Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (5): 981-994.doi: 10.3864/j.issn.0578-1752.2023.05.013

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Lipid-Lowering Effects and Its Regulation Mechanism of Buckwheat Polyphenols in High-Fat Diet-Induced Obese Mice

YAO YiJun(), JU XingRong, WANG LiFeng()   

  1. College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety, Nanjing 210023
  • Received:2022-04-26 Accepted:2022-11-25 Online:2023-03-01 Published:2023-03-13

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Abstract:

【Background】At present, relevant animal experiments have confirmed that buckwheat had a good intervention effect on obesity and lipid metabolism disorder caused by high-fat diet. At the same time, studies have shown that the adaptive thermogenesis of brown fat could effectively improve the body’s energy metabolism. Therefore, increasing the activity of brown fat and promoting the browning of white fat could be used as an effective way to prevent obesity and improve energy metabolism diseases.【Objective】The raw buckwheat and four kinds of main polyphenols identified from buckwheat were prepared according to the content proportion to study the lipid-lowering effects of buckwheat polyphenols in high-fat diet-induced obese mice via browning of white adipocytes.【Method】Phenolic compounds in buckwheat polyphenol extract were identified by UHPLC-Q-Orbitrap mass spectrometry combined with database. And the main polyphenol compound was prepared according to the content proportion in the raw buckwheat. In order to explore the regulating effects of buckwheat on body weight, organ index and blood lipid level in C57BL/6J mice, the dietary intervention was carried out with 10%, 20% and 40% proportion of buckwheat to replace basic feed and the 2.5 mg∙mL-1 of mixture buckwheat polyphenol standard substance. Last but not least, Western-blot and q-PCR were used to investigate the effects of buckwheat feed and polyphenol intervention on the expression of heat-producing proteins and genes in white and brown fat in mouse subcutaneous adipose tissue.【Result】Twenty phenolic compounds in buckwheat polyphenol extract were identified by UHPLC-Q-Orbitrap mass spectrometry, and the content of four phenolic compounds with the highest content (hydroxycinnamic acid, quercetin，scoparone, and rutin) accounted for more than 80% of the total phenolic compounds, which were the main polyphenols in buckwheat. The results showed that after 39 d in the diet intervention, compared with high-fat diet group, adding buckwheat feed and buckwheat polyphenol intervention on body weight in mice, the liver index and serum triglyceride (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-c), aspartate aminotransferase (ALT) and third transaminase (AST) increased significantly inhibitory effect; with the increase of amount of buckwheat, the inhibition increased. At the same time, adding buckwheat feed and buckwheat polyphenol could protect and improve the decrease of renal index and HDL-c in mice. It was also the high dose 40% buckwheat and polyphenol group that had the best effect, which showed that phenolic substances played a major role in the improvement of abnormal lipid metabolism in high-fat diet-induced obese mice. UCP1, PRDM-16, and PGC-1α protein and gene expression levels were significantly decreased in the high-fat diet group compared with the normal diet group, while Tcf21 and HOXC8 expression levels were significantly increased (P<0.05). After dietary intervention, protein expression levels of UCP1, PRDM-16 and PGC-1α in the buckwheat intervention group were significantly up-regulated, while protein expression levels of Tcf21 and HOXC8 were significantly decreased (P<0.05). In addition, the 40% buckwheat and polyphenol intervention group significantly increased the expression of UCP1, PRDM-16 and PGC-1α genes in mouse visceral adipose tissue (P<0.05), and which down-regulated the expressions of Tcf21 and HOXC8 genes.【Conclusion】Buckwheat intervention could effectively improve the lipid metabolism disorder in high-fat diet-induced obese mice, and it was confirmed that buckwheat polyphenols played a major role. The mechanism was mainly buckwheat polyphenols could significantly improve the thermogenic activity of brown fat, reduce the content of white fat, and regulate the energy metabolism balance. This study provided a new idea and theoretical basis for the utilization of buckwheat and the development of functional foods about lipid-lowering products.

Key words: buckwheat, polyphenols, high-fat diet-induced mice, lipid-lowering effects, browning of white adipocytes

Table 1

Chromatographic gradient elution procedure"

时间Time (min) A (%) B (%)
0 98 2
1 98 2
17 50 50
17.5 50 50
18 98 2
20 98 2

Table 2

Main composition of basic feed"

成分指标 Index of composition 含量 Content (g∙kg-1) 成分指标 Index of composition 含量 Content (g∙kg-1)
水分 Water 100 钙 Calcium 10-18
粗蛋白 Crude protein 180 总磷 Phosphorus 6-12
Oligo T (50 μmol∙L-1) 60 赖氨酸 Lysine 13.2
粗纤维 Coarse fibre 50 蛋氨酸+胱氨酸
Methionine + Cystine		 7.8
粗灰分 Coarse ash 80

Table 3

Main ingredients of high-fat diet group and rice bran intervention group feed"

原料
Raw material		 对照组
Control group		 基础高脂组
High fat group		 10%荞麦组
10% Buckwheat group		 20%荞麦组
20% Buckwheat group		 40%荞麦组
40% Buckwheat group
基础饲料 Basic feed 0.805 0.655 0.555 0.455 0.255
猪油 Lard 0 0.15 0.15 0.15 0.15
蔗糖 Sucrose 0.10 0.10 0.10 0.10 0.10
蛋黄粉 Egg yolk powder 0.08 0.08 0.08 0.08 0.08
胆固醇 Cholesterol 0.01 0.01 0.01 0.01 0.01
胆酸钠 Sodium cholate 0.005 0.005 0.005 0.005 0.005
荞麦 Buckwheat 0 0 0.1 0.2 0.4
合计 Total 1 1 1 1 1

Table 4

Sequences of primers used for real-time qPCR"

基因Genes 上游引物 Upstream primer 下游引物 Downstream primer
PRDM-16 CTGGACCTGAGCATCGGCAG GACGGCCGCAGGTACTTCTC
UCP1 CCCAGAGCCATCTGCATGGG AGGGGACGTCATCTGCCAGT
PGC-1α GAGCACGAGAAGCGGGAGTC CTGTCCGCGTTGTGTCAGGT
Tcf21 AGGAGGAAGGCGCCCACTAA GACGCCAGCCTGAGAGTGTC
HOXC8 CCATGGATGAGACCCCACGC GGGCCCCAGGCAGTTTATCC

Table 5

Composition and identification of polyphenols in buckwheat extract by UHPLC-Q-Orbitrap MS"

保留时间
Retention time		 峰面积
Area peak		 分子量 Molecular weight 误差值
Error value		 分子式
Formula		 数据库来源 Annotation source 化合物
Compound
实测值
Measured value		 理论值
Theoretical value		 mzCloud mzVault ChemSpider
1 1.317 36571473.05 164.158 164.04735 0.11065 C9H8O3 √ 羟基肉桂酸
Hydroxycinnamic acid
2 8.927 6169014.93 302.2357 302.0422 0.1935 C15H10O7 √ √ 槲皮素 Quercetin
3 11.668 5856748.00 206.195 206.05785 0.13715 C11 H10O4 √ √ 滨蒿内酯 Scoparone
4 7.446 5688758.95 290.27 290.07866 0.19134 C15H14O6 √ √ √ 儿茶素 Epicatechin
5 8.928 3132034.98 610.518 610.15286 0.36514 C27H30O16 √ √ 芦丁 Rutin
6 12.059 2292049.71 416.3781 416.11025 0.26785 C21H20O9 √ 大豆苷 Daidzin
7 7.403 1199629.88 180.16 180.04219 0.11781 C9H8O4 √ √ √ 咖啡酸 Caffeic acid
8 9.284 919736.70 272.25 272.06795 0.18205 C15H12O5 √ 柚皮素 Naringenin
9 11.36 896927.74 178.185 178.06294 0.12206 C10H10O3 √ 甲氧基肉桂酸 Methoxycinnamic acid
10 8.887 776919.85 192.168 192.04226 0.12574 C10H8O4 √ 东莨菪素 Scopoletin
11 12.997 723078.22 576.504 576.12641 0.37759 C30H24O12 √ 原花青素 Procyanidin A2
12 13.099 637134.93 182.17 182.04731 0.12269 C9H10O4 √ 二羟基苯基丙酸Dihydroxyphenylpropionic acid
13 11.237 546215.75 446.41 446.12111 0.28889 C22H22O10 √ 黄豆黄素苷 Glycitin
14 8.929 529856.93 464.3763 464.09526 0.28104 C21H20O12 √ √ √ 金丝桃苷 Hyperoside
15 8.052 401414.00 274.27 274.08381 0.18619 C15H14O5 √ √ √ 根皮素 Phloretin
16 6.109 369541.11 452.41 452.13163 0.27837 C21H24O11 √ 虾青素 Aspalathin
17 8.323 249449.72 610.55 610.18694 0.36306 C28H34O15 √ 橙皮甙 Hesperidin
18 7.458 237259.65 290.28 290.06056 0.21944 C15H14O6 √ 儿茶酸 Catechin
19 8.3 91517.24 448.3769 448.09996 0.27694 C21H20O11 √ 槲皮甙 Quercitrin
20 9.492 52981.16 316.262 316.05763 0.20437 C16H12O7 √ 异鼠李素 Isorhamnetin

Fig. 1

Effects of different content of buckwheat and polyphenol mixture supplementation on the body weight of high-fat diet- induced mice The same as below"

Fig. 2

Effects of different content of buckwheat and polyphenol mixture supplementation on the liver (A), kidney (B), heart (C), pancreas organ (D) index of high-fat diet-induced mice The same as below"

Fig. 3

Effects of different content of buckwheat and polyphenol mixture supplementation on the T-CHO (A), TG (B), LDL-c (C), HDL-c (D), ALT (E), AST (F) of high-fat diet-induced mice The same as below"

Fig. 4

Histological analysis for liver of mice Con: Basic diet group; HFD: High-fat diet group; HPE: High-fat diet group was given polyphenols; 10%: Feed containing 10% buckwheat; 20%: Feed containing 20% buckwheat; 40%: Feed containing 40% buckwheat"

Fig. 5

Effects of different content of buckwheat and polyphenol mixture supplementation on the expression levels of thermogenesis- related proteins in subcutaneous adipose tissues"

Fig. 6

Effect of different content of buckwheat and polyphenol mixture supplementation on the expression levels of thermogenesis-related genes in subcutaneous adipose tissues"

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