Bioactive Potential And Mechanisms Of Proanthocyanidins From Kiwi Leaves

Proanthocyanidins is a kind of polyphenols widely existing in plants.A large number of studies have proved that proanthocyanidins have strong antioxidant and antidiabetic activity,but its antioxidant mechanism is not clear yet.At the same time,the poor biological stability and low bioavailability of proanthocyanidins also limit its development and utilization prospects.As a featuring characteristic fruit in China,kiwi has a widely international market.Due to the abundance of vitamin C,kiwi is favored by consumers,and China accounts for about 50%of the world’s production.The branches and leaves of kiwi pruned as waste during the growth,which is not fully utilized.It has been proved that kiwi leaves are rich in polyphenolic bioactive compounds,which consider as potential resource of proanthocyanidins with development application.This paper studied the extracted technology of proanthocyanidins from kiwi leaves,further identified its chemical structure and composition,and explored the antioxidant effect and mechanism of purified kiwi leaves proanthocyanidins using Caco-2 oxidative damage model.Using nanoliposomes to encapsulate purified kiwi leaves proanthocyanidins as a drug delivery system,to improve its biological stability and bioavailability.This study finally researched the effect of purified kiwi leaves proanthocyanidins on gut microbiota.The main research results are as follows:(1)Ultrasonic-assisted extraction of proanthocyanidins from kiwi leavesSingle factor and response surface interaction experiment were used to explore the optimal ultrasonic-assisted parameters.The optimal ultrasonic extraction condition were exhibited under the ultrasonic amplitude 40%,ultrasonic time 15 min,ultrasonic temperature 70℃ and liquid to material ratio 30 mL/g,with the yield of proanthocyanidins under 122.19 ± 5.71 mg PC/g DW.HPLC-QTOF-MS/MS results showed that ultrasonic-assisted extraction could significantly increase(increased by 30.6%)the content of proanthocyanidins when compared with traditional extraction.(2)To purify and identify the chemical structure and compositions of extracted proanthocyanidins from kiwi leavesUsing AB-8 macroporous resin and Sephadex LH-20 column to purify the ultrasonic extracted sample,and methanol eluted phase and acetone eluted phase were obtained.And FT-IR,RP-HPLC-QTOF-MS/MS,MALDI-TOF MS and 13C-NMR were used to systematically identify the chemical structure and composition of purified kiwi leaves proanthocyanidins.The results showed that the purified kiwi leaves proanthocyanidins contained procyanidins and prodelphinidins,even including the rarely reported propelargonidins.Among them,(epi)catechin exist as terminal and extension units,while(epi)afzelechin and(epi)gallocatechin exist as extension units.In addition,the mean degree of polymerization of purified kiwi leaves proanthocyanidins obtained by various elution phases was different,and methanol eluted phase and acetone eluted phase were 3.2 and 5.9,respectively,and methanol eluted phase including small amount of flavonoids.(3)The antioxidative mechanism of purified kiwi leaves proanthocyanidinsTo establish H2O2-induced oxidative stress on Caco-2 cells,and the normal culture group,H2O2 negative treatment group,catechin positive treatment group and purified kiwi leaves proanthocyanidins experimental group were set up respectively.The oxidative damage was verified by measuring the cell viability,intracellular ROS,MDA,GSH px,CAT and T-SOD values.The expression of genes related to Nrf2 signalling pathway was determined by qRT-PCR,western blot,single cell immunofluorescence and other analysis,to further explore the antioxidant mechanism of purified kiwi leaves proanthocyanidins.The experimental results showed that purified kiwi leaves proanthocyanidins could significantly reverse the transcription and expression levels of relative antioxidant genes,such as:SOD-1,HO-1,Bcl-2,Bax,NQO-1 and CAT,thereby attenuation oxidative stress.Purified kiwi leaves proanthocyanidins showed stronger antioxidant activity than catechin,meanwhile,methanol eluted phase showed more powerful antioxidant activity due to its smaller relative molecular weight and the mean degree of polymerization.(4)Preparation the nanoliposome to enhance the biostability and bioavailability of purified kiwi leaves proanthocyanidinsThe optimal preparation technology with pH 3.2 of sodium acetate acetic acid buffer,5%(wt.)mass ratio of lecithin,270 W of ultrasonic power,5 min of the ultrasonic time,which exhibited the optimal stability and encapsulation efficiency.A simulated digestion experiment was established to verify the biological stability of nanoliposome by measuring its surface potential,particle size and microstructure changes.The results showed that nanoliposome could be used as drug delivery system to release purified kiwi leaves proanthocyanidins until small intestine.Nanoliposomes could protect purified kiwi leaves proanthocyanidins from external factors and maintaining good physical and storage stability.And the concentration of purified kiwi leaves proanthocyanidins in loaded nanoliposomes was 2.28 to 3.07 times higher than purified kiwi leaves proanthocyanidins in an aqueous solution during the intestinal digestion stage.In addition,the pharmacokinetics experiment in rats showed the concentration of purified kiwi leaves proanthocyanidins nano-encapsulated by lecithin in the plasma was significantly higher than that in an aqueous solution,which reached the maximum value after 8 h,33.25 ± 8.38 μg/mL and 12.17 ± 1.85 μg/mL,respectively.(5)The influence of purified kiwi leaves proanthocyanidins on gut microbiotaBy feeding various treatment of proanthocyanidins,the relationship between purified kiwi leaves proanthocyanidins,gut microbiota and body function was preliminarily explored.The experimental results exhibited that purified kiwi leaves proanthocyanidins could not improve the diversity and abundance of gut microbiota,while significantly decline the F/B value.Akkermansia,Lachnospira and Muribaculaceae increased significantly after treated by nanoliposomes loaded purified kiwi leaves proanthocyanidins,while reduce the relative abundance of pathogenic Enterococcus,thereby improvement the gut microbiota.Tax4Fun function predicts that purified kiwi leaves proanthocyanidins will affect the body metabolism,such as accelerating the metabolism of starch,sucrose and other polysaccharides and promoting the expression of phosphotransferase.