A Study On The Extraction,Separation And Purification Of Sour Orange (Citrus Aruantium L.)PMFs And Its Effects On Alleviating Radiation-induced Intestinal Injury In Mice

Sour orange(Citrus aurantium L.)is one of the primary ancestral plant of zhishi and zhiqiao,which has a high utility value.Its quality evaluation index primarily looks for three substances: neohesperidin,naringin,and synephrine.However,neither the sour orange specific markers nor the secondary metabolites with the largest content of sour orange fruit can be found in the aforementioned compounds.Additionally,Sour orange has variable phenotypes,leading to a common sypotom of misterming.Because of this,even if sour orange has a special medical benefit,it is impossible to distinguish or assess its authenticity under the present quality standards,which severely limits the utilization of sour orange resources.Sour orange contains a particular family of flavonoids called polymethoxyflavones(PMFs).Sour orange can be distinguished from other citrus materials by distinct PMFs content and composition,which have certain characteristics.However,there is currently a lack of study on the composition and amount of PMFs features across different varieties of sour orange,which restricts the use of PMFs in the assessment of sour orange resources.Intestinal tract is an essential digestive and immune system organ that is vulnerable to ionizing radiation.A radioactive intestine damage could result from a nuclear leaking accident or from pelvic radiation therapy given to a patient with a tumor.This injury could manifest as an electrolyte disturbance,a loss of absorption function,or even death.There isn’t currently a specific medication for radiation induced intestinal injury.PMFs have been proven to have multiple bioactivities,for instance,antioxidant,anti-inflammatory.In addition,zhishi or zhiqiao showed positive preventative or therapeutic effects of a number of intestinal illness models.Sour orange PMFs’ impact on radiation-induced intestinal damage has not been documented.In order to provide information for the evaluation of citrus resources using PMFs,this study first determined the composition and content of PMFs in the peel of 12 distinct sour orange cultivars.It next investigated the features of PMFs in various citrus varieties.Secondly,the PMFs mixture of sour orange was obtained by using HPD750 for PMFs enrichment.And served as the foundation for the activity study,with the adsorption properties of several macroporous adsorption resins on the major flavonoid compounds of sour orange being screened and discussed;The possible target of a blend of sour orange PMFs was then predicted using network pharmacology to treat mice with radiation-induced intestinal damage;Finally,an animal model of radiation-induced intestinal illness and cytotoxicity provided preliminary evidence that sour orange PMFs protect against radiation-induced intestinal injury and cytotocxity was investigated.The main results were as follows:(1)Extraction of PMFs from Fruits of Different Sour Orange Cultivars and Analysis of Their Composition CharacteristicsThe main flavonoids,especially PMFs,in 12 sour orange cultivars were extracted and detected.Among them,the total flavonoid content of different sour orange cultivars is Within the range of 955～4327.5 μg/g DW,the variety with the lowest total flavonoid content is Lubidukesisuancheng,and the cultivar with the highest total flavonoid content is Goutoucheng.In the samples of different sour orange cultivars,the content of PMFs ranges from 32(Moluogesuancheng)to 48(Lianhesuancheng)μg/g DW.The monomers of PMFs of sour orange are mainly nobiletin,heptamethoxyflavone,5,7,4 ’-trimethoxyflavone,tangeretin and isosinensetin.The results of cluster analysis showed that the components and contents of PMFs in different varieties of sour orange were similar,which could be significantly distinguished from Dahongpao mandarin,Newhall navel orange and Liangping pomelo.In the interior of sour orange,the characteristics of 12 varieties of PMFs can be divided into three main branches.The contents of naringin and neohesperidin in sour orange had no significant correlation with the total amount of polymethoxyflavones,but the contents of naringin 5,7,3 ’,4’-tetramethoxyflavones were significantly negatively correlated with heptamethoxyflavone,5,7,4 ’-trimethoxyflavone and hesperidin;Neohesperidin was significantly negatively correlated with the contents of isoorange flavone,heptamethoxyflavone,5,7,4 ’-trimethoxyflavone and hesperidin,and significantly positively correlated with the contents of 5,6,7,4’-tetramethoxyflavone.(2)Enrichment of Sour orange Polymethoxy Flavonoids MixtureA high-performance liquid chromatography(HPLC)method for the determination of the main flavonoids in citrus was established.The adsorption characteristics of different macroporous adsorption resins on the main flavonoid monomers of citrus were discussed,and the PMFs mixture of citrus was prepared by enrichment.The results showed that the established HPLC method for the determination of citrus flavonoids was accurate,reliable,and suitable for the qualitative and quantitative determination of 9 citrus flavonoids;The solubility experiment showed that the solubility order of four flavonoids(naringenin,hesperidin,nobiletin,and tangeretin)in absolute ethanol was naringenin>nobiletin>hesperidin>tangeretin,respectively.It was hopeful to separate hesperidin from other compounds by adjusting the volume fraction of ethanol.Six macroporous resins with preference and one macroporous resin with universal adsorption specificity for four flavonoids were screened through static adsorption experiments.Through adsorption thermodynamics experiments,it was found that:(1)Among the seven selected macroporous resins,ADS-7,HPD 300,and CAD 40 macroporous resin adsorption of naringenin was an endothermic process,and HPD 750,HPD 400,NKA-9 and DA 201 macroporous resin adsorption of naringenin was an exothermic process;However,the adsorption of hesperidin,tangeretin and nobiletin on 7 kinds of macroporous resins is exothermic.Increasing the adsorption concentration of hesperidin,tangeretin and nobiletin and cooling down is conducive to the adsorption reaction.(2)The adsorption mode of naringenin and hesperidin on HPD 300 and HPD 400 resins was more consistent with the Freundlich model,which was multi-layer physical adsorption;The adsorption on the other five resins conforms to Langmuir model.The adsorption process of nobiletin on seven kinds of macroporous resins conforms to the Freundlich thermodynamic model,while the adsorption process of tangeretin conforms to the Langmuir model;Therefore,the macroporous adsorption resin HPD750 was used to separate the PMFs and flavanones under the adsorption difference of different components in the crude extract of sour orange flavonoids,and a mixture of PMFs with a relative content of 67.44% was obtained by enrichment,which can be used for the subsequent biological activity study.Compared with the crude extract,the main components of the enriched product did not change,but the relative content of tangeretin was significantly increased,while the relative content of sinensetin and heptamethoxyflavone was not significantly increased.(3)Pharmacological Analysis of Potential Target Network of Sour orange PMFs for Alleviating Radiation-induced Intestinal InjuryThe published transcriptome data and database were used to identify the potential targets of nobiletin,tangeretin,sinensetin and isosinensetin,and the network pharmacological analysis was conducted to obtain the potential core target of sour orange PMFs to alleviate radiation-induced intestinal injury.The function enrichment analysis showed that the gene expression involved in intestinal cell adhesion,cell secretion factors,amino acid metabolism,blood coagulation,and other pathways was significantly downregulated after irradiation;The expression of genes involved in cell cycle,synapse formation,and other pathways was significantly up-regulated.The enrichment results of KEGG pathway showed that irradiation significantly affected PI3K-Akt,p53,glucose metabolism,and other related pathways.Sour orange PMFsmixture may regulate intestinal epithelial homeostasis and maintain intestinal function through 3 biological subnetworks.The relevant targets in the 3 subnetworks were analyzed by protein-protein interaction(PPI)network topology,and 11 core targets(Creb1,Mapk1,Mapk14,Met,Mmp9,Mtor,Nos2,Ppar,Ptk2,Rela,Tlr4)were obtained by measuring degree,betweenness centrality,and closeness centrality.(4)Study on the Preventive Effect of Sour orange PMFs on Radiation-induced Intestinal Injury in MiceUtilize 60 Co source γ The KM mice model of radiation-induced intestinal injury was established by abdominal irradiation of 12 Gy with gamma rays.The preventive effect of sour orange PMFs pretreatment on radiation-induced intestinal injury was observed.The intestinal tract of mice was seriously damaged,and their weight decreased rapidly,and they died 10-15 days after irradiation.The preventive effect of sour orange PMFs on radiation-induced intestinal injury was observed in mice by gavage with 50 mg/Kg,200 mg/Kg and 800 mg/Kg of sour orange PMFs respectively.The results showed that 200 mg/Kg of sour orange PMFs could reduce the body weight loss,alleviate organ damage and regulate fat index of KM mice after irradiation,while 50 mg/Kg and 800 mg/Kg had no effect on slowing the changes of body weight and organ index after irradiation.200 mg/Kg sour orange polymethoxyflavone treatment alleviates the damage of ileum and colon mucosa caused by radiation,reduces the infiltration of inflammatory cells,maintains the number of ileal crypts,reduces the abnormal distribution of ileum and colon mucus layer,and maintains the number of normal intestinal crypts after irradiation,but has no significant impact on proliferation.Real time quantitative PCR(RT q PCR)showed that polymethoxyflavones may maintain intestinal epithelial homeostasis and promote intestinal damage repair by regulating the expression of Mmp9,etc.In addition,the treatment of sour orange polymethoxyflavone can alleviate the increase of AST,ALT,CRE and UA levels in irradiated mice,and reduce the level of plasma interleukin-1 in mice β(IL-1 β)And interleukin-6(IL-6)levels.In vitro cytotoxicity study showed that the IC50 value of sour orange PMFs on human normal intestinal epithelial cell line CCD 841 Co N was 42.23 μ G/m L,the IC50 value of hesperidin on CCD 841 Co N cells is 51.58 μ G/m L,the inhibition rate of hesperidin and sweet orange flavone on cell viability did not exceed 50% in the maximum solubility range.In addition,sour orange polymethoxyflavone can also alleviate the colitis induced by dextran sulfate sodium(DSS)in mice,slow down the rise of Disease Active Index(DAI),alleviate colon edema and shorten,maintain liver morphology,and reduce the inflammatory factor IL-1 in plasma β、 The level of IL-6 reduces the level of AST and ALT in plasma.The above results indicate that sour orange PMFs can alleviate the intestinal epithelial barrier dysfunction caused by radiation injury,reduce the level of inflammation,and prevent liver and kidney damage caused by bacterial translocation infection.This process may be achieved by regulating the expression of Mmp9.