Studies On The Structure Analysis, And The Relationship Between Structure And Antioxidant Activities In Vitro Of Carotenoids In Citrus Fruits

Citrus is one of the major fruits in worldwide, with bright colour, delicious taste, pleasant flavor and aboundant nutrients. There are plenty citrus fruits resources in China with first cultivated area and first outcome of whole world, it would be meaningful for the exploration of citrus processing. The studies in our lab showed that there are abundant carotenoids in the citrus fruits. Carotenoids are the principal pigments of citrus fruits and the content and profile differ in different species and tissues. Carotenoids not only can present the color of citrus fruits, but also play an important role in health care such as antioxidation activities and the relationship between the structure and bioactivies are rare and unsolved. So it has very important strategic significance to make comprehensive studies on carotenoids of citrus fruits, besides we have excellent reserch traditions in this area.The objective of this study was to gain insight into the composition and content of carotenoids in citrus fruits cultivated in Hubei Province, and the relationship between the major carotenoids and colour parameters, the stability and isomerization of carotenoids during thermal processing and the relatioship between carotenoids structure and antioxidant activity in vitro were also observed, furthermore, microcapsulation ofβ-cryptoxanthin was applied to improve its theraml stability. In this study, modern apparatus means of chromatogram and spectrum and the chemical analysis are used to investigate carotenoids and carotenoid esters in citrus fruits, the relationships between the colour parameters (L*, a* and b*) and the major content of cartenoids in citrus juices were also observed and the regression equations were used to predicte the carotenoids contents. And photosenstive peroxidation in oil assays were used to study the relationship between isomerization of carotenoids and their antioxiant activties, and three chemiluminescence assays were applied to determine the effects of esterification with fatty acid ofβ-cryptoxanthin on its thermal stability and antioxidant activities in chemiluminescence assays in vitro. The results are as follows: 1 The complex patten of carotenoid and carotenoid esters, and the relationship between colour paremeters and content in citrus fruitsCarotenoids in citrus fruits were extracted with a mixture of methanol, acetone, and petroleum ether (1:1:1, v/v/v). Total unsaponified carotenoids extracts from citrus fruits (UCE) and saponified carotenoids extracts (SCE) with methanolic KOH overnight at room temperature in the dark were obtained separately. Carotenoids were separated and analyzed along a C30 column by reverse-phase HPLC-DAD-APCI-MS.The composition and content differed in citrus fruits. The relative contents of various carotenoids in citrus fruits (fresh weight) were examined byβ-carotend standard curve. The major composition and content of carotenoids and carotenoid esters were analysis in mandarin(Citrus unshiu cv. Guoqing NO.1, Citrus unshiu Marc.cv.Miyagawa wase, and Citrus unshiu cv. Owari), sweet orange(Citrus sinensis cv. Jincheng, Citrus sinensis cv. Hamlin and Citrus sinensis cv. Valencia), red grapefruit(Citrus paradisi macfad.), pale yellow citrus fruits including bitter orange(Citrus auantium), lemon (Citrus limon) and white grapefruit(Citrus paradisi Macf.).Carotenoid composition of saponified samples of sweet orange was more complex than mandarin, though the total content of them were lower relatively. The total content of three varieties of unsaponified carotenoids extracts of satsuma mandarin were 5.03±0.25,4.88±0.18 and 5.09±0.16, respectively, which were higher than their saponified carotenoids extracts with 4.38±0.39, 4.30±0.27 and 4.48±0.30 mg/1 (fresh weight), respectively. The main features of the carotenoid profile of saponified sweet orange samples were auroxanthin, violaxanthin, antheraxanthin and zeaxanthin, whereas in the native samples, violaxanthin laurate, violaxanthin myristate, violaxanthin palmitate and violaxanthin diesters were the major composition, and around 23 carotenoids were detected in these samples, which indicated that such food stuff is one of the most intricate sources of those pigments. Meanwhile, theβ-cryptoxanthin and its esters were the major carotenoids in the investigated native or the saponified samples of mandarin, which is quite different from sweet orange and the total contents were much higher than sweet orange. Tristimulus Colorimetry was applied to characterize the color of red grapefruit juices, satsuma mandarin juices and sweet orange juices. The correlations between the color parameters L*, a*, b*, Cab* and hab and the carotenoids content were explored by partial least squares. The results obtained have shown that it is possible to obtain equations, by means of multiple regression models, which allow the determination of the individual carotenoid levels from the CIELAB color parameters, as the observed values and predicted values were not not significant different (p<0.05). In this sense, These equations could be used, as a first step, in the qualitycontrol of juices showing quite constant color during the whole season, although further studies should be carried out to extend the applicability of tristimulus colorimetry to estimate the carotenoids content in juices differing greatly in color characteristics.2 Thermal stabiltiy and isomerization of carotenoids in orange juice in microwave conditionsThe main features of the carotenoid profile were the absence or occurrence at low levels of certain isomers of the 5,6-epoxycarotenoids antheraxanthin and violaxanthin,which are major carotenoids in fresh or slightly processed juices. This fact is connected with the higher quantitative importance of their 5,8-epoxy derivatives mutatoxanthin and auroxanthin, from which it has been suggested that a rough estimation of the age of the juices can be made by considering their epoxycarotenoids content, the decrease of antheraxanthin and violaxanthin indicating that the juice has been processed for a relatively long period in microwave conditions. As for the quantitative analysis, the total carotenoid contents of the Jincheng orange juices analyzed fell within the bracket 1.37-5.89 mg/l, the levels of individual carotenoids being lower in comparison with those reported in the literature for slightly processed orange juices. The results showed that microwave processing almost had no influence on vitamin C, hesperidin, pH value and titratable acid in the juice, while, other parameters such as browning index increased. The increase of browning index and the blue shif of 5,6-epoxycarotenoids to 5, 8-epoxycarotenoids would influence the colour of orange juice, which would suggest the nonthermal processing was an alternative option.3 Antioxidation activities of carotenoids in citrus fruits in vitroThen fractions of lycopene andβ-carotene were separated by open column chromatography from red grapefruit and fraction of P-cryptoxanthin purified from satsuma mandarin Guoqing NO.1 by thin layer chromatography (TLC), respectively.(1) The effects of lycopene and P-carotene on lipid peroxidation in oil were also studied. Both lycopene andβ-carotene could inhibit the photosensitive oxidation induced by methylene blue, and the antioxidation effects were in dose-effect relationship. And the effect of compound of lycopene and P-carotene was better than each of them. The isomerization and degradation of lycopene andβ-carotene occurred during the procedure, the contents of isomers of 15-cis,13-cis,9-cis and 5-cis of lycopene and 13-cis and 9-cis isomers ofβ-carotene increased, meanwhile the antioxidantive effects were not influenced by the decrease of total carotenoid contents, which imply that the isomerization would not affect the antioxidative ablities in the detected assay.(2) The antioxidant activities of FCX and its esters on superoxide anion, hydroxide radical and singlet oxygen were investigated by the pyrogallol-luminol system, the CuSO4 phenanthroline-Vc-H2O2 system, and OH-NaClO-H2O2 system, respectively, using chemiluminescence (CL) methods in vitro. The antioxidant activities ofβ-cryptoxanthin fatty acid esters were not significantly different in all assays (p<0.05), furthermore, no synergetic effects among them were obtained. These results suggest that esterification of OH groups with fatty acids might not affect antioxidant activity ofβ-cryptoxanthin but stabilizeβ-cryptoxanthin against heat degradation.4 preparation and thermal stability of the inclusion complex ofβ-cryptoxanthin withβ-cyclodextrinThe inclusion complex ofβ-cryptoxanthin withβ-cyclodextrin (β-CD) was prepared. The orthogonal test results showed that the optimum conditions of process were as follows:water amount 40mL, temperature 30℃, time 4h,β-cryptoxanthin:β-cyclodextrin 0.3:200 (mg/mg), and in the optimal condition, the rate of retention in the microcapsule was 81.92±1.35%. Infrared spectroscopy (IR) proved the formation of the inclusion complex. The water solubility of the inclusion complex was>1.0 mg/ml, which is much better than that of P-cryptoxanthin. The stability of the inclusion complex in solution was also tested. Forming of the inclusion complex greatly enhanced the stability of P-cryptoxanthin against heat, which indicated that the release ofβ-cryptoxanthin from the inclusion complex was controlled. Furthermore, analysis of kinetic data suggested a first-order reaction for theβ-cryptoxanthin and its inclusion complex with the half-lives of to in 4,25,50 and 75℃.There are few reports about analysis of carotenoids and carotenoid esters in citrus fruits, the isomerization in the peroxidation assays, and effects of esterification ofβ-cryptoxanthin on its thermal stability and antioxidant activity by chemiluminescence method in vitro and the preparation and thermal stability of the inclusion complex ofβ-cryptoxanthin withβ-cyclodextrin.