown that Tartary buckwheat flour is capable of reducing plasma cholesterol. The present study was to examine the effect of rutin and Tartary buckwheat protein on plasma total cholesterol (TC) in hypercholesterolemia hamsters. In the first animal experiment, 40 male hamsters were divided into four groups fed either the control diet or one of the three experimental diets containing 8.2 mmol rutin, 8.2 mmol quercetin, or 2.5 g kg−1 cholestyramine, respectively. Results showed that only cholestyramine but not rutin and its aglycone quercetin decreased plasma TC, which suggested that rutin was not the active ingredient responsible for plasma TC-lowering activity of Tartary buckwheat flour. In the second animal experiment, 45 male hamsters were divided into five groups fed either the control diet or one of the four experimental diets containing 24% Tartary buckwheat protein, 24% rice protein, 24% wheat protein, or 5 g kg−1 cholestyramine, respectively. Tartary buckwheat protein reduced plasma TC more effectively than cholestyramine (45% versus 37%), while rice and wheat proteins only reduced plasma TC by 10−13%. Tartary buckwheat protein caused 108% increase in the fecal excretion of total neutral sterols and 263% increase in the fecal excretion of total acidic sterols. real-time polymerase chain reaction and Western blotting analyses showed that Tartary buckwheat protein affected the gene expression of intestinal Niemann-Pick C1-like protein 1 (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), and ATP binding cassette transporters 5 and 8 (ABCG5/8) in a down trend, whereas it increased the gene expression of hepatic cholesterol-7α -hydroxylase (CYP7A1). It was concluded that Tartary buckwheat protein was at least one of the active ingredients in Tartary buckwheat flour to lower plasma TC, mainly mediated by enhancing the excretion of bile acids via up-regulation of hepatic CYP7A1 and also by inhibiting the absorption of dietary cholesterol via down-regulation on intestinal NPC1L1, ACAT2 and ABCG5/8. KEYWORDS: cholesterol, rice protein, Tartary buckwheat protein, wheat protein
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INTRODUCTION High blood total cholesterol (TC) is a risk factor for coronary heart disease (CHD). Cholesterol in blood is mainly present in two lipoproteins namely low-density lipoprotein (LDL) and high-density lipoprotein (HDL).1 Research has clearly demonstrated that when TC and LDL cholesterol (LDL-C) are abnormally elevated, cholesterol and other lipids may build up in the walls of arteries and cause atherosclerosis and lead to development of CHD. In contrast, HDL helps remove cholesterol from the arteries and transports it back to the liver for elimination. Thus, high concentration of HDL cholesterol (HDL-C) is protective against CHD. Current medicines including statins and cholestyramine in treatment of hypercholesterolemia suffer from some side effects.2 Therefore, there is a growing interest in using functional foods and nutraceuticals to manage the hypercholesterolemia. In this regard, Tartary buckwheat flour is among the list. Chinese have a long history of consuming Tartary buckwheat flour as a functional food in treatment of diabetes and cardiovascular disease as well wounds and ulcers.3,4 Research has shown that incorporation of Tartary buckwheat into diet is effective in reducing plasma TC and LDL-C. In humans, consumption of Tartary buckwheat has been shown to be inversely correlated with plasma TC and LDL-C.5−7 In animals, addition of © XXXX American Chemical Society
buckwheat into diet significantly lowers TC, LDL-C, and triacylglycerols (TG), while it increases HDL-C.8−12 We have previously studied the cholesterol-lowering activity of Tartary buckwheat flour compared with that of wheat and rice flours and found that Tartary buckwheat flour was effective in reducing plasma TC.13 However, the underlying mechanism and active ingredients responsible for such effect remain largely elusive. Compared with gross compositions of wheat and rice flours (Supporting Information Table 1),13 Tartary buckwheat flour contains 30-fold the amount of rutin as that in wheat and rice flours. In view of the hypocholesterolemic activity of rutin reported in rats,14 it is speculated that rutin or its aglycone quercetin may be one of the active ingredients in Tartary buckwheat flour responsible for its cholesterol-lowering activity.13 Another possible active ingredient in Tartary buckwheat flour may be its protein. Previous reports have demonstrated that L-arginine (Arg) may decrease while Lmethionine (Met) and L-lysine (Lys) may increase plasma TC.15,16 The amino acid analysis has demonstrated that Tartary Received: Revised: Accepted: Published: A
January 6, 2017 February 13, 2017 February 15, 2017 February 15, 2017 DOI: 10.1021/acs.jafc.7b00066 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
by Tomotake et al.17 In brief, Tartary buckwheat flour (108 kg) was defatted using petroleum ether and then suspended in distilled water. The suspension was adjusted to pH 8.0 using 0.1 mol/L NaOH and then centrifuged at 7500g for 20 min to remove precipitate. The supernatant was adjusted to pH 4.5 using 0.1 mol/L HCl and centrifuged at 7500g for 20 min to isoelectrically precipitate protein. The precipitate was resuspended in deionized water, adjusted to pH 7.0 using 0.1 mol/L NaOH, and then freeze-dried, which produced 2.2 kg of crude Tartary buckwheat protein. Crude Tartary buckwheat protein consisted of 63.1% protein, 34.5% carbohydrate, 1.4% fat, and 1.1% ash. Amino Acid Analysis. The amino acid profile of casein, Tartary buckwheat protein, rice protein, and wheat protein was determined according to the standard method of GB/T 5009.1242003.18 In brief, protein samples were hydrolyzed in 6 mol/L HCL under vacuum for 22 h before analysis. Hydrolyzed protein samples were then quantified using an amino acid analyzer (Sykam S-433d, Eresing, Germany). Tryptophan was determined according to the standard method of GB/T 182462000.19 Protein samples were hydrolyzed in 4 mol/L LiOH under vacuum for 20 h before the analysis. Tryptophan was quantified using an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA). Tartary buckwheat protein was characterized by having a highest percentage of arginine (Table 1). Animal Experiment 1: Effect of Rutin and Quercetin on Plasma TC. Four diets were prepared. A high cholesterol control diet (C) was prepared by mixing the following ingredients of 508 g of corn starch, 242 g of casein, 119 g of sucrose, 50 g of lard, 40 g of mineral mix, 20 g of vitamin mix, 1 g of DL-methionine, and 2 g of cholesterol (Table 2).13 The two experimental diets were prepared by adding 8.2 mmol (5.5
buckwheat protein has a higher content of Arg, while it has relatively lower proportions of Met and Lys (Table 1). Table 1. Amino Acid Composition (%) of Casein, Tartary Buckwheat Protein (TB-P), Rice Protein (R-P), and Wheat Protein (W-P) Asp Glu Thr Leu Cys Gly Val Ile Arg Lys Ala Met Ser His Pro Tyr Phe Trp total
casein
TB-P
R-P
W-P
6.56 21.44 4.00 8.79 0.35 1.76 5.89 4.67 3.41 7.37 2.89 2.80 5.36 2.01 10.35 5.10 4.80 2.47 100
9.64 18.07 3.22 6.81 5.12 5.23 6.00 4.40 9.41 4.97 4.91 1.64 4.44 2.30 3.80 2.56 5.33 2.16 100
10.79 18.98 3.52 7.67 5.31 3.87 4.84 4.69 7.18 5.99 4.50 1.19 4.47 2.11 4.96 3.21 5.31 1.39 100
3.07 35.42 2.25 6.73 4.16 3.17 4.48 4.18 3.08 1.91 2.92 1.96 3.90 1.78 12.58 2.56 4.74 1.11 100
Table 2. Composition of Diets in Animal Experiment 1a ingredients
The objectives of the present study were (i) to test if rutin and its aglycone quercetin were able to decrease plasma TC in hypercholesterolemia hamsters and (ii) to investigate effect of Tartary buckwheat protein on plasma TC compared with that of casein, rice, and wheat proteins. The present study was the first report of its kind to examine the interaction of Tartary buckwheat protein with genes involved in cholesterol absorption and metabolism including intestinal 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) as well as hepatic sterol regulatory element binding protein 2 (SREBP2), liver X receptor alpha (LXRα), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA-R), LDL receptor (LDL-R), and cholesterol-7α-hydroxylase (CYP7A1).
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g/kg Dry Diet corn starch casein gelatin sucrose lard mineral mixture-AIN 76 vitamin mixture-AIN 76A DL-methionine cholesterol rutin quercetin cholestyramine
C
R
Q
508 242 20 119 50 40 20 1 2 0
508 242 20 119 50 40 20 1 2 5.5
508 242 20 119 50 40 20 1 2 0 2.5
P-C 508 242 20 119 50 40 20 1 2 0 2.5
a
Hamsters were fed the control diet (C) or one of the three experimental diets containing 8.2 mmol rutin (R), 8.2 mmol quercetin (Q), and 2.5 g kg−1 of cholestyramine as a positive control drug (P-C).
MATERIALS AND METHODS
Materials. Quercetin and cholestyramine were purchased from Sigma (St. Louis, MO), while rutin was purchased from Shanghai DND Pharm-technology Co. Inc. (Shanghai, China). Rice protein, wheat protein, and casein were purchased from Huangchao Chem-tech Co., Ltd. (Zhengzhou, Henan, China), Baixing Biotech Co., Ltd. (Wuhan, Hubei, China), and ENVIGO Co., Ltd. (Indianapolis, IN), respectively. Casein contained 91.9% protein, 7.4% carbohydrate, 0.4% fat, and 0.4% ash. Rice protein consisted of 92.2% protein, 3.0% carbohydrate, 0.4% fat, and 4.3% ash, while wheat protein consisted of 76.2% protein, 21.9% carbohydrate, 0.6% fat, and 0.7% ash. The antibodies of LXRα, CYP7A1, SREBP2, MTP, ABCG5, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, Texas). The antibodies of LDL-R and HMGCoA-R (1 mg/mL each) were purchased from Merck Millipore (Billerica, MA). The antibodies of NPC1L1 and ABCG8 were purchased from Novus Biologicals (Littleton, CO). The primary antibody of ACAT2 was purchased from ABCAM (Cambridge, MA). Isolation of Tartary Buckwheat Protein. Tartary buckwheat flour was purchased from Shanxi Longeal Biotechnology Limited Company (Linfen, Shanxi, China). The flour contained 10.5% protein. Tartary buckwheat protein was prepared using the method previously described
g kg−1) of rutin (R) and 8.2 mmol (2.5 g kg−1) of quercetin (Q) into one kilogram of the control diet, respectively. The positive control diet (PC) was prepared by adding 2.5 g kg−1 of cholestyramine into the control diet. The powder diets were then added into a 10% gelatin solution in a ratio of 1 kg diet to 200 mL. The diets were cut into cubes (10 g) after the gelatin had set. The rationale for adding 6.5 g kg−1 rutin into the diet was that Tartary buckwheat contained about 20 g kg−1 of rutin,20,21 which was equivalent to about 6 g kg−1 of rutin if 30% Tartary buckwheat flour was added into diet. Male Golden Syrian hamsters (n = 40) were divided into four groups and housed in wire-bottom cages (n = 2 per cage) at 23 °C with a 12-h light−dark cycle in an animal room. They were fed one of four diets C, R, Q, or P-C for 8 weeks. Hamsters were given fresh diets every 2 days, and uneaten portion was measured and discarded. The hamsters were allowed to access the food and water ad libitum. Food consumption was recorded every 2 days. Body weights were measured, and feces were collected weekly. Blood sample was collected from the retro-orbital sinus under light anesthesia using a mixture of ketamine, xylazine, and saline (v/v/v; 4:1:5) at the end of week 8 after overnight fasting. After B
DOI: 10.1021/acs.jafc.7b00066 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry the 3 day recovery, all hamsters were sacrificed without fasting under carbon dioxide suffocation. Animal Experiment 2: Effect of Tartary Buckwheat, Rice, and Wheat Proteins on Plasma Lipids. Five diets were prepared. The control diet (C) was prepared as described above by mixing 508 g of corn starch, 242 g of casein, 119 g of sucrose, 50 g of lard, 40 g of mineral mix, 20 g of vitamin mix, 1 g of DL-methionine, and 2 g of cholesterol (Table 3). Three experimental diets were similarly prepared as the
distilled water. The fatty streak was scanned and quantified using a computer image analyzing program. Measurement of Liver Cholesterol. Cholesterol in the liver was analyzed as previously described.22,23 In brief, 1.5 mg of 5α-cholestane as an internal standard was added into 100 mg of liver sample, and total lipids were extracted using chloroform−methanol (2:1, v/v). The other lipids were removed by saponification, and the remaining cholesterol was converted to its corresponding TMS-ether derivative followed by GC analysis on a SAC-5 (30 m × 0.25 mm, i.d.; Supelco, Inc., Bellefonte, PA) in a Shimadzu GC-2010 equipped with a FID detector (Shimadzu, Tokyo, Japan). Cholesterol was identified according to its retention time of authentic standard and quantified according to the amount of 5αcholestane added in the liver sample. Fecal Sterol Analysis. Fecal sterols consist of neutral and acidic sterols. They were quantified as previously described.22,23 In brief, 5αcholestane (0.5 mg) and hyodeoxycholic acid (0.6 mg) were added into 300 mg fecal samples as internal standards for quantification of total neutral and acidic sterols, respectively. First, the fecal samples were saponified with 8 mL of 1 M NaOH in 90% ethanol, and the neutral sterols were extracted using cyclohexane. Second, the bottom aqueous layer after cyclohexane extraction was saponified again with 10 M NaOH followed by acidification using 25% HCl. The total acidic sterols were then extracted using diethyl ether. Both neutral and acidic sterols were converted to their corresponding TMS-ether derivatives and then analyzed in a Shimadzu GC-2010 gas chromatograph equipped with a SAC-5 column described above. Individual fecal neutral and acidic sterols were identified according to their retention times of authentic standards and quantified according to the amount of the corresponding internal standards added in the fecal sample before the extraction. Real-Time Polymerase Chain Reaction Analysis. mRNA for each target gene was quantified as previously described.22,24 In brief, the total RNA from the liver and intestine was extracted and transcribed to complementary DNA (cDNA) using a high capacity cDNA reverse transcription kit (Invitrogen, CA) on a thermocycler (Gene Amp PCR system 9700, Applied Biosystems). The gene expression of NPC1L1, ACAT2, ABCG5, ABCG8, MTP, LXRα, CYP7A1, PPARα, LDL-R, HMG-CoA reductase, and SREBP2 was normalized with β-actin. Realtime PCR was performed using a SYBRGreen Fast Universal PCR Master Mix. Data were analyzed using the Sequence Detection Software version 1.3.1.21 (Applied Biosystems, Waltham, MA). Western Blot Analysis. The Western blot analysis of each target protein was carried out as previously described.22,24 The frozen liver samples and intestine were homogenized, the extract was centrifuged, and the supernatant was collected. A portion of the total protein in supernatant was then centrifuged at 150 000g with the pellet being saved, then resuspended in the homogenizing buffer, and the total protein was collected. The protein was size-fractionated on SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was incubated in blocking solution (3% nonfat milk in TBST) followed by overnight incubation in the same solution containing the primary antibody of each target protein. The membrane was washed in TBST and incubated with the corresponding second antibody. The membrane was developed with ECL enhanced chemiluminescence agent and subjected to autoradiography on SuperRX medical X-ray film (Fuji, Tokyo, Japan). Densitometry was quantified using the BioRad Quantity One (Bio-Rad, Hercules, CA). Data on protein abundance were normalized with β-actin. Statistical Analysis. The data were expressed as mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) followed by student’s t test was used to detect any significant difference between any two groups. Significance was defined as p-value less than 0.05.
Table 3. Composition of Diets in Animal Experiment 2a ingredientsb g/kg Dry Diet corn starch casein crude buckwheat protein crude rice protein crude wheat protein gelatin sucrose lard mineral mixture vitamin mixture cholesterol cholestyramine % Energy protein carbohydrate fat
C
TB-P
R-P
W-P
P-C
508 242 0 0 0 20 119 50 40 20 2 0
397 0 353 0 0 20 119 50 40 20 2 0
509 0 0 241 0 20 119 50 40 20 2 0
458 0 0 0 292 20 119 50 40 20 2 0
508 242 0 0 0 20 119 50 40 20 2 5
24 65 11
24 65 11
24 65 11
24 65 11
24 65 11
a
Hamsters were fed the control diet (C) or one of the four experimental diets containing Tartary buckwheat protein (TB-P), rice protein (R-P), or wheat protein (W-P), and 5 g kg−1 cholestyramine as a positive control drug (P-C). bCasein consists of 91.9% protein, 7.4% carbohydrate, 0.4% fat, and 0.4% ash; crude buckwheat protein consists of 63.1% protein, 34.5% carbohydrate, 1.4% fat, and 1.1% ash; crude rice protein consists of 92.2% protein, 3.0% carbohydrate, 0.4% fat, and 4.3% ash; crude wheat protein consists of 76.2% protein, 21.9% carbohydrate, 0.6% fat, and 0.7% ash. control diet by replacing 242 g of casein with the same amount of Tartary buckwheat protein (TB-P), rice protein (R-P), or wheat protein (W-P), respectively. The positive control diet (P-C) was prepared by adding 5 g kg−1 of cholestyramine into the control diet (Table 3). As prepared in experiment 1, 1 kg powder diets were added into 200 mL of 10% gelatin solution and cut into cubes (10 g) after the gelatin had set. Male hamsters (n = 45) were randomly divided into five groups and similarly housed as in experiment 1. Hamsters were fed one of the five diets C, TB-P, R-P, W-P, or P-C for 6 weeks. Food intake and body weights were similarly recorded as in experiment 1. Feces were collected weekly. Blood from the retro-orbital sinus was similarly collected at the end of week 6. After the 3 days recovery, all hamsters were sacrificed without fasting under carbon dioxide suffocation. Liver was removed, 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 and stored in a −80 °C freezer. Thoracic aortas were collected and stored in DEPC-PBS buffer at −4 °C before analysis. The experimental protocols described in both experiments 1 and 2 were approved by the Animal Experimental Ethical Committee, The Chinese University of Hong Kong (ref. No. 15−066-MIS). Analysis of Plasma Lipids. Plasma TC, HDL-C, non-HDL-C, and TG were measured using the commercial enzymatic kits from Infinity (Waltham, MA) and Stanbio Laboratories (Boerne, TX), respectively.13 Analysis of Fatty Streak. Fatty streak in endothelial layer of thoracic aorta was measured as previously described.22 In brief, aorta from each hamster was dissected, cleaned, and cut open under a microscope (Olympus, Shinjuku, Tokyo, Japan). Aorta was stained with saturated oil red and washed with 2-propanol followed by rinsing with
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RESULTS Effect of Rutin and Quercetin on Plasma Cholesterol. There were no significant differences in food intakes and body weights among the four groups. Cholestyramine is a cholesterollowering medicine via a mechanism of increasing the bile acid excretion. Compared with C diet, P-C diet significantly decreased plasma TC and non-HDL-C by 16% and 24% (p < C
DOI: 10.1021/acs.jafc.7b00066 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
inconsistent. TB-P diet was equally effective as P-C diet in reducing liver cholesterol by 59%. In contrast, R-P and W-P diets had no effect on liver cholesterol (Table 5). Compared with C diet, TB-P, R-P, W-P, and P-C diets had no effect on formation of fatty streak in the aorta (Table 5). Effect of Tartary Buckwheat, Rice, and Wheat Proteins on Excretion of Fecal Sterols. TB-P diet increased the excretion of fecal cholesterol by four-fold compared with C diet, whereas, R-P, W-P, and P-C diets had no significant effect on excretion of fecal cholesterol (Table 6). TB-P and R-P diets increased the excretion of total neutral sterols by 108% and 116%, respectively, compared with C diet, while W-P and P-C diets had no significant effect on the excretion of total fecal neutral sterols. TB-P and P-C diets increased the excretion of primary bile acid chenodeoxycholic acid compared with C diet, while R-P and W-P diets had no effect (Table 6). Quantitatively, TB-P and P-C diets increased the excretion of total bile acids by 263% and 294%, respectively, compared with C diet, whereas, R-P and W-P diets had no significant effect on excretion of total bile acids. Effect of Tartary Buckwheat, Rice, and Wheat Proteins on mRNA and Protein Mass of Intestinal NPC1L1, ACAT2, MTP, and ABCG5/8. TB-P diet decreased both mRNA and protein mass of intestinal NPC1L1 compared with C diet, while the other three diets had no effect on intestinal NPC1L1 (Figure 1). TB-P diet significantly down-regulated mRNA of ACAT2 and ABCG8 compared with C diet. At translational level, TB-P diet showed a trend of down-regulating the protein mass of ACAT2 and ABCG8. TB-P diet down-regulated both mRNA and protein mass of ABCG5 compared with C diet, whereas, R-P and W-P diets had no significant effect. P-C diet down-regulated mRNA of ABCG5; however, it had no significant effect on protein mass of ABCG5. TB-P and W-P diets decreased MTP protein expression, but mRNA results were inconsistent. Effect of Tartary Buckwheat, Rice, and Wheat Proteins on mRNA and Protein Mass of Liver SREBP2, LXRα, HMGCoA-R, LDL-R, and CYP7A1. TB-P and P-C diets up-regulated both mRNA and protein mass of hepatic CYP7A1, while R-P and W-P diets had no significant effect compared with C diet (Figure 2). TB-P, W-P, and P-C diets down-regulated the mRNA but not protein mass of HMG-CoA-R compared with C diet. Four experimental diets had no effect on both mRNA and protein mass of hepatic SREBP2, LXRα, and LDL-R (Figure 2).
0.05), respectively. R diet had no significantly effect on plasma TC and non-HDL-C (Table 4). Q diet did not affect plasma TC; Table 4. Change in Body Weight, Plasma Total Cholesterol (TC), Triacylglycerols (TG), High-Density Lipoprotein Cholesterol (HDL-C), Non-HDL-C, Liver Cholesterol, and Aortic Atherosclerosis in Animal Experiment 1a Body Weights initial final Plasma Lipids TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) Non-HDL-C (mg/dL)
C
R
Q
P-C
116 ± 9 131 ± 12
110 ± 9 122 ± 11
112 ± 8 131 ± 11
113 ± 7 131 ± 12
259 ± 41a 202 ± 60a 112 ± 12a 147 ± 19a
241 ± 23a 149 ± 33b 114 ± 15a 127 ± 16a
250 ± 25a 163 ± 46ab 132 ± 16b 118 ± 37b
218 ± 18b 149 ± 20b 105 ± 10a 113 ± 12b
a
Hamsters were fed the control diet (C) or one of the three experimental diets containing 8.2 mmol rutin (R), 8.2 mmol quercetin (Q), and 2.5 g kg−1 of cholestyramine as a positive control drug (P-C) at week 8. Data were expressed as mean ± SD; n = 10. Mean values within a row having unlike superscript letters (a,b,c) were significantly different (p < 0.05).
however, it significantly decreased non-HDL-C by 19%, while it increased HDL-C by 18%. P-C diet significantly lowered plasma TG compared with the control group (P < 0.05). Although there was a trend of decreasing plasma TG in hamsters given R and Q diets, the effect was not statistically significant (Table 4). Effect of Tartary Buckwheat, Rice, and Wheat Proteins on Plasma Cholesterol. Feeding TB-P, R-P, W-P, and P-C diets had no significant effect on body weight gain in hamsters compared with C diet (Table 5). P-C diet decreased plasma TC by 37% compared with the control diet. TB-P, R-P, and W-P diets significantly decreased plasma TC compared with C diet. However, TB-P diet reduced plasma TC by 45%, while R-P and W-P diets reduced it only by 10−13% (Table 5). Similarly, TB-P diet decreased plasma non-HDL-C by 60%, while R-P and W-P diets only decreased it by 13%. TB-P and R-P diets were equally effective in reducing plasma TG by 26%, while W-P diet had no significant effect on plasma TG (Table 5). Effect of Tartary Buckwheat, Rice, and Wheat Proteins on Liver Cholesterol and Fatty Streak. The effect of TB-P, RP, and W-P diets on liver cholesterol concentration was
Table 5. Changes in Body Weights, Plasma Total Cholesterol (TC), Triacylglycerols (TG), High-Density Lipoprotein Cholesterol (HDL-C), and Non-HDL-C in Animal Experiment 2a C Body Weights (g) initial final Plasma Lipids TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) non-HDL-C (mg/dL) fatty streak (%) liver cholesterol (mg/g)
TB-P
R-P
W-P
P-C
114 ± 8 124 ± 7
115 ± 10 124 ± 10
114 ± 8 131 ± 7
114 ± 5 128 ± 6
113 ± 9 125 ± 9
292 ± 23a 160 ± 37a 141 ± 12a 151 ± 27a 28 ± 13 37 ± 6a
162 ± 16d 117 ± 38b 101 ± 9d 61 ± 17c 20 ± 16 15 ± 6b
262 ± 19b 177 ± 41a 130 ± 5b 132 ± 16b 28 ± 18 38 ± 5a
253 ± 18b 164 ± 48a 122 ± 13bc 131 ± 18b 18 ± 12 37 ± 5a
185 ± 23c 111 ± 29b 113 ± 6c 72 ± 20c 24 ± 11 18 ± 4b
a
Hamsters were fed the control diet (C) or one of the four experimental diets containing Tartary buckwheat protein (TB-P), rice protein (R-P), or wheat protein (W-P), and 5 g kg−1 cholestyramine as a positive control drug (P-C). Data were expressed as mean ± SD; n = 9. Mean values within a row having unlike superscript letters (a,b,c) were significantly different (p < 0.05). D
DOI: 10.1021/acs.jafc.7b00066 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 6. Fecal Neutral and Acidic Sterol Excretion (mg/hamster/day) in Animal Experiment 2a Neutral Sterols coprostanol coprostanone cholesterol dihydrocholesterol total Acidic Sterols lithocholic acid deoxycholic acid chenodeoxycholic acid cholic acid total
C
TB-P
R-P
W-P
P-C
0.57 ± 0.31b 0.02 ± 0.01 0.12 ± 0.10b 0.14 ± 0.12bc 0.83 ± 0.49bc
0.83 ± 0.15ab 0.05 ± 0.02 0.58 ± 0.47a 0.31 ± 0.16ab 1.73 ± 0.71a
1.07 ± 0.46a 0.05 ± 0.02 0.36 ± 0.24ab 0.36 ± 0.20a 1.80 ± 0.87a
0.81 ± 0.28ab 0.04 ± 0.02 0.24 ± 0.13b 0.44 ± 0.18a 1.51 ± 0.56ab
0.66 ± 0.22b 0.04 ± 0.02 0.19 ± 0.11b 0.16 ± 0.09b 1.03 ± 0.40b
0.03 ± 0.02c 0.05 ± 0.03bc 0.03 ± 0.01b 0.06 ± 0.03 0.16 ± 0.08b
0.18 ± 0.07a 0.14 ± 0.10b 0.22 ± 0.09a 0.05 ± 0.03 0.58 ± 0.23a
0.06 ± 0.03c 0.03 ± 0.02c 0.04 ± 0.01b 0.03 ± 0.01 0.16 ± 0.04b
0.05 ± 0.02c 0.10 ± 0.06bc 0.04 ± 0.02b 0.04 ± 0.02 0.22 ± 0.08b
0.13 ± 0.07b 0.26 ± 0.16a 0.21 ± 0.12a 0.04 ± 0.02 0.63 ± 0.25a
a
Hamsters were fed the control diet (C) or one of the four experimental diets containing Tartary buckwheat protein (TB-P), rice protein (R-P), or wheat protein (W-P), and 5 g kg−1 cholestyramine as a positive control drug (P-C). Mean values with different letters differ significantly at p < 0.05.
Figure 1. mRNA and protein levels of intestinal NPC1L1, ACAT2, MTP, ABCG5 and 8 in animal experiment 2. Hamsters were fed the control diet (C) or one of the four experimental diets containing Tartary buckwheat protein (TB-P), rice protein (R-P), or wheat protein (W-P), and 5 g kg−1 cholestyramine as a positive control drug (P-C). Values were expressed as mean ± SD. (a,b,c) Mean values with different letters differed significantly at p < 0.05.
Figure 2. mRNA and protein levels of hepatic SREBP2, HMG-CoA-R, LDLR, LXRα, and CYP7A1 in animal experiment 2. Hamsters were fed the control diet (C) or one of the four experimental diets containing Tartary buckwheat protein (TB-P), rice protein (R-P), or wheat protein (W-P), and 5 g kg−1 cholestyramine as a positive control drug (P-C). Values were expressed as mean ± SD. (a,b,c) Mean values with different letters differed significantly at p < 0.05.
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DISCUSSION The first animal experiment showed the supplementation of rutin and its aglycone quercetin in diet had no effect on plasma TC (Table 4), thus eliminating the possibility that rutin was the active ingredient responsible for plasma TC-lowering activity of Tartary buckwheat flour. However, it was noteworthy quercetin
but not rutin decreased non-HDL-C, while it increased HDL-C. The observation for such effect of quercetin on plasma lipoprotein was in agreement with that reported in rabbits25 and humans.26 We suggest the two possibilities to explain why quercetin not rutin changed the ratio of non-HDL-C/HDL-C. First, this might be due to the poor bioavailability of rutin in the E
DOI: 10.1021/acs.jafc.7b00066 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
primary bile acids were also converted to the secondary bile acids by gut bacteria. In this connection, the total acidic sterols should be summed up to reflect the excretion of total bile acids. We speculated that Tartary buckwheat protein enhanced the excretion of bile acids in a way similar to that of cholestyramine. As an anionic resin, cholestyramine binds bile acids, increases the excretion of bile acids, and thus reduces plasma cholesterol. We found Tartary buckwheat protein caused 263% increase in the excretion of total bile acids, comparable to 294% increase caused by cholestyramine. We further targeted the interaction of Tartary buckwheat proteins with gene expression of hepatic SREBP2, HMG-CoA-R, LDLR, LXRα, and CYP7A1. HMG-CoA-R is a regulatory enzyme in cholesterol synthesis pathway, while LDLR is to mediate the removal of LDL-C from blood into the liver. SREPB2 is a transcription factor that regulates the gene expression of HMG-CoA-R and LDL-R. The present study found that Tartary buckwheat protein had no effect on protein level of SREBP2, HMG-CoA-R, and LDL-R, which suggested that the cholesterol-lowering activity of Tartary buckwheat protein was unlikely mediated by its effect on cholesterol synthesis pathway and the removal of LDL cholesterol. CYP7A1 is an enzyme that catalyzes the rate-limiting reaction in bile acid synthesis pathway. Cholesterol can stimulate CYP7A1 transcription via activation of oxysterol receptor LXRα. Similar to cholestyramine, Tartary buckwheat protein could up-regulate the gene expression of CYP7A1 without activation of hepatic LXRα (Figure 2), which suggests that Tartary protein could decrease plasma TC via the same mechanism as cholestyramine by enhancing the conversion of cholesterol to bile acids and thus lead to reduction in cholesterol level in both plasma and liver. In fact, addition of Tartary buckwheat protein into diet was associated with 45% reduction in plasma TC and 59% reduction in liver cholesterol (Table 5). In summary, Tartary buckwheat protein in replacement of casein in diet was more effective than rice and wheat proteins in reducing plasma cholesterol. Plasma cholesterol-lowering activity of Tartary buckwheat protein was mainly mediated by enhancing the excretion of total fecal bile acids via up-regulation of hepatic CYP7A1 and also by inhibiting the absorption of dietary cholesterol via down-regulation of intestinal NPC1L1, ACAT2, and ABCG5/8. It was concluded that Tartary buckwheat protein was the active ingredient or at least one of the active ingredients responsible for plasma TC-lowering activity of its flour.
intestine. It has been reported that the quercetin absorption from rutin is only about 6%, while that of free quercetin is about 36− 53%.27 Second, it is possible that quercetin could inhibit cholesteryl ester transport protein (CETP), which is a plasma protein transporting a cholesteryl ester from HDL to LDL with exchange of a triacylglycerol, thus leading to reduction in the ratio of non-HDL-C to HDL-C. We plan to investigate the effect of quercetin on plasma CETP activity in the future study. The second animal experiment clearly demonstrated that Tartary buckwheat protein was very effective in reducing plasma TC with the following observations. First, Tartary buckwheat protein in replacement of casein in diet was much more potent than rice and wheat proteins in reducing plasma TC (45% versus 11−13% reduction) (Table 5). The present result was in agreement with that of Tomotake et al.,11 who found that Tartary buckwheat protein at 20% in replacement of casein caused 25% reduction in plasma TC in rats fed a cholesterol diet. Second, Tartary buckwheat protein was more effective than cholestyramine in reducing plasma TC under the current experimental conditions. Third, Tartary buckwheat protein was capable of decreasing plasma TG, while rice and wheat proteins had no significant effect. Results from experiment 2 proved that Tartary buckwheat protein was the active ingredient, or at least one of the active ingredients, accountable for plasma TC-lowering activity of its flour.13 To explore the underlying mechanism by which Tartary buckwheat protein was able to reduce plasma TC, we proposed that Tartary buckwheat protein could inhibit the cholesterol absorption. Cholesterol in the large intestine is converted to coprostanol, coprostanone, and dihydrocholesterol by gut bacteria. In this regard, total neutral sterols should be summed to reflect total cholesterol excretion. We had the following evidence to support the hypothesis. The fecal sterol analysis clearly showed Tartary buckwheat protein could increase the excretion of total neutral sterols, while rice and wheat proteins had no or little effect. This was in agreement with the report of Metzger et al.,28 who found that buckwheat protein reduced the incorporation of cholesterol into micelles and decreased the cholesterol uptake in Caco-2 cells in a dose-dependent manner. The present study was the first report of its kinds to investigate the interaction of Tartary buckwheat protein with genes of transporters, enzymes, and proteins involved in cholesterol absorption pathway. Cholesterol absorption starts with intestinal sterol transporter NPC1L1, which transfers the cholesterol from the lumen of intestine to enterocytes, while ACAT2 is an enzyme in enterocytes, which converts free cholesterol to cholesteryl ester (CE) before CE can be assembled into chylomicrons by MTP. ABCG5 and 8 are also sterol transporters, which transfer the unabsorbed cholesterol from enterocytes back to the lumen of intestine in a way of avoiding the accumulation of excessive cholesterol in enterocytes. In this connection, Tartary buckwheat protein had an inhibitory trend on the gene expression of intestinal NPC1L1, ACAT2, and ABCG5/8 (Figure 1). At the molecular level, the cholesterol-lowering activity of Tartary buckwheat protein was mediated by down-regulation of gene expression of these sterol transporters and enzyme in the enterocytes. The second mechanism for the cholesterol-lowering activity of Tartary buckwheat protein was mediated by increasing the fecal excretion of bile acids. The removal of excessive cholesterol usually starts in the liver where cholesterol was converted to bile acids followed by elimination via bile duct. Similar to the microbial conversion of cholesterol in the large colon, the
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00066.
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Composition of Tartary buckwheat flour, wheat flour, and rice flour (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: (852) 3943-6382. Fax: (852) 2603-7246. ORCID
Zhen-Yu Chen: 0000-0001-5615-1682 Notes
The authors declare no competing financial interest. F
DOI: 10.1021/acs.jafc.7b00066 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
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ACKNOWLEDGMENTS This project was supported by a grant from the Health and Medical Research Fund, The Food and Health Bureau, The Government of the Hong Kong Special Administrative Region, China (Project No. 13140111).
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ABBREVIATIONS USED ABCG 5/8, ATP binding cassette transporters 5 and 8; ACAT2, acyl CoA:cholesterol acyltransferase 2; CE, cholesteryl ester; CYP7A1, cholesterol-7α-hydroxylase; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; LDL-R, low density lipoprotein receptor; HMGCoA-R, 3-hydroxy-3-methylglutaryl-CoA reductase; LXRα, liver X receptor alpha; non-HDL-C, non-high density lipoprotein cholesterol; NPC1L1, Niemann-Pick C1-like protein 1; MTP, microsomal triacylglycerol transport protein; R-P, rice protein; SREBP2, sterol regulatory element binding protein 2; TB-P, Tartary buckwheat protein; TC, total cholesterol; TG, triacylglycerols; W-P, wheat protein
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DOI: 10.1021/acs.jafc.7b00066 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
