| Obesity has become a serious public health challenge.Obesity caused by longterm high-fat and high-cholesterol diets can lead to disturbances in lipid metabolism and cholesterol metabolism.Phytochemicals found in plant-based functional foods show the ability to modulate lipid metabolism and cholesterol metabolism via various pathways,thus playing a typical role in combating obesity.Lotus roots are rich in phenolic compounds,which have significant antioxidant and lipid metabolism regulating effects.However,it is precisely due to its high content of polyphenols that lotus roots are prone to browning.Lotus root browning is normally considered as a deterioration of lotus root quality,resulting in significant waste of lotus root resources and economic losses.Actually,during the browning process,the oxidation and polymerization of phenolic compounds may alter the functional bioactivity of plant extracts.Therefore,it is necessary to have a correct understanding of enzymatic browning in lotus roots,and to understand the specific recognition of polyphenol oxidase(PPO)on specific substrates as well as the molecular mechanisms.Furthermore,it is crucial to analyze what factors determine the precise recognition and specific binding of PPO to specific substrates with minor differences.More importantly,it is necessary to understand the changes in the functional components of lotus roots during the browning process.What bioactive components are still retained or newly generated in the browning lotus root? Therefore,this study focuses on fresh lotus root and browning lotus root,on the one hand,to investigate the browning mechanism of lotus root and,on the other hand,to compare the differences in bioactive components between lotus root and browning lotus root.Furthermore,this study explores the potential mechanisms underlying their regulation of cholesterol metabolism and lipid metabolism.The main research findings are as follows:(1)The optimal extraction method for lotus root PPO was homogenate stirring water extraction method.The optimal extraction process for lotus root PPO was an extraction time of 3.1 h,a liquid-to-material ratio of 4.0 m L/g,and a p H of 7.0.The specific activity of PPO extracted by this process was 4.19 times that of the extraction method,and the specific activity of lotus root PPO was increased to 417.87 U/mg.The lotus root PPO was purified by DEAE Sepharose Fast Flow anion exchange column and Sephadex G-75 gel column.Two highly homologous PPO were identified in lotus root,with molecular weights of 56.8 k Da(PPO 1)and 67.2 k Da(PPO 2),respectively.Lotus root PPO exhibited the highest catalytic activity at 35°C and p H 6.5,and oxalic acid was the most effective inhibitor of lotus root PPO.(2)Nine phenolic compounds were identified in lotus root,with the highest content being(+)-catechin,followed by(-)-epigallocatechin.Substrate specificity studies showed that(-)-epigallocatechin had the lowest Km,while(+)-catechin had the highest Vmax in 9 substrates.Molecular docking studies confirmed that compared to(+)-catechin,PPO formed more hydrogen bonds with(-)-epigallocatechin and exhibited lower docking energy.Additionally,(+)-catechin had one fewer hydroxyl group than(-)-epigallocatechin and entered the catalytic cavity of PPO more rapidly.Therefore,both(-)-epigallocatechin and(+)-catechin are key substrates for enzymatic browning of lotus root,which is a major reason for the rapid browning of lotus root.(3)Compared with fresh lotus root(total phenolic content: 4.52 ± 0.04 mg GAE/g DW;total flavonoid content: 4.64 ± 0.02 mg CAE/g DW),browning lotus root showed higher total phenolic content(8.96 ± 0.06 mg GAE/g DW)and total flavonoid content(11.79 ± 0.04 mg CAE/g DW),and exhibited stronger antioxidant capacity.A total of39 phytochemicals were identified in the extract of fresh lotus root,while 34 phytochemicals were identified in the extract of browning lotus root.The most abundant phenolic compounds in the extract of fresh lotus root were catechin,epigallocatechin,and caffeic acid,while the active components in the extract of browning lotus root were mainly theaflavin and forsythoside A.After in vitro simulated digestion and intestinal microbiota fermentation,the major active components in both fresh extract and browning lotus root extract significantly decreased,while theaflavin showed 50.82% residual content after the intestinal microbiota fermentation.(4)Using a model of cholesterol metabolism disorder induced by a highcholesterol and high-fat diet in mice,the study investigated the mechanisms by which extracts from fresh and browning lotus root regulate cholesterol metabolism in vivo.Both fresh lotus root extract and browning lotus root extract improved cholesterol metabolism in the mouse liver,promoted the synthesis of bile acids(BAs)in the liver,and increased the excretion of BAs in the feces(p<0.05).Furthermore,both extracts effectively inhibited the expression of key genes and proteins in the FXR-FGF15 pathway and reduced the levels of BAs reabsorption-related enzymes(p<0.05).Compared to fresh lotus root extract,browning lotus root extract had a stronger effect on promoting BAs synthesis in the liver and relieving the inhibitory effect of the intestinal FXR-FGF15 signaling pathway on hepatic bile acid synthesis(p<0.05),thereby increasing BAs production and reducing cholesterol deposition in the liver,effectively regulating cholesterol metabolism.(5)Using a model of obesity induced by a high-fat diet(HFD)in mice,the study investigated the mechanisms by which extracts from fresh and browning lotus root regulate lipid metabolism in vivo.Both fresh lotus root extract and browning lotus root extract significantly reduced body weight and TG levels as well as improved liver damage and lipid deposition caused by HFD(p<0.05).Both fresh lotus root extract and browning lotus root extract activated AMPK and ACC phosphorylation,thereby suppressing FAS expression and promoting downstream CPT1β expression(p<0.05).Notably,compared to fresh lotus root extract,browning lotus root extract had a more significant effect on inhibiting fatty acid synthesis and promoting fatty acid β-oxidation(p<0.05).Additionally,browning lotus root extract significantly increased adiponectin levels,promoted the expression of adiponectin m RNA genes(p<0.05),and significantly increased the abundance of the probiotic Akkermansia(p<0.05).This alleviated obesity and lipid metabolism disorders induced by HFD.(6)The main active components in browning lotus root were identified as theaflavin and forsythoside A,corresponding to the precursors of catechin and caffeic acid found in fresh lotus root.The oxidation product of catechin is theaflavin,while the derivative of caffeic acid is forsythoside A.A comparative study was conducted to investigate the regulatory effects of theaflavin,forsythoside A,catechins,and caffeic acid on lipid metabolism in obese mice induced by HFD.It was found that theaflavin,forsythoside A,catechin,and caffeic acid all effectively improved lipid metabolism in obese mice,with theaflavin having a more significant effect(p<0.05).Moreover,theaflavin not only activated ARL15 expression but also significantly upregulated FGF21 m RNA expression(p<0.05),thereby increasing adiponectin levels and more effectively activating AMPK phosphorylation and downstream CPT1β expression(p<0.05),regulating lipid metabolism.Additionally,theaflavin significantly increased the abundance of the gut microbiota Akkermansia(p<0.05),improving gut microbiota composition and ameliorating lipid metabolism disorders in HFD-fed mice.Research has confirmed that the main component that plays a lipid-lowering role in browning lotus root is theaflavin.In summary,this study systematically investigated the enzymatic browning mechanism of lotus root PPO and analyzed the specific substrate selection mechanism of PPO for structurally similar substrates,providing molecular-level insights into why lotus root is prone to browning.Further research showed that both fresh and browning lotus root extracts effectively improved lipid metabolism disorder and cholesterol metabolism disorder induced by high-fat and high-cholesterol diet,while the browning lotus root extract has a more significant effect.Compared to fresh lotus root extract,browning lotus root extract more effectively relieved the inhibitory effect of the intestinal FXR-FGF15 signaling pathway,increasing BAs production and excretion,thereby effectively regulating cholesterol metabolism.Additionally,browning lotus root extract had a significant improvement effect on obesity induced by HFD,promoting adiponectin production,activating AMPK and ACC phosphorylation,and increasing fatty acid β-oxidation.Browning lotus root extract also significantly increased the abundance of the probiotic Akkermansia,ameliorating gut microbiota composition and relieving obesity and lipid metabolism disorders.Further experimental validation confirmed that the key active component in browning lotus root was theaflavin.The functions of browning lotus root were reported for the first time.The research findings provided important scientific evidence for the development of anti-obesity functional foods,and they hold significant practical value for the development and utilization of browning lotus root resources,reducing waste of lotus resources. |
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