| The naturally dropped fruits of six representative citrus cultivars were collected from Zhejiang province on May17, May27, June16, July6and July16, including Citrus poonensis Hort. ex Tanaka (PG), Citrus suavissima Hort. ex Tanaka (OG), Citrus unshiu var. praecox Tanaka (ZS), Citrus unshiu Marc.cv Yamada (WS), and Citrus paradise Macf. Changshanhuyou (HY) and Citrus sinensis Osbeck (TC). In this dissertation major functional compounds and antioxidant activity in above6mandarin varieties (four mandarins, one sweet orange and one pummelo) were investigated to provide useful information for the utilization of citrus resources. The main results were presented as follows:(1) Optimization of synephrine extraction from young fruits of Citrus poonensis Hort. ex Tanaka (PG) by response surface methodology. An efficient ultrasonic extraction technique was developed to extract synephrine from young fruits of Citrus poonensis Hort. ex Tanaka. A series of single-factor experiments were made. Based on the experiment results, a response surface design was generated by the Design-Expert software according to the Central Composite Design and experimental data obtained were subjected to quadratic regression analysis. The results showed that the optimum extraction conditions were as follows:methanol concentration70.46%,ultrasonic extraction temperature34.45℃,ultrasonic extraction time45min, liquid to solid ratio24.84mL/g. The predictive maximal yield of synephrine was10.88mg/g DW. Validation experiments were carried out at these optimum conditions and the results indicated that the synephrine yield was10.85mg/g DW. The results were basically corresponding to model prediction.(2) Orthogonal design was used to optimize the microwave-assisted extraction of total flavonoids from young fruits of Citrus poonensis Hort. ex Tanaka. The total flavonoid yield was investigated with respect to four process conditions including microwave power, ethanol concentration, solvent-to-solid ratio and microwave time. The results showed that the optimum extraction conditions were as follows:616W of microwave power,50%of ethanol concentration,40s of microwave treatment time, and30mL/g of solvent-to-solid ratio. Validation experiments were carried out at these optimum conditions and the results indicated that the total flavonoid yield was24.9452mg RT/g DW. The influence of the four factors on the flavonoid yield extraction was ethanol concentration>solvent-to-solid ratio>microwave time> microwave time> microwave power. The microwave-assisted extraction of total flavonoid proved highly efficient.(3) The main bioactive substances were analyzed, including seven phenolic acids, ten flavanones, seven flavones, synephrine, the total flavonoid content and the total phenolic content with the development of fruit. Phenolic acids were determined by HPLC, which included three hydroxybenzoic acids:protocatechuic,p-hydroxybenzoic and vanillic, and the four hydroxycinnamic acids:caffeic, p-coumaric, ferulic, and sinapic. The contents of cinnamics were11.27times higher than that of the benzoics. The predominant phenolic acids of the two types were ferulic acid and p-hydroxybenzoic respectively. The order of the contents of the seven phenolic acids in all mandarin fruits was ferulic acid> p-coumaric acid> caffeic acid> p-hydroxybenzoic acid> vanillic>sinapic acid> protocatechuic acid. The average contents of ferulic and protocatechuic acids were3144.23ug/g DW and15.59μg/g DW. It was observed that phenolic acid contents vary according to the citrus species and the growing periods. With the development of fruit, the content of protocatechuic acid, caffeic acid and sinapic acid increased gradually, while the content of p-hydroxybenzoic, vanillic and ferulic increased gradually, and then declined. The order of the total contents of seven phenolic acids in young mandarin fruits collected on the same date was that OG>PG>ZS>WS>TC>HY.The total content of ten flavanones in all young fruits was higher than that of seven flavones. Neohesperidin was found to be the major flavonoid and followed by naringin (472.32mg/g DW), narirutin (308.61mg/g DW), and hesperidin (258.32mg/g DW). The highest concentration of neohesperidin was present in young fruits of Ougan. Their concentrations ranged from150.09-316.56mg/g DW. The contents of tangeretin were the lowest (0.46mg/g DW). The highest contents of flavonoid were found in Ougan and Changshanhuyou. The contents of ten flavanones seven flavones showed a downward trend overall, which decreased from358.38mg/g DW to203.68mg/g DW and from268.80mg/g DW to85.27mg/g DW in different growth periods of Ougan and Changshanhuyou respectively. The results indicate that the two young mandarin fruits collected before June16th were very good source material for extracting flavanones and flavones.The order of average content of synephrine in different citrus varieties was PG>OG>WS>ZS>TC>HY. The highest synephrine content was found in Ougan (21.93mg/g DW); while the lowest in Changshanhuyou (0.52mg/g DW).The results showed that the contents of synephrine were significantly degressive trend with the increase of fruit maturity. Significant higher levels of total flavonoids and total phenolic acids were found in young fruits than that in the subsequently mature fruits.With the development of fruit, the content of the two declined gradually. The highest total flavonoids content was found in Ougan (41.25mgRT/g DW), followed by Changshanhuyou(34.28mgRT/g DW),Ponkan(30.59mgRT/g DW), Shantian Satsuma(26.84mgRT/g DW),Gongchuan Satsuma(22.96mg RT/g DW) and Sweet oranges(22.61mg RT/g DW). The highest phenolic acids content was found in Ougan(101.01mg GAE/g DW), followed by Changshanhuyou, Sweet oranges, Shantian Satsuma, Ponkan, and Gongchuan Satsuma, with contents of80.73,46.82,43.39,39.91, and7.13mg GAE/g DW respectively. (4) The present study applied four tests of antioxidant potency including ABTS free radical-scavenging activity, DPPH free radical-scavenging activity, ferric reducing antioxidant power (FRAP) and total oxygen radical absorbance capacity (ORAC) to evaluate the antioxidant capacity of extract of mandarin fruits. Antioxidant potency composite index (APC) was determined by all assays an equal weight to assess the antioxidant capacities. The relevance between the contents of total phenolic acids, total flavonoids, synephrine and the antioxidant capacities were analyzed. The order of TEAC in the same mandarin fruits collected on the same date was ORAC assays>ABTS assays>FRAP assays>DPPH assays. The highest APC was found in Ougan (69.66), followed by Changshanhuyou (49.28), Sweet oranges (46.72) Ponkan (25.12), Shantian Satsuma (23.34), and Gongchuan Satsuma (21.55).The results showed that mandarin fruits had considerable antioxidant capacities. Changes in antioxidant potency composite index and ABTS assay showed similar pattern during fruit development. With the development of fruit, the APC of mandarin fruits except for Changshanhuyou increased gradually. The APC showed the highest level was that collected on May27th or June16th, then slightly declined later.The results indicated that the correlation coefficients of the antioxidant capacity detected by three methods (ABTS, DPPH, FRAP) with total phenolic acids, total flavonoids were extremely significant (p<0.01). The higher the content of total phenolic acids and total flavonoids were, the stronger the antioxidant capacity was. But the synephrine content had a lower or even negative relationship with antioxidant activity. |
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