| Epidemiological and clinical studies have associated the consumption of large amount of fruits,vegetables and grains rich in phytochemicals with lower risks of different chronic diseases,such as cancer,cardiovascular diseases and diabetes.The bioactivities of these phytochemicals will be influenced by plant development,food processing and preparation.The study on the changes of phytochemicals and their bioactivities during comprehensive utilization of plant-derived food will contribute to development of nutraceuticals and functional food.The dried peel of Citrus retivulata Blanco and its cultivars are used as Pericarpium Citri Retivulatae(in Chinese Chenpi),which have been used in China and southeast Asia for hundreds of years.As an edible and medicinal ingredient,Pericarpium Citri Reticulatae is widely used in soup and congee as dietary condiment,in tea with Puer or Oolong,and in miscellaneous snacks.It is also used as a traditional medicine to remedy indigestion and respiratory disease.Pericarpium Citri Reticulatae can be classified as Chenpi and Guangchenpi(Pericarpium Citri Reticulatae 'Chachiensis'),and the latter is listed as National Protection of Geographical Indications due to its outstanding medicinal and health benefits.Pericarpium Citri Reticulatae 'Chachiensis' is orginated from pericarps of Citri Reticulatae Blanco cv.Chachiensis grown in Xinhui district of Guangdong Province.In the present study,the effect of Citri Reticulatae Blanco cv.Chachiensis ripening on the phytochemical profiles,antioxidant and antiproliferative activities were investigated.Then the phenolic compounds and bioactivities of Pericarpium Citri Reticulatae 'Chachiensis' during preparation and aging process were assessed.Furthermore,the digestibility of phenolic compounds in Pericarpium Citri Reticulatae 'Chachiensis',along with the antioxidant activity and anti-inflammatory property were compared by in vitro digestion.Then,genome-wide transcriptome expression profiling by digital gene expression(DGE)was performed to identify how hesperetin is responsible for inhibitory effect on human breast cancer cells.Furthermore,the inhibitory mechanism of hesperetin was compared between estrogen receptor-dependent and independent breast cancer cells.The main results are as follows:1.Regardless of different ripening stages of Citrus reticulata Blanco cv.Chachiensis,peel had the highest amounts of phenolics and flavonoids,strongest antioxidant activities and antiproliferative activities.The increased content of total phenolics hesperidin and naringin,and elevated antioxidant capacities were detected in peel during maturity,whereas highest level of nobiletin and strongest antiproliferative activities were observed in immature peel.In flesh the vitamin C content elevated during ripening,while lower phenolic content,smallest ORAC value and weakest antiproliferative activity were detected in mature flesh.The flavonoid content and antioxidant capacity in seed were comparable with flesh and relatively constant during ripening.2.During aging process,higher phenolic and flavonoid contents,more individual phenolics,and stronger antioxidative activities were observed in the free phenolic fraction compared with the bound phenolic fraction.However,the bound phenolics and flavonoids intensified the contribution to the total benefit of aged Pericarpium Citri Reticulatae'Chachiensis',which exhibited higher phenolic and flavonoid content,and superior antioxidant activity.A synergistic effect of the free and bound phenolics on antiproliferation against SiHa and HepG2 cells suggested simultaneous application of free and bound fractions.The multivariate data analysis distinguished the samples during preparation and attributed the special qualities of aged Pericarpium Citri Reticulatae 'Chachiensis' to the accumulation of bound phenolic compounds.During the first two years of aging,total phenolics,flavonoids and characteristic flavonoids,such as hesperidin,elevated dramatically in the PCR01 sample,especially in the bound fraction,thus implying the active transformation of phenolics during aging process.3.The total phenolic content was increased during in vitro digested process of Pericarpium Citri Reticulatae 'Chachiensis',although the lower phenolic and flavonoid content after enzymatic digestion.Among the phenolic compounds,individual phenolic acids and hesperetin were accumulated significantly during enzymatic digestion.The antioxidant activities were highly correlated with the phenolic contents.It was noteworthy that Pericarpium Citri Reticulatae 'Chachiensis' after in vitro digestion suppressed the expression of LPS-induced iNOS,TNF?,IL-1? and IL-6,which were comparable to the effect of chemical extracts.4.Hesperetin had potential antiproliferative activity in human estrogen receptor-dependent breast cancer MCF-7 cell.The digital gene expression profile analysis of MCF-7 cells treated with hesperetin for 4 h and 24 h indicated that 94 genes expressed differentially in cells after 4 h-treatment of hesperetin(43 up-regulated and 51 down-regulated)and 491 genes expressed differentially in cells after 24 h-treatment of hesperetin(292 up-regulated and 199 down-regulated).The GO and KEGG enrichement of differential expressed genes indicated that endoplasmic reticulum stress,MAPK pathway and hormone signaling were associated with the inhibitory effect of hesperetin on the MCF-7 cells viability.The network of protein-protein interaction identified the hub genes including EGR1,SOCS1,EGR3,NR4R1,NFATC,GATA4 for 4 h-treatment and POTTE,MYC,VEGFA,EGF,EPRS,HSPA1 A,CD44 for 24 h-treatment.The common differential expressed genes in MCF-7 cells exposed to hesperetin for 4 h and 24 h were compared and subjected to GO analysis.The up-regulated GO term was cellular calcium ion homeostasis,whereas the down-regulated GO terms were related to nucleus,response to glucose,transcription,DNA-templated and response to insulin.The genes including EGR1 MAFA and TXNIP involved in most biological processes were further discussed to identify their roles in the inhibitory effect of hesperetin on the growth of MCF-7 cells.Compared with MCF-7 cells,similar antiproliferative effect of hesperetin was found in MDA-MB-231 cells.The relative expression of selected genes in MDA-MB-231 cells response to hesperetin treatment for 1 h,4 h,24 h and 48 h indicated that genes involved in endoplasmic reticulum stress and MAPK pathway were changed significantly at the later period(24 h and 48 h),as consistant to the results of MCF-7 cells.However,hesperetin inhibited the expression of CYP1A1 and CYP1B1,whereas induced the expression of INSIG1,which showed opposite changes compared with those in MCF-7 cells. |
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