An On-line HPLC-FRSD System For Rapid Evaluation Of The Total Antioxidant Capacity Of Citrus Fruits And Their Derived Products

Many human diseases have been directly or indirectly linked to the capacity of the antioxidant system in the human body. Similarly, the deterioration of food caused by oxidation is also closely related to the antioxidant capacity of the food. People can improve their own capacity of antioxidant system through taking in the antioxidants. And adding the man-made or natural antioxidants is benefit for prolonging the keeping time and the shelf-life of foods. However, there has big debate about the safety of the man-made antioxidants doubting that will threat the human health for a long time. Therefore, the research about finding the strong natural antioxidants or species of plants has become a research hotpot in the antioxidative field.Citrus(Citrus L., Rutaceae) was an important fruit crop consumed around the world, and citrus fruits have been proved to be a good source for natural antioxidants. Until now, many detection methods of antioxidant capacity have been built, such as off-line DPPH method, ABTS method, FRAP method and ORAC method, and on-line HPLC-DPPH, HPLC-ABTS method, and so on. However, the off methods (DPPH method, ABTS method, FRAP method and ORAC method) used for antioxidant capacity detection are always characterized by time-consumed and high labor intensity; on line methods reported focused on the antioxidant capacity of a single compound in a complex plant matrix, which cannot be used for analyzing total antioxidant capacity (TAC) of whole mixture. However, the analysis of total antioxidant capacity of whole samples was always the first step for many researches. It is very important for many antioxidant studies to quickly analyze the total antioxidant capacity of samples. The establishment of fast and efficient detection methods for analyzing antioxidant capacity can promote the antioxidant capacity studies, and will be very beneficial for fast and systematically analyzing antioxidant capacity of plant resources, and will be meaningful for fast analyzing the antioxidant capacity of foods.Aimed to establish and apply fast and efficient detection method of antioxidant capacity, the study combined high performance liquid chromatography (HPLC) system with post column (free radical scavenger detector, FRSD) system to build a new on-line system (on-line HPLC-FRSD system), optimized the experiment condition of on-line HPLC-FRSD system. And two free radical solutions (DPPH and ABTS free radicals) were studied in FRSD system to establish on-line HPLC-DPPH-FRSD and HPLC-ABTS-FRSD methods. Then the new one-line system was verified by parameters containing repeatability, stability, recovery ratio, etc. Based on the above mentioned work, antioxidant capacities of peels, segment membranes, seeds and juices of35genotype citrus fruits were assayed by on-line HPLC-DPPH/ABTS-FRSD methods. And the correlation of the new on-line HPLC-DPPH/ABTS-FRSD methods and off-line DPPH/ABTS methods was analyzed to value the application of new on-line methods for fast analyses of samples in different matrixes. Meanwhile, the total phenolic contents and total flavonoid content in four parts of citrus fruits were all tested. The HPLC-FRSD system, used for correlation study between antioxidant capacity and total phenolic/flavonoids content, was also analyzed. Compared with the reported on-line antioxidant capacity detection methods, the new on-line HPLC-FRSD system established was one sign of technological advance, which will have important theoretical and practical values for evaluation of citrus resources and research of citrus fruits nutriology in future. The main results are as follows:1. The establishment of the on-line HPLC-FRSD system for detecting the antioxidant capacity1.1Comparison of antioxidant capacity of phenolic standards in citrusIn order to prepare the antioxidant standards for establishing the new on-line methods, the study tested antioxidant capacity of15citrus phenolic compounds by off-line DPPH method, ABTS method, FRAP method, reported on-line HPLC-DPPH method and HPLC-ABTS method. And the antioxidant potency composite (APC) index was used for evaluating the antioxidant capacity of phenolic standards in citrus. The order for antioxidant capacity of phenolic compounds in citrus was as following(APC index,%): gallic acid (92.32%)> caffeic acid (85.29%)> chlorogenic acid (69.75%)> ferulic acid (50.97%)> eriodictyol (39.38%)> eriocitrin (39.36%)> rutin (27.42%)> hesperitin (4.88%)> naringenin (4.78%)> diosmin (3.24%)> hesperidin (2.33%)> nobiletin (1.18%)> sinensetin (1.16%), naringin (1.16%)> tangeretin (0.41%). Based on the above results of the antioxidant capacity, rutin, ferulic acid, gallic acid, chlorogenic acid and caffeic acid were used as antioxidant standards for establishing the new on-line HPLC-FRSD system.1.2The optimization of on-line HPLC-FRSD systemIn order to establish the new system for fast and efficient detection of samples’ antioxidant capacity, the research optimized the factors which influenced the on-line HPLC-FRSD system. And results of the analysis condition optimized were showed as followings:In this study, an on-line high performance liquid chromatography-free radical scavenging detection (HPLC-FRSD) system was developed. It consisted of a Waters e2695HPLC system [a Waters quaternary pump system, a column oven, an autosampler, a Waters X-Terra MS C18guard column and Waters2998photodiode array detector (PAD)] and a free radical scavenging detector system (peek reaction coil, temperature control model, pump and2489UV/Visible detector). The whole system was supported by Waters Empower TM2Chromatography Data Software (Waters Corporation, Milford, MA, USA).25μL of each sample was injected into the on-line HPLC-FRSD system. The positive chromatographic peaks of compounds in the samples were separated by Waters e2695HPLC system with a Waters X-Terra MS C18guard column (3.9mm×20mm,5μm). Water/acetic acid (999:1, v/v) as mobile phase A and HPLC-grade methanol as mobile phase B were used as mobile phases. Elution conditions was0-5min of37%A and63%B (isocratic elution) at a flow rate of0.91mL min-1using a detector, Waters2998PAD detector array at245nm and283nm wavelengths simultaneously. The temperature of the column oven was maintained at30℃. The phenolic compounds eluted from the HPLC system were reacted with the free radical solutions in post-column system. For the on-line HPLC-DPPH-FRSD assay, the flow rate of DPPH free radical solution reagent (0.20mM) was set at0.80mL min-1, and the induced bleaching was detected as a negative peak at517nm by the Waters2489UV/Visible detector. For the on-line ABTS radical scavenging assay, the flow rate of ABTS free radical solution reagent (an absorbance of0.70±0.02AU at400nm) was set to be0.80mL min-1, and the induced bleaching was monitored as a negative peak at400nm by the Waters2489UV/Visible detector. The temperature of the on-line post-column system was maintained at30℃. Running time for each sample was5min. The TAC values of samples were all quantified by reference L-ascorbic acid calibration curve and expressed as L-ascorbic acid equivalent antioxidant capacity(AAEAC)[mg g-1Dry Weight (DW) or mg L-1, Fresh Weight (FW)].1.3Verification of the new on-line HPLC-FRSD systemTo verify our new on-line methods, the performance parameters of the on-line HPLC-FRSD system, including linearity, limits of detection (LODs), limits of quantification (LOQs), recovery, precision, reproducibility and stability were established using5phenolic standards.The results showed that the on-line HPLC-DPPH-FRSD method and the on-line HPLC-DPPH-FRSD method both have good linearity (r<0.999), low limits of detection (0.001-0.010mg mL-1) and quantification (0.005-0.020mg mL-1), excellent recovery rate (90.44-115.72%), stability (RSD<15.80%), reproducibility (RSD<2%), and precision (RSD<2%). The results of parameters validation proved the system were qualified.2. Application of on-line HPLC-FRSD system for analyzing the total antioxidant capacity of citrus fruitsIn order to prove whether the new on-line HPLC-FRSD system can be used for fast and efficiently analyzing samples in different matrixes or not, the total antioxidant capacity (TAC) of the35mature citrus fruit (peels, segment members, seeds, citrus juice) extracts were tested by new on-line and off-line methods; and Pearson’s correlation coefficients between new on-line and conventional off-line methods was analyzed. The research showed that the correlation between on-line and off-line are significant correlation (P<0.05); the on-line methods can be used for antioxidant capacity fast and efficient detection of samples in different matrixes. Among them, the HPLC-DPPH-FRSD method can be used for antioxidant capacity detection of citrus segment members, seeds and citrus juices; the HPLC-ABTS-FRSD method can be used for antioxidant capacity detection of citrus peels, seeds, citrus juices.3. Application of on-line HPLC-FRSD system for study about analyzing correlation between total antioxidant and total phenolic/flavonoid contentThe antioxidant capacity was caused by antioxidants. The total phenolic compound content and total flavonoids content were tested by Folin-Ciocalteu method and AICl3method. Application of on-line HPLC-FRSD system for research about analyzing correlation between total antioxiidant and total phenolic/flavonoid content was studies. The results showed that the on-line HPLC-DPPH-FRSD can be used for analyzing correlation between total antioxidant capacity and total phenolic/flavonoid content of citrus fruits; the on-line HPLC-ABTS-FRSD can be used for analyzing correlation between total antioxidant and total phenolic/flavonoid content of citrus peels and juices.4. The antioxidant capacity, total phenolic and flavonoid contents of citrus fruits4.1The antioxidant capacity results of35mature citrus fruits The citrus peel samples with higher antioxidant capacity contained Xin Sheng Xi No.3Ponkan and Liu Sheng Gan; the segment membrane with higher antioxidant capacity contained Gou Tou Cheng; the seeds with higher antioxidant capacity contained Miho-core, Kara Mandarin; the juice with higher antioxidant capacity contained Ling Nan Sha Tian You, Parson Special mandarin, Parson’s Special Mandarin, Bai Ju. The citrus samples with lowest antioxidant capacity contained seeds and juice of Sha Gan, juice of Guo2701-1Li Meng, juice, segment membrane and seed of Rangpur Lime. In general, the citrus peel and juice were the main contributors to the antioxidant capacity of citrus fruits.4.2The results of total phenolic content of35mature citrus fruits The higher total phenolic content for peel contained Gou Tou Cheng and Dai Dai; the higher total phenolic content for segment membrane contained Gou Tou Cheng and Dai Dai; the higher total phenolic content for seed contained Miho-core, Kara Mandarin, Red Blush, Man Ju and Rio Red Grapefruit; the higher total phenolic content for citrus juice contained Liu Sheng Gan, Gou Tou Cheng, Xin Sheng Xi No.3Ponkan and Red Blush.4.3The results of total flavonoid content of35mature citrus fruits The higher total flavonoid content for peel contained Gou Tou Cheng, Dai Dai and Rio Red Grapefruit; the higher total flavonoid content for segment membrane contained Gou Tou Cheng, Star Ruby Grapefruit, Dai Dai, Red Blush and Zhu Luan; the higher total flavonoid content for seed contained Kara Mandarin, Bai Ju and Hayaka; the higher total flavonoid content for citrus juice contained Rio Red Grapefruit and Gou Tou Cheng.