Research On Processes Of Ehretia Macrophylla Wall. Fruit And Structural Identifications Of Separated Phenolic Compounds

Yepipa(wild loquat)is the fruit of Ehretia macrophylla Wall. under Ehretia genus, Boraginaceae family. The processed fruit is edible with medicinal usages such as relieving pharyngitis and cough in the folk. Previous studies have found that plants under same genus as Ehretia macrophylla Wall. are rich in natural active ingredients, which are proved to have strong antioxidant, anti-inflammatory and anti-histamine and other physiological activity. Previous studies has mainly focused on the branches and leaves of Ehretia plants, however, the fruit of Ehretia macrophylla Wall., Yepipa is rarely reported for its active ingredients. Therefore, the main objectives of present study were to investigate the total phenolic and flavonoid contents distribution in distinct tissues in Yepipa, and to optimize the process mothods by phenolic contents and antioxidant activity values. At last the alcohol extract of Yepipa was separated and purified to give monomeric compounds with certain activity.Firstly, nitrogen protective and non-nitrogen conditions were applied on the separation of the peel, pulp and seed tissues, which were extracted with cold acetone to give phenolic extracts. Total phenolics and flavonoids contents were measured by Folin-reagent method and sodium borohydride/chloranil-based assay(SBC) respectively and the result showed that that: extracts obtained under nitrogen protection had higher total phenolic and total flavonoid content. Peel had the highest total phenolic and total flavonoid contents at 6909.17±176.85 GAE/100 g, DW and 3066.96±169.06 CAE/100 g, DW respectively. Antioxidant activities were evaluated by PSC and ORAC method. Peel extracts also showed strongest antioxidant activity with PSC and ORAC values were 859.85±70.32 μmol vit. C equiv./g, DW, 2877.01±163.80 μmol trolox equiv./g. A significant correlation was found between the phenolic contents and the antioxidant activity.Then the effect of four different processes(fresh fruit, steamed fruit, dried fruit, dried steamed fruit) on total phenolic, total flavonoid contents and antioxidant activities were compared. Yepipa fruit heated by steaming had the highest phenolic content of 4884.89±69.78 mg GAE/100 g, higher flavonoid content 2117.41±55.57 mg CAE/100 g, DW, and the strongest antioxidant activity. Heat treatment could remarkablely reduce the loss of phenolic compounds in the fruit in the following drying process. Further studies were to investigate two common heating treatments(blanching and steaming) on Yepipa samples. After heating, the total phenolic content and antioxidant activity of Yepipa increased to various degrees, but no significant effect was found on the content of total flavonoid. Steaming for 5 min was determined to be the best condition for retaining higher phenolic and flavonoid contents and stronger antioxidant activity.Finally, as raw material, the steamed dried fruits of Yepipa were reflux extracted with ethanol to obtain ethanol extract which was followed by systematic solvent extraction. 7 monomeric compounds were separated and purified from the ethyl acetate phase extract, by means of column chromatography on silica gel and Sephadex LH-20 dextran gel as well as recrystallization. Analyzed with LC-HRMS and NMR spectroscopy, those monomeric compounds were identified as follows: methyl rosmarinate, rosmarinic acid, methyl caffeate, caffeic acid and protocatechuic acid, which were reported to have antioxidant, anti-inflammatory, anti-proliferative and other biological activities.