| Citrus is one of the most important fruit crops in the world. It is widely grown in the tropical and subtropical areas of the world. The genus Citrus L. was established in 1753 by Carl Linnaeus, it belongs to the subfamily Aurantioideae of the family Rutaceae. The genus Citrus L., Poncirus Raf., Fortunella Swingle, Microcitrus Swingle, Eremocitrus Swingle, Clymenia Swingle were classified into the true citrus fruits group based mainly on the structure of pulp-vesicles of the genera and number of stamen. The fruit of Citrus and its relatives contain ample bioactive compounds including flavonoids, carotenoids, limonoids and coumarins. Citrus fruits have been reported to exhibit important bioactivities, including antioxidant, anti-inflammatory, anti-obesity, anti-cardiovascular and antitumor abilities. Citrange is a generic hybrid between Poncirus trifoliata Raf. and Citrus senesis Osbeck. It is known for its well-developed root system being highly resistant to cold and pathogens. In horticulture, citrange was mainly used as rootstock for citrus variety. In previous studies, both parents of citrange, P. trifoliate Raf.and C. senesis Osbeck., have been shown to have abundant bioactive compounds, especially flavonoids. However, the the flavonoid composition of citrange fruits and the bioactive ability such as anti-oxidant and anti-obesity effects have not been reported yet.In this study, four citrange genotypes: cv. Carrizo(C), cv.Moxico(M), cv.Rusk(R), cv.Anlu(A); and the potential parents of Carrizo: trifoliate orange(Poncirus trifoliata)(T) and Washington orange(Citrus sinensis cv.Washington)(W) wereanalyzed for their flavonoid compounds and anti-oxidant effects.First,the flavonoid compounds of peel extract(PE) and flesh extract(FE) of citrange were identified by Ultra Performance Liquid Chromatography-Q time of flight mass spectrometry(UPLC-Qtof-MS). Then, the antioxidant flavonoid components were screening by High Performance Liquid Chromatography(HPLC) coupling with Free Radical Scavenger Detector(FRSD). Also, the total anti-oxidant capacities were detected in a high efficiency way by on-line HPLC-ABTS+, these results, together with the total anti-oxidant capacities detected by off-line methods, are used for the analysis of the relationship of anti-oxidant ability and the contents of total flavonoid and polyphenol.To evaluate the anti-obesity effect of citrange fruit, we used the high fat diet induced obesity mouse as animal model, investigated whether long-term supplementation of citrange fruit extracts would have a role in the prevention of obesity and its related metabolic diseases was investigated. In addition,Real Time PCR was used to explore the MOA of the anti-obesity effect, the expresstion levels of peroxisome proliferator-activated receptor γ( PPARγ)and liver X receptor β(LXRβ) were analyzed.The main results obtained are as follows: 1. Identification of the Flavonoid compounds of Citrange fruitTo identify the flavonoid composition in the peels and flesh methanol extracts of 4 citranges and potential parent genotypes, all the samples was analyzed by UPLC-Qtof-MS. The signals were identified by the fragmentation rules of MS and comparing with standards.Methods:The chromatograph column was operated at 25 °C, and 2 μL extract was injected into HPLC. The mobile phase consisted of 100% methanol(A) and water containing 0.1% acetic acid(B) at a flow rate of 0.4 m L/min. The gradient profile was as follows: 0—0.6 min 90%—80% A;0.6—6.5 min 80%—40% A;6.5—7 min 40%—10% A;7—8 min 10% —90%A. The mass spectrometer was operated in positive ion mode with a capillary voltage of 3 k V, sampling cone voltage of 40 V, desolvation gas flow of 800 L/h, desolvation temperature of 300 oC, source temperature of 100 oC, collision energy of 20 V. Mass spectra were collected at the rate of 1 spectrum/s and the inter-scan delay was 0.02 s.Totally, 34 chemical compounds were identified. There are 20 kinds of flavonoid was 26 in Carrizo peel extract and is the most numerous among all the extracts. The number of flavonoid compounds in potential parents trifoliate and Washington orange are 8 and 9, respectively. This showed the heterosis of the Carrizo on the quantity of the kinds of flavonoids. Also, almost all the flavonoids detected in trifoliate and Washington orange can also been detected in Carrizo.The number of flavonoid compounds were all higher in the peel extract than in flesh extract of citrange. No obvious variation was existed among the flavonoid composition of 4 citrange, the main compounds were neoeriocitrin, naringin, neohesperidin and ponrincin。2. Analysis of the anti-oxidant ability of citrange fruit2.1 On-line HPLC-ABTS+ screening anti-oxidant components in citrange fruitMethods:(1)HPLC: The chromatograph column(C18 Column) was operated at 25 oC, and 30 μL extract was injected into HPLC. The mobile phase consisted of 100% methanol(A) and water containing 0.1% acetic acid(B) at a flow rate of 0.7 m L min-1. The gradient profile was as follows: 0—20 min 37%—50% A;20—35 min 50%—80% A;35—40 min 80%—100% A;40—50 min 100% A;50—60 min 37% A,;(2)FRSD:The flow rate of ABTS+ solution is 0.40 m L min-1,the column was operated at 40 o C, the induced bleaching was detected as a negative peak at 414 nm.The results showed that the main anti-oxidant flavonoid compound in citrange peel extracts is neoeriocitrin; neohesperidin and ponrincin also has minor anti-oxidant abilities. However, no flavonoid compound in citrange flesh extracts, trifoliate and Washington orange fruit extracts showed the scavenging ability of ABTS+.2.2 On-line detecting the total anti-oxidant capacity of citrange fruitMethods:(1)HPLC: The mobile phase consisted of 100% methanol(A) and water containing 0.1% acetic acid(B) at a flow rate of 0.91 m L min-1. The gradient profile was as follows::0-4 min 63%A,37%B. The temperature of the column oven was maintained at 30 °C. 30 μL extract was injected into HPLC.(2) FRSD:The flow rate of ABTS+ free radical solution reagent was 0.60 m L min-1, and the ABTS+ antioxidant capacity wasdetected as a negative peak at 414 nm by the Waters 2489 UV/Visible detector. The temperature of the on-line post-column system was 40 °C.The order for antioxidant capacity of citrange fruit extracts from high to low was as following(VE equivalent): MPE(85.00 ± 0.24)> APE(84.27 ± 1.18)>CPE(83.45 ± 0.97)> RPE(80.45 ± 0.63)>TPE(77.59 ± 0.72)>WPE(74.60±0.45)>WFSE(73.10±0.07)>MFSE(71.73±APE1.47)>CFSE(70.23±0.06)> AFSE(69.94±0.15)> RFSE(65.53±1.06)>TFSE(58.87±0.45)2.3 Off-line detecting the total anti-oxidant capacity of citrange fruitThe total anti-oxidant capacity of citrange fruit measured by off-line DPPH method, ABTS method, FRAP method. Using antioxidant potency composite(APC) index for analysing the antioxidant ability of flavonoids compounds in citrange fruit.The order for antioxidant capacity of citrange fruit extracts from high to low was as following(APC index): MPE(97.03%)>APE(94.95%)>CPE(86.67%)> RPE(84.38%)>TPE(76.75%)>WPE(73.20%)>WFSE(56.46%)>CFSE(55.07%) > MFSE(54.13%)> RFSE(52.22%)>AFSE(47.28%)>TFSE(40.15%)。2.4 Correlation between total antioxidant capacity and total phenolic/flavonoid contents of citrus fruitsThe total phenolic compounds and total flavonoids content were tested by Folin-Ciocalteu method and Al Cl3 method. And the correlation between antioxidant capacity and total contents of phenolic compounds and flavonoids was also studied for revealing the possible existing antioxidant compounds. The results were as followings:(1)The total phenolic contents of citrange fruits: MPE has the highest phenolic contents among the peels extracts, while in flesh extracts, the WFE and MPE are the highest.(2) The total flavonoid contents of citrange fruits: MPE and CPE have the highest flavonoid contents among the peels extracts, while in flesh extracts, the WFE is the highest.(3)The correlation analysis showed that the citrus antioxidant capacity was significant positive correlation with the contents of total phenolic compounds and totalflavonoids(P<0.05).3. Evaluation of the anti-obesity ability of citrange fruitFemale C57BL/6 mice were weighed and randomly separated into four groups(seven mice/group): chow diet(10% of calories derived from fat, Research Diets; D12450B; New Brunswick, NJ, USA), HF diet(60% of calories derived from fat, New Brunswick, NJ, USA, Research Diets; D12492), HF diet supplemented with 1% w/w CPE(HF + CPE) and HF diet supplemented with w/w CFSE(HF + CFSE) for 8 weeks.The results showed that CPE and CFSE supplement diet significantly decreased the body weight gain of HF diet-induced mice by 13% at the end of treatment(p = 0.012, 0.012). Also in HF+CPE and HF+CFSE groups, the blood glucose, serum total cholesterol(TC) and low density lipoprotein cholesterol(LDL-c) levels were significantly(p < 0.05) reduced relative to those in the HF group.To explore the mechanisms of action of CPE and CFSE on the metabolism of glucose and lipid, related genes’ expressions in liver were assayed. In liver tissue, the expression level of peroxisome proliferator-activated receptor γ(PPARγ) and its target genes were down-regulated by CPE and CFSE supplementation as revealed by q PCR tests. In addition, both CPE and CFSE decreased the expression level of liver X receptor(LXR) α and β, which are involved in lipid and glucose metabolism.The results obtained in our study clearly demonstrated that:There is no significant variation in the flavonoid compound and anti-oxidant capacity among the citrange fruit extracts. Compared to its potential parents, Carrizo citrange showed heterosis on the anti-oxidant ability. Carrizo fruit extracts administration could ameliorate obesity and related metabolic disorders in HF diet-induced obesity mice probably through the inhibition of PPARγ and LXRs gene expressions. |
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