Study On Anti-aging Active Components,Molecular Mechanism And In Vitro Simulated Digestion And Absorption Of Radish Leaves

Based on free radical theory,screening safe and non-toxic active ingredients from natural products,which exhibit an anti-aging activity,has become a research hotspot.Modern pharmacological studies have shown that radish leaves have a various biological activities.Previous studies showed that ethanol extract from radish leaves has strong free radical scavenging and antioxidant activities,and could effectively prolong the lifespan of Drosophila melanogaster.However,the chemical composition of anti-aging active fraction and its molecular mechanism as well as the digestion and absorption rule of radish leaves are still not clear.Therefore,in this study,a polyphenols-rich extract from radish leaves were prepared by subcritical water extraction firstly.Then,an active fraction from radish leaves extract was screened using the H₂O₂-induced MRC-5 cell oxidative stress senescence model,meanwhile the molecular mechanism of the anti-aging effect was also studied at cellular level.At the same time,the changing rules during in vitro simulated digestion and the transmembrane transport rules of active components consist in active fraction were also investigated.The research results will provide theoretical basis and scientific foundation for the utilization of radish leaf resources and the development of food and health products for delaying aging.The main research contents and results are as follows:（1）Extraction of polyphenols from radish leaves by subcritical waterIn this study,polyphenols extraction by subcritical water from radish leaves were optimized using response surface methodology.Based on the single factor test results,Box-Behnken design was used to optimize the extraction variables:extraction temperature（137℃）,solvent to solid ratio（34:1）mL/g,and extraction time（13 min）with NaHSO₃ addition of 0.3%（m/v）for the extraction yield of total polyphenols.Under the optimal condition,the experimental yield of polyphenols is（7.11±0.03）mg GAE/g dw,which is agreed closely with the predicted yield.The yield of polyphenols by subcritical water extraction is 16.60%higher than that of hot water extraction.In the range of test concentration,the antioxidant activity of radish leaves polyphenols extracted by subcritical water（SWEP）was significantly higher than that of radish leaves polyphenols extracted by hot water（HWEP）.The IC₅₀0 of DPPH and ABTS radical scavenging and EC₅₀0 of reducing power for SWEP were 30.18%,18.80%and 18.27%lower than that of HWEP,respectively.（2）Screening and composition analysis of anti-aging active fraction from radish leavesBased on the aging free radical theory,a premature senescence MRC-5 cell model was established by exposure to 200μM of H₂O₂.In this study,using total polyphenol contents（TPC）,total flavonoid contents（TFC）,antioxidant activity,and cell viability of model control cells as indicators,the active fraction for delaying cell senescence were screened from radish leaves extract.The results showed that ethyl acetate fraction（EAF）in different polarity fractions from radish leaves extracts exhibited the highest TPC,TFC,antioxidant activity and the best cell protective effect.Further screening tests found that F2 fraction,separated from EAF by AB-8 macroporous resin chromatographic column,had the highest TPC of（477.95±5.31）mg GAE/g dw and TFC of（172.91±4.88）mg CE/g dw,as well as the best anti-aging activity（cell viability is 31.94%higher than model group at 100μg/mL）.Furthermore,the main component of F2 fraction were identified as caffeoylmalic acid,p-coumaroylmalic acid,feruloylmalic acid and kaempferitrin by LC-ESI-MS or/and comparison of the mass spectrometric data,retention time,infrared spectroscopy with that of commercial standards.In addition,the content of the four components were quantified as（9.67±0.65）,（11.01±0.16）,（22.26±0.72）and（176.54±1.02）mg/g dw,respectively.（3）The protective effect of F2 on H₂O₂-induced MRC-5 cell senescence and its mechanismIn this study,the effect of F2 on the aging phenotypes and redox system in H₂O₂induced MRC-5 senescence cells were investigated.The possible molecular mechanism of anti-aging activity of F2 was further investigated by Western blot technology.The results showed that pretreatment of F2 at 50 g/mL for 12 h exhibited the best protective effect on MRC-5 cells.Compare with model control,F2pretreatment decreased in the senescence associatedβ-galactosidase（SA-β-gal）activity and senescence associated heterochromatic foci（SAHF）positive cell rate significantly,stimulated cells enter into S phase from G1 phase and promoted cell proliferation.Furthermore,pretreatment of F2 increased the activity of intracellular antioxidant enzymes（SOD and CAT）,eliminated the excess ROS,maintained the balance of redox system,and then reduced the level of MDA,kept the integrity of mitochondria and DNA,thereby playing a role in delaying cell senescence.In addition,F2 shows the delaying cell senescence effect maybe in connection with inhibiting apoptosis,activating PI3K/Akt/Fox O1 pathway.（4）Recovery of active components in radish leaves during in vitro digestionIn this study,the aim of this study was to evaluate the influence of the in vitro simulated digestion（mouth,stomach,intestines）on the recovery and bioaccessibility indexes of polyphenols and flavonoids,antioxidant activities,and anti-aging active components in digestive juices from radish leaves.The obtained recovery indexes revealed that digestion could remarkably increase the releasing of polyphenols（54.91±0.96%）and flavonoids（25.26±0.55%）from radish leaves,and so as antioxidant activities.After digestion,the bioaccessibility index of polyphenols and flavonoids in radish leaves were（14.62±0.38）%and（11.95±0.77）%,respectively.Furthermore,the anti-aging active fraction of digestive juices（GIDF2）still exhibited an excellent anti-aging effect against MRC-5 cells induced by H₂O₂.（5）Transmembrane transport of GIDF2 and kaempferitrin of radish leaves.A Caco-2 cell monolayer model was established with TEER>500Ω·cm²,the ratio of alkaline phosphatase（AKP）in apical to basolateral(AKP_AP/AKP_BL)of 2.8,and the apparent permeability coefficient(P_app)of lucifer yellow（LY）<1×10^-66 cm/s,indicating that the model can be used for to study the transmembrane transport rules of the main component in GIDF2.It was found that the transport of each component in GIDF2 was time-and dosage dependent.At the concentration of 400μg/mL,the efflux rates（ER）caffeoylmalic acid,p-coumaroylmalic acid,feruloylmalic acid,and kaempferitrin were 2.111.49,1.68 and 1.69,respectively.The results showed that the decrease in temperature caused a reduction in P_{app（AP→BL）}and P_{app（BL→AP）}of each components in GIDF2,suggesting an active transport model.P_apppp and ER of caffeoylmalic acid,p-coumaroylmalic acid and feruloylmalic acid did not changed significantly with additional MK571,but additional VP significantly increased the P_{app（AP→BL）}and decreased the ER.For kaempferitrin,the transport was dramatically affected by addition of VP and MK571.As a result,P_{app（AP→BL）}increased,P_{app（BL→AP）}decreased,and ER decreased after additional VP and MK571.Therefore,it can be speculated that the secretory transport of kaempferitrin can be mediated by P-gp and MRP2,while the other three components is mediated by P-gp.