Comparison Of Spectrophotometry Of Citrus Flavonoids And Evaluation Of Their Antioxidant And Pancreatic Lipase Inhibitory Activities

Citrus,as the world's largest fruit,is a group of fruit crops belonging to the genus Citrus L.of the Rutaceae family.A large amount of processing waste such as citrus peel is produced every year,which is a good source for recovering flavonoids and other active components.Up to now,there are no universal colorimetric methods suggested for estimating flavonoids,which may lead to the misuse of quantitative methods.On the other hand,the various flavonoid profiles in different citrus species lead to the discrepancy of biological activities,while there is a lack of research on the relationship between the prevention of oxidative stress as well as obesity related metabolic syndrome and the rational utilization of citrus varieties.In the present study,we comprehensively evaluated the applicability and deficiencies of four widely used colorimetric methods,then a reliable and rapid quantitative method was established with HPLC method as the standard to avoid methods misuse.Moreover,eight representative citrus peel extracts were chosen as the samples.From the perspective of citrus species classification as well as taking oxidative stress and obesity,which are two important risk factors associated closely with metabolic syndrome,into account,the citrus species with the best overall characteristics was selected.Finally,the specific interaction mechanism between hesperidin(the main pancreatic lipase inhibitory active component in citrus peel extract)and pancreatic lipase was elucidated by the methods of fluorescence quenching,CD spectra and molecular docking.The main conclusions are revealed as follows:1.Four common colorimetric methods have their own applicability and deficiencies,and the modified Davis method is relatively reliable for citrus flavonoids determination.The NaNO2-Al(NO3)3-NaOH method was suitable for the determination of flavonoids with catecholic moieties in B-ring and no C-3 hydroxyl group,but had no specificity;the AlCl3 method could be used for the determination of flavonols and flavones with C-5 hydroxyl group,but connot make sufficient determination.The inaccuracy of these two methods was mainly owing to the unsuitability of their principles,which resulted in the results lower than the reality.The 2,4-DNPH method was suitable for the determination of flavanones and polymethoxylated flavones,while this method may be strongly affected by other unknown components containing carbonyl groups in extracts.The modified Davis method(by choosing naringin as the standard at the wavelength of 420 nm for grapefruit and pomelo,and hesperidin as the standard at the wavelength of 360 nm for other species)which could be interchangeable with the HPLC method to some extent and compensate for the depancrecy of the original Davis method,was considered a rapid,convenient and reliable method for the determination of citrus flavonoids.2.Ponkan peel extract has the highest total phenolic content and strongest overall antioxidant capacity in eight representative citrus peel extracts(CPEs).After extensive ethanol extraction and purification by D-101 resin,the purity of flavonoids in CPEs was enhanced by 3-9 times.Hesperidin,naringin,neohesperidin.narirutin and eriocitrin were identified as the major flavonoids,the amount of which varied a lot in different CPEs.The highest total phenolic content was found in ponkan peel extract,while the highest total flavonoid content was found in grapefruit peel extract.Based on the results obtained from four chemical antioxidant assays,ponkan peel extract had the strongest overall chemical antioxidant activity.Besides,results from cellular antioxidant activity assay showed that hesperetin could exert certain antioxidant activity in the intracellular environment,which reflected the in vivo antioxidant potential of ponkan peel extract to some extent.3.The interaction mechanism between hesperidin,which is the main pancreatic lipase inhibitor in CPEs,and pancreatic lipase for the first time.Pancreatic lipase(PL)activity is closely related to the formation of obesity.Eight kinds of CPEs had strong PL inhibitory capacity,from which sweet orange peel extract was the strongest and the inhibitory pattern belonged to non-competitive inhibition.Hesperidin has been determined as the main active PL inhibitor in CPEs and could quench the lipase intrinsic fluorescence effectively via static quenching,where the glycosylation on 7-C of hesperidin played a vital role.In the spontaneous exothermic interaction process of lipase and hesperidin,hydrogen bonds and van der Waals forces played the major roles,and only one binding site existed in a place away from the catalytic sites of lipase.Particularly,the secondary structure of lipase would not clearly change after interaction with hesperidin.The results of molecular docking,which showed that hesperidin could form hydrogen bonds with amino acid residues like Thr221,Argl91,Ser195 and His224 in the non-catalytic region of lipase,were consistent with that of experiments.