| Immature pomelo fruit,a traditional Chinese medicine,is a dried immature fruit of Citrus grandis(L.)Osbeck and its varieties with fragrant smell,bitter and irritating taste.Immature pomelo fruit is rich in nutrients and various active ingredients including essential oil,naringin,pectin,limonin analog,etc.Moreover,the anti-oxidation,anti-inflammatory,anti-tumor activity,make it has high healthy and medicinal value.At present,the development and utilization of pomelo fruit processing is limited,and the traditional extraction process of naringin has some disadvantages,such as environmental pollution.Therefore,it is of far-reaching significance to promote the application of new extraction technology in the deep processing of pomelo fruit.Currently,there are a large number of researches on the immunological activity of plant polysaccharides have been reported.While there is less research on immunomodulatory activity about pectin,because researchers are mainly focused on the gel characteristics of pectin recently.In this study,a systematic study was carried out focused on the extraction of essential oil and naringin of immature pomelo fruit by continuous phase transition extraction technology,and the structural characteristics,chain conformation,immunomodulatory activity of HPP-1 purified from pomelo fruit pectin.Immunomodulatory activity was evaluated by macrophage model in vitro.The main research results are as follows:1.Analysis of basic components of pomelo fruit.The dry weight content of naringin in pomelo fruit in April was 34.53%,which was significantly higher than 17.67%in May.There was no significant difference in pectin content,which was 10.14%and 15.60%respectively.So the immature pomelo fruit in April was selected as the research object.The main component of pomelo fruit are naringin,pectin,essential oil,water-insoluble dietary fiber and others,which accounts for 34.53%,10.77%,5.03%,50.31%.2.The continuous phase transition extraction technology was used to extract the essential oil from the pomelo fruit,and the yield of essential oils was 1.12%.The components of essential oil were analyzed by GC-MS.The result indicated that a total of 43 chemical compounds were identified.D-limonene was the highest detected by 10.24%,followed by osthole 8.69%,2,4-di-tert-butylphenol 7.29%,trans-?,?-5-trimethyl-5-vinyltetrahydro-2-furan methanol 6.44%and camphor 6.41%.3.The extraction of naringin by continuous phase transition extraction technology.After extracting the essential oil,the three factors of extraction temperature,extraction time and raw material particle size were studied for extraction rate of naringin in de-esterified pomelo fruit.The best extraction conditions were temperature at 75?,time for 4 h and raw material granularity of 40 mesh,through the response Surface experiment optimization.Under this condition,the extraction rate of naringin was 99.47%,and the purity of naringin in the extract was 61.21%.It indicates that the continuous phase transition extraction of naringin in pomelo fruit is excellent,which show the characteristics of high efficiency and the advantages of environmental protection in industry.4.Purification and structural analysis of active pectin component HPP-1.The immature pomelo fruit pectin was obtained from the pomace after extracting naringin,by extracting,concentrating,desalting,alcohol precipitation and freeze-drying.The yield was20.23%.The total sugar content,galacturonic acid content,protein content and ash content in the crude pectin was 71.17%,59.90%,1.93%and 4.33%,respectively.The homogeneous component HPP-1 was purified by cellulose DEAE-52 anion exchange column chromatography and Sephadex G-200 column chromatography.The structural characterization revealed that HPP-1 was no protein-induced absorption peak in the UV spectrum and had an average molecular weight of 5.9×10~4 Da.The neural monosaccharide of HPP-1 was composed of rhamnose,arabinose,fucose,mannose and galactose at a molar ratio of 1.00:11.12:2.26:0.56:6.40.The main glycosidic linkage types of HPP-1 consisted of1,4-D-Gal A,1,6-?-D-Gal,1,6-?-D-Man,1,3-?-L-Ara 1and 1,2-?-L-Rha by FTIR,periodate oxidation-Smith degradation and NMR analysis.The structure of HPP-1 contains regions of RG I and RG II,which was a typical pectic polysaccharides.5.The mice macrophage RAW264.7 was used as the cells model to evaluate the immunity of HPP-1 of pomelo fruit pectin.The results indicated that at the concentration of 10-1000?g/m L,HPP-1 was not toxic to macrophages,and show the ability to promoted the proliferation and phagocytosis of macrophages.Moreover,it can significantly increase the secretion of NO,TNF-?and IL-6.Meanwhile,the m RNA expression of i NOS,TNF-?and IL-6 in macrophages analyzed by RT-PCR can be also enhanced by HPP-1,which showed a significant dose effect. |
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