Studies On The Biochemical And Molecular Foundation Of Punicalagin Metabolism In Pomegranate

Pomegranate is rich in various bioactive substances and abundant in germplasm resources,which had a long cultivation history in our country.The health function of pomegranate is strong,which development prospect is very bright.Punicalagin belong to ellagitannins,is a unique polyphenol in pomegranate.The content of punicalagin is highest in pomegranate,which has strong antioxidant and plays a vital role in the health functions of pomegranate.At present,the metabolic mechanism of punicalagin is unclear in pomegranate fruit,which greatly limiting the cultivation regulation of functional quality and breeding work in pomegranate.Therefore,the content of punicalagins and related synthetic substances determined by HPLC optimization system were established in Chinese dominant pomegranate cultivars.The change regularity and relationships of punicalagins and related synthetic substances were evaluated in different pomegranate cultivars and parts during development phases.The differences of phenolic compounds and antioxidant capacity of different pomegranate cultivars were comparative analyzed in different producing areas at mature stage.The cDNA fragments of punicalagin synthesis related enzyme genes were cloned,which bioinformatics and expression level were analyzed by real-time quantitative PCR.It laid the biochemical and molecular basis for revealing the mechanism of functional substance punicalagin in pomegranate fruit.The main results for this study were as follows:（1）The content determination of punicalagins and related synthetic substances were optimized and established by HPLC methods in peel,juice,seed and leaf of ‘Taishanhong’ and ‘Taishansanbaitian’ pomegranate.The mobile phases of punicalagin,gallic acid and ellagic acid were was acetonitrile and 2% glacial acetic acid solution with the ratio of 20:80.Their determines wavelength all were 270 nm.The mobile phase of shikimic acid and 3-dehydroshikimic acid was methanol and 1% phosphoric acid with the ratio of 5: 95.Their determine wavelengths both were 214 nm.The mobile phase of pentagalloylglucose was acetonitrile and 0.5% phosphoric acid with the ratio of 20:80.Its determine wavelength was 275 nm.The standard recovery rates of six substances were more than 98%.The relative standard deviationas（RSD）were 1.2%².2%.There were significant differences in the contents of punicalagin,gallic acid,ellagic acid shikimic acid,3-dehydroshikimic acid,pentagalloylglucose in different pomegranate cultivars and parts.（2）The variation tendency of punicalagins and related synthetic substances of different pomegranate cultivars were different in different parts during development period.The relationships of punicalagin,gallic acid,shikimic acid,3-dehydroshikimic acid,anthocyanin,total flavonoids,total phenols and DPPH radical scavenging activity all were significant and positive in leaves.Shikimic acid and 3-dehydroshikimic acid were closely related to gallic acid metabolism.The metabolism of gallic acid could produce pentagalloylglucose.Ellagic acid was the degradation product of punicalagin.Pentagalloylglucose mainly existed in pomegranate leaf.Gallic acid and pentagalloylglucose metabolized into punicalagin might be mainly in pomegranate fruit.Punicalagin,gallic acid,ellagic acid,shikimic acid,3-dehydroshikimic acid and pentagalloylglucose all were the bioactive substances in pomegranate.The contents of these bioactive substances were closely related to their antioxidant capacity.Pomegranate peel was rich in phenolic compounds,strongest in antioxidant capacity,which had development and utilization potential.（3）The 3’ end cDNA sequence of DHD/SDH and DAHPS gene,conservative sequence of DHQS gene were amplified by RT-PCR methods in pomegranate peel.The sequence length of DHD/SDH gene was 1585 bp,which contained 1265 bp CDS sequence and encoded 421 amino acids.The DHD/SDH protein had DHQase I and SDH（AroE）conserved domains.The GenBank accession was KU133479.The sequence length of DAHPS gene was 1373 bp,which contained 1180 bp CDS sequence and encoded 393 amino acids.The DAHPS protein had Cys-Ser-Ala-His and typical DAHP_Synthe₂ super family conserved domain.The GenBank accession was KU198932.The conservative sequence length of DHQS gene was 273 bp,which encoded 91 amino acids.The DHQS protein had DHQ_Fe_ADH super family conserved domains.The GenBank accession was KY200850.The bioinformatics indexes of physical and chemical properties,hydrophobicity/hydrophilicity,transmembrane structure,signal peptide,secondary structure and tertiary structure of DHD/SDH and DAHPS gene were analyzed.（4）The relative expression levels of DAHPS and DHQS decreased significantly,and the DHD/SDH increased gradually with the increase of development days.The expression levels of DAHPS,DHD/SDH and DHQS in ‘Taishanhong’ were higher than that in ‘Taishansanbaitian’ at the whole fruit development period.The expression level of DAHPS in two pomegranate cultivars was obviously higher than DHD/SDH and DHQS.Correlation analysis indicated that DAHPS and DHQS were significantly and positively correlated with punicalagin,gallic acid,ellagic acid and shikimic acid,significantly and negatively correlated with 3-dehydroshikimic acid and pentagalloylglucose.There was significant negative correlation between DHD/SDH and punicalagin,gallic acid,ellagic acid and shikimic acid,and positive correlation between DHD/SDH and 3-dehydroshikimic acid,pentagalloylglucose.（5）The differences of fruit quality,phenolic compounds and antioxidant capacity were significant in different dominant pomegranate cultivars at different producing areas.The contents of punicalagin,gallic acid,ellagic acid,anthocyanin,total flavonoids,total phenols and DPPH radical scavenging activity were highest in peel,which were second highest in juice,and lowest in seed.There was certain correlation among fruit quality indexes,fruit quality and phenolic compound.The contents of punicalagin,gallic acid,ellagic acid,anthocyanin,total flavonoids and total phenols were closely related to their antioxidant capacity.‘Kashitian’,‘Qianzihong’,‘Taishanhong’,‘Hongpisuan’ and ‘Damayatian’ had excellent comprehensive fruit quality,which were worth popularization,development and utilization.The tissue,organ and development specificity of punicalagin and its related substances were analyzed in the study.The cDNA fragment sequence of punicalagin related regulatory enzyme genes were cloned,which bioinformatics and expression level were analyzed in pomegranate.The fruit quality,phenolic compound content（included punicalagin）and antioxidant ability of 24 main cultivars were analyzed,which were from different regions of China.The results laid a theoretical foundation for revealing the source-sink relationship of punicalagin metabolism in leaf and fruit,the cultivation regulation of functional quality,digging into the depth of fruit nutrient value and the creation of new pomegranate germplasm.The study in the metabolic mechanism of functional substance punicalagin was propelled in pomegranate.