新橙皮苷二氢查尔酮(neohesperidin dihydrochalcone, NHDC)是非营养型甜味剂,可用于无糖食品。该研究以化学方法合成NHDC并探究其体外的胰脂肪酶抑制效果。以廉价易得的柚皮苷为原料,经碱性条件开环得到分解产物根皮乙酰苯-4′-新橙皮苷(phloroacetophenone-4′-neohesperidoside, PN),通过Claisen-Schmidt缩合反应合成中间体新橙皮苷,再以廉价的雷尼镍替代昂贵的Pd/C催化氢化反应,合成目标产物NHDC。以高分辨质谱仪、核磁共振波谱仪鉴定产物结构。通过体外胰脂肪酶(pancreatic lipase, PL)抑制实验、荧光猝灭实验探究其降脂活性与动力学作用机制。结果表明,PN的合成中,NaOH与柚皮苷的最佳质量比为1.7,合成新橙皮苷的催化剂吡咯烷与乙酸的最佳摩尔比为2∶1,合成NHDC的最佳NaOH质量分数为7%,雷尼镍催化能力强于Pd/C,最佳添加量为20%。合成工艺改进后,NHDC的总收率为70.6%。NHDC能抑制PL的活性,半数抑制浓度(IC50)为0.71 mg/mL,抑制类型为可逆、反竞争性抑制,NHDC与PL结合后,猝灭PL的内源荧光,改变酶结构及微环境,从而降低PL活性,荧光猝灭类型为混合猝灭。因此,NHDC拥有作为胰脂肪酶抑制剂的应用潜力,具有减少脂质吸收的作用。
Neohesperidin dihydrochalcone (NHDC) is a non-nutritive sweetener that can be used in sugar-free foods.This study involved the chemical synthesis of NHDC and an investigation of its pancreatic lipase inhibitory effect in vitro.The intermediate neohesperidin was synthesized using inexpensive and readily available naringin as a raw material.The decomposition product, phloroacetophenone-4′-neohesperidoside (PN), was obtained through alkaline condition ring-opening, and neohesperidin was synthesized via a Claisen-Schmidt condensation reaction.The catalytic hydrogenation reaction was carried out by replacing the expensive Pd/C with cheap Rainey nickel, resulting in the synthesis of the target product NHDC.The structure of the product was identified using high-resolution mass spectrometry and a nuclear magnetic resonance spectrometer.In vitro, pancreatic lipase (PL) inhibition assay and fluorescence quenching assay were used to explore its lipid-lowering activity and kinetic mechanism.The results indicated that the optimum mass ratio of sodium hydroxide to naringin for the synthesis of PN was 1.7, the optimum molar ratio of pyrrolidine to acetic acid for the catalyst for the synthesis of neohesperidin was 2∶1, the optimum sodium hydroxide concentration for the synthesis of NHDC was 7%, and the optimum amount of Raney nickel, which exhibited stronger catalytic ability than Pd/C, was added at a level of 20%.The total yield of NHDC was 70.6% after the improved synthesis process.NHDC was found to inhibit the activity of PL, with a half inhibitory concentration (IC50) of 0.71 mg/mL, and the inhibition type was reversible, anti-competitive inhibition.After NHDC combined with PL, it quenched the endogenous fluorescence of PL and changed the enzyme structure and microenvironment, thereby reducing PL activity.The fluorescence quenching type was identified as mixed quenching.Thus, NHDC has the potential to be applied as a pancreatic lipase inhibitor with the ability to reduce lipid uptake.
[1] TAO F Y, WANG P, DAI Z R, et al.Neohesperidin dihydrochalcone improves hyperglycemia and insulin resistance in diabetic zebrafish[J].ACS Food Science & Technology, 2023, 3(9):1548-1558.
[2] AHMED M, DAVIS J, AUCOIN D, et al.Structural conversion of neurotoxic amyloid-beta(1-42) oligomers to fibrils[J].Nature Structural & Molecular Biology, 2010, 17(5):561-567.
[3] XIA X M, FU J L, SONG X F, et al.Neohesperidin dihydrochalcone down-regulates MyD88-dependent and-independent signaling by inhibiting endotoxin-induced trafficking of TLR4 to lipid rafts[J].Free Radical Biology & Medicine, 2015, 89:522-532.
[4] YOUNES M, AQUILINA G, CASTLE L, et al.Re-evaluation of neohesperidine dihydrochalcone (E 959) as a food additive[J].EFSA Journal, 2022, 20(11):e07595.
[5] TOMÁS-BARBERÁN F A, CLIFFORD M N.Flavanones, chalcones and dihydrochalcones - nature, occurrence and dietary burden[J].Journal of the Science of Food and Agriculture, 2000, 80(7):1073-1080.
[6] DWIVEDI R S.Dihydrochalcones Flavonoid Super Sweet Principles[M].Singapore:Springer Nature Singapore, 2022.
[7] FRYDMAN A, WEISSHAUS O, HUHMAN D V, et al.Metabolic engineering of plant cells for biotransformation of hesperedin into neohesperidin, a substrate for production of the low-calorie sweetener and flavor enhancer NHDC[J].Journal of Agricultural and Food Chemistry, 2005, 53(25):9708-9712.
[8] HOROWITZ R M, GENTILI B.Flavonoids of Citrus—VI:The structure of neohesperidose[J].Tetrahedron, 1963, 19(5):773-782.
[9] HOROWITZ R M, GENTILI B.Taste and structure in phenolic glycosides[J].Journal of Agricultural and Food Chemistry, 1969, 17(4):696-700.
[10] 钟艺琼. 从柚皮苷制新橙皮苷二氢查尔酮的研究[D].厦门:厦门大学, 2022.
ZHONG Y Q.Preparation of neohesperidin dihydrochalcone from naringin[D].Xiamen:Xiamen University, 2022.
[11] YUN J W.Possible anti-obesity therapeutics from nature:A review[J].Phytochemistry, 2010, 71(14-15):1625-1641.
[12] FRANCO R R, MOTA ALVES V H, RIBEIRO ZABISKY L F, et al.Antidiabetic potential of Bauhinia forficata Link leaves:A non-cytotoxic source of lipase and glycoside hydrolases inhibitors and molecules with antioxidant and antiglycation properties[J].Biomedicine & Pharmacotherapy, 2020, 123:109798.
[13] DU X P, BAI M L, HUANG Y, et al.Inhibitory effect of astaxanthin on pancreatic lipase with inhibition kinetics integrating molecular docking simulation[J].Journal of Functional Foods, 2018, 48:551-557.
[14] CARDULLO N, MUCCILLI V, PULVIRENTI L, et al.Natural isoflavones and semisynthetic derivatives as pancreatic lipase inhibitors[J].Journal of Natural Products, 2021, 84(3):654-665.
[15] YU B, TANG Q M, FU C X, et al.Effects of different particle-sized insoluble dietary fibre from Citrus peel on adsorption and activity inhibition of pancreatic lipase[J].Food Chemistry, 2023, 398:133834.
[16] 廖家乐, 方甜, 范艳丽.枸杞叶黄酮对胰脂肪酶活性的抑制作用[J].中国食品学报, 2022, 22(5):43-53.
LIAO J L, FANG T, FAN Y L.Inhibitory effects of Lycium barbarum leaves flavonoids on pancreatic lipase activity[J].Journal of Chinese Institute of Food Science and Technology, 2022, 22(5):43-53.
[17] 纪慧杰, 朱彩平.石榴皮多糖的提取及组成、体外降脂活性研究[J].食品与发酵工业, 2023, 49(4):161-168.
JI H J, ZHU C P.Study on the extraction and composition of pomegranate peel polysaccharide and hypolipidemic activity in vitro[J].Food and Fermentation Industries, 2023, 49(4):161-168.
[18] CHEN M C, LIU T T, WANG J P, et al.Strong inhibitory activities and action modes of lipopeptides on lipase[J].Journal of Enzyme Inhibition and Medicinal Chemistry, 2020, 35(1):897-905.
[19] 郑丽婷, 周鸿, 刘奕明, 等.黄柏碱对α-葡萄糖苷酶的体外抑制作用[J].南京中医药大学学报, 2020, 36(6):853-858.
ZHENG L T, ZHOU H, LIU Y M, et al.Inhibitory effect of phellodendrine on α-glucosidase in vitro[J].Journal of Nanjing University of Traditional Chinese Medicine, 2020, 36(6):853-858.
[20] 高航, 单雪玉, 高延芬, 等.莲子红衣多酚对α-葡萄糖苷酶的抑制作用[J].食品科学技术学报, 2016, 34(6):36-40; 45.
GAO H, SHAN X Y, GAO Y F, et al.Inhibitory effect on α-glucosidase activity of polyphenol from red-skin of Lotus seed[J].Journal of Food Science and Technology, 2016, 34(6):36-40; 45.
[21] URBIZO-REYES U, LICEAGA A M, REDDIVARI L, et al.Enzyme kinetics, molecular docking, and in silico characterization of canary seed (Phalaris canariensis L.) peptides with ACE and pancreatic lipase inhibitory activity[J].Journal of Functional Foods, 2022, 88:104892.
[22] DAS S, HAZARIKA Z, SARMAH S, et al.Exploring the interaction of bioactive kaempferol with serum albumin, lysozyme and hemoglobin:A biophysical investigation using multi-spectroscopic, docking and molecular dynamics simulation studies[J].Journal of Photochemistry and Photobiology B:Biology, 2020, 205:111825.
[23] LI Y Q, YANG P, GAO F, et al.Probing the interaction between 3 flavonoids and pancreatic lipase by methods of fluorescence spectroscopy and enzymatic kinetics[J].European Food Research and Technology, 2011, 233(1):63-69.
