| Citrus medica L.is a medicinal and edible resource of Citrus in Rutaceae.There were no reports about its pharmacological activity.Polysaccharides have attracted much attention due to their wide biological activity and low toxicity.However,citron polysaccharide has not been studied.In this paper,citron fruit was used as raw material to analyze the structure and biological activity of its polysaccharide.The details were as follows:(1)Single factor analysis and response surface methodology were used to optimize the main factors affecting the hot water extraction of citron polysaccharide.The theoretical optimal parameters were obtained as follows: extraction times 3,solid-liquid ratio 1:25,extraction temperature 90.63 ?,single extraction time 120 min,and the theoretical optimal yield was5.65 %.According to the actual situation: extraction times 3,solid-liquid ratio 1:25,extraction temperature 90?,single extraction time 120 min,and the actual yield was 5.59 % ± 0.024 %.(2)The hot-water extractions of citron were separated and purified,and physicochemical properties of the purified components were analyzed.After decolorization,deproteinization,alcohol precipitation and freeze-drying,the crude polysaccharides(CMs)were obtained.Then,two kinds of CM-1 and CM-2 with relatively homogeneous molecular weight were obtained by anion exchange chromatography column DEAE-Sepharose Fast Flow and Sephadex column Sephadex G-100 respectively.The total sugar content of CM-1 and CM-2 was 88.74 % and93.28 % respectively,and the protein content was 0.03 % and 0.09 % respectively,which could be ignored.CM-1 and CM-2 did not contain nucleic acid,protein and endotoxin through 190nm-500 nm UV spectrum scanning and chromogenic matrix Limulus amebocyte lysate kit.TGDSC analysis showed that the thermal degradation behaviors of CM-1 and CM-2 were quite different,and CM-2 was more stable.(3)The structures of CM-1 and CM-2 were analyzed by HPGPC,FT-IR,GC,methylation analysis and 1D/2D NMR.FT-IR spectra of CM-1 and CM-2 showed the characteristic absorption peaks of polysaccharides.GC analysis showed that CM-1(Mw = 21520 Da)was mainly composed of arabinose,xylose,mannose and glucose,with the molar ratio of10.78:11.53:1.00:1.70;CM-1 was arabinoxylan,with(1?4)-?-D-Xylp as the linear backbone substituted by ?-L-Araf unit at O-3 position.CM-2(Mw = 22303 Da)was mainly composed of arabinose,mannose,glucose and galactose in the molar ratio of 25.46:1.45:1.00:6.57;CM-2was galactoacetan with(1?5)-linked ?-L-Araf backbone substituted by ?-D-Galp units at O-2and/or O-3 positions.SEM showed that CM-1 and CM-2 had different surface morphology.(4)The bioactivities of citron polysaccharides were evaluated and the structure-activity relationship between CM-1 and CM-2 was tried to establish.The results showed that citron polysaccharides had potential immune activity: inducing RAW 264.7 deformation;enhancing its pinocytic ability,and promoting the release of TNF-?,IL-6 and NO by up-regulating the expression of TNF-?,IL-6 and i NOS m RNA.Citron polysaccharides could inhibit the proliferation of human breast cancer cells MDA-MB-231 and MCF-7,which might be related to the promotion of apoptosis.Citron polysaccharides could inhibite ?-amylase,?-glucosidase and improve glucose consumption of Hep G2-IR cells to exect hypoglycemic activity.Citron polysaccharides had anti-skin photoaging activity.CM-1 was more effective than CM-2 in immunostimulatory and anticancer activities,and CM-2 was better than CM-1 in hypoglycemic and anti-skin photoaging activities.The difference of biological activity between CM-1 and CM-2 might be related to the difference of molecular weight,glycosidic bond type and skeleton structure.In conclusion,CM-1 and CM-2 were new polysaccharides;citron polysaccharides had potential immune stimulation,anti-cancer,hypoglycemic and anti-skin photoaging activities.These results might lay a foundation for the further study of citron polysaccharides,and also provide an effective idea for the development and utilization of citron pharmacological activity. |
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