Evaluation Of Phenolic Compounds In Acer Truncatum Leaves And Characterizations Of Its Flavonoids Biosynthesis Pathway

Acer truncatum Bunge,belonging to Aceraceae,is a kind of deciduous tree endemic in China.As an economic and greening tree species,it also has a variety of biological functions due to its unique chemical components in its leaves,kernel and seed coat.Acer truncatum leaf possess potent development prospect owing to its large annual biomass and peculiarity of natural renewal.At the same time,Acer truncatum leaf is rich in phenols,flavonoids,tannins and other chemical components with a variety of biological activities.However,current researches on the phenolic composition and antioxidant activities,as well as their basic processing in Acer truncatum leaf,such as the harvesting time,extraction method,and thermal treatment processing,are not yet systematic and in-depth,thus need to be improved.In addition,flavonoids are not only important constituents of phenolic in Acer truncatum leaf,but also the main contributor to its antioxidant activity.However,studies on the flavonoid biosynthesis pathway in it have not appeared,and the regulatory mechanism of transcription factors is still vacant.This study will systematically bridge the above gaps in the study of Acer truncatum leaf,laying a foundation for the in-depth research and development of Acer truncatum and providing scientific support.The main results are as follows:(1)The seasonal variation pattern of phenolic components and antioxidant capacity in Acer truncatum leaf during its whole growth period was investigated.It was found that Acer truncatum leaf at different harvest times all contained a large amount of total phenols,total tannins and total flavonoids,and possessed strong antioxidant capacity determined with DPPH,ABTS and ORAC assays.Among them,leaves collected in April and November had higher total phenol,total tannin,total flavonoids,1,2,3,4,6-penta-O-galloyl-?-D-glucose,methyl gallate,quercetin-3-O-L-rhamnoside,DPPH' and ABTS+ free radical scavenging capacity than leaves collected in other intermediate months(May to October).Correlation analysis showed that total phenols,total tannins and total flavonoids were the main components contributing to the antioxidant capacity,while 1,2,3,4,6-penta-O-galloyl-?-D-glucose,methyl gallate,and quercetin-3-O-L-rhamnoside were the main phenolic compounds contributing to the antioxidant capacity.High Performance liquid chromatography(HPLC)confirmed that leaves collected in April and November contained a large amount of 1,2,3,4,6-penta-O-galloyl-?-D-glucose and quercetin-3-O-L-rhamnoside(all greater than 800 mg/100g).The strong antioxidant capacity of leaves collected in April and November was further verified by cell antioxidant assay,and the correlation showed that flavonoids compounds contributed the most to the antioxidant capacity determined by cell antioxidant assay.(2)Extraction method based on phenolics and antioxidant capacity in Acer truncatum leaf were optimized.Extraction solvent and extraction time were selected through single factor experiment,and.a four-factors-three-levels response surface methodology was applied to optimize the solvent concentration(X1),material-liquid ratio(X2),extraction temperature(X3),and ultrasonic power(X4)for phenol yield(Y1)and antioxidant capacity(Y2).Results show that the best optimization extraction method is:ethanol:water(v:v)66.21%,and solid-liquid ratio 1:15.31 g/mL,ultrasonic 60?C temperature,ultrasonic power 267.30 W,extracting time 30 min,extract repeated three times to get maximum phenol yield(7593.62 mg gallic acid equivalent/100 g d.w.)and antioxidant capacity(74241.61 mu mol Trolox equivalent/100 g d.w.).And the above extraction methods were verified,and the results proofed that they were reliable.Furthermore,29 phenolics were identified in ATL extract obtained under the optimized conditions,among which,21 were identified in ATL for the first time.These results indicated that gallates,gallotannins,quercetin,myricetin and chlorogenic acid derivatives were the main phenolic components in ATL.(3)Effect of thermal treatment on phenolic components and antioxidant capacity of extract from Acer truncatum leaf and flower.Phenolic components of Acer truncatum flower were investigated for the first time by Ultra-high Performance Liquid Chromatography-Diode Array Detector-Quadrupole Time Of Flight-Mass Spectrometry/Mass Spectrometry(UPLC-DAD-QTOF-MS/MS)and compared with those of Acer truncatum leaf.An optimized high performance liquid chromatography fingerprint was then established,and 10 major phenolics existing in both ATL and ATF were quantified.Gallic acid derivatives and flavonol-3-O-glycosides were found to be their dominant phenolic constituents,with the former being key constituents which was affected by thermal treatments and further influencing the variations of total phenols.Moreover,the mechanism underlining the changes of phenolics in ATL and ATF by the treatments was characterized as a thermol hydrolysis process.During thermal treatments,polymerized gallotannins were hydrolysed to 1,2,3,4,6-penta-O-galloyl-?-D-glucose,ethyl gallate and gallic acid,resulting in more than five-fold and two-fold increase of their contents in ATL and ATF,respectively.By contrast,contents and antioxidant contributions of flavonol-3-O-glycosides gradually decreased during the process.(4)Characteristic ATL samples S1,S5,S8 were selected based on total phenols and antioxidant activities for further metabolomic and transcriptomic analyses.Metabolomic data revealed that there was 27 differently expressed phenolic metabolites.Among which,17 were flavonoids.Moreover,gallic acid derivatives and flavonoids were positively correlated with total phenols and antioxidant activities while caffeoylquinic acids were negatively correlated with them.Transcriptomic analyses characterized 20 structure genes correlated with flavonoids pathway with high expression levels,CHS2 and FLS1 were main structure genes upregulated flavonols.Based on all those indexes,AtrMYB 12,a transcription factor belonging to MYB family with characteristic domain in S7 subgroup,were screened.Correlation analysis revealed it upregulated flavonols biosynthesis.