Extraction Of The Main Active Ingredients In Platycodon Grandiflorus And Immunological Activity Studies Of Its Polysaccharides

Platycodon grandiflorus(Jacq.)A.DC.is a traditional medicinal and edible plant in China,With extremely high economic value.It is enriched with chemical constituents containing saponins,polyenes,polysaccharides and other effective ingredients and has pharmacological activities such as expectorant and cough relieving,antibacterial and anti-inflammatory,antitumor,hypoglycemic and lipid-lowering,immune regulation and antioxidant.In order to fully utilize the resources of P.grandiflorus,in this paper took P.grandiflorus as the research object and the efficient extraction process of platycodin D and lobetyolin was optimized,then a systematic study was conducted on the efficient extraction and purification of P.grandiflorus polysaccharide from the residue and their effects on immune function for mice,with a view to providing reference for the maximum comprehensive utilization of P.grandiflorus polysaccharide and the subsequent development of P.grandiflorus polysaccharide health food or medicine.The main study results as follows:The quality evaluation of multiple indexes of water,total ash and ethanol extracts of P.grandiflorus from four places of origin,namely,Zibo,Shandong,Chifeng,Inner Mongolia,Taihe,Anhui and Zitong,Sichuan.The results showed that the water,total ash and alcohol extract content complied with the pharmacopoeia regulations.There are significant differences in the contents of platycodin D,lobetyolin and total polysaccharide.The content of platycodin D and polysaccharide in P.grandiflorus from Chifeng is the highest,and the content of lobetyolin in Taihe is the highest.In general,the quality of P.grandiflorus in Chifeng,Inner Mongolia was better.Homogenate pretreatment followed by ultrasonic-microwave synergistic extraction of platycodin D and lobetyolin from the P.grandiflorus.Using single factor experiment and response surface experiment,the optimal extraction process was homogenization pretreatment for 3 min,ethanol volume fraction 60%,solid-liquid ratio 1:20 g/mL,microwave power 580 W,extraction time was 15 min,and temperature 50 ℃.(5.24±0.13)mg/g and(0.24±0.01)mg/g,respectively;higher than Soxhlet extraction(4.72±0.24 mg/g;0.20±0.03 mg/g)and ultrasonic extraction(4.93±0.15 mg/g;0.21±0.02 mg/g).From the P.grandiflorus residue of platycodin D and lobetyolin,the polysaccharide was extracted by compound enzyme and ultrasonic extraction.Through single factor sequential optimization experiment,the best extraction process was the solid-liquid ratio of 1:30 g/mL,the mass ratio of papain and cellulase was 1:2,the compound enzyme was 5%of P.grandiflorus mass,the ultrasonic power was 150 W,the ultrasonic time was 40 min,and the ultrasonic temperature was 50℃.After Sevage deproteinization from polysaccharide of P.grandiflorus by Sevage mothod,then a single polysaccharide PGP-a was purified by used DEAE-52 cellulose chromatography column and Sephadex-G100 gel chromatography column.PGP-a contained 87%total sugar,without uronic acid,nucleic acid and protein.Gel permeation chromatograms showed a high purity of PGP-a with a Mn of 5805 Da and Mw of 6539 Da.PGP-a is mainly composed of a large amount of fructose and a small amount of glucose and arabinose(molar ratio:9.82:1.73:1).The FT-IR analysis showed that PGP-a fits the polysaccharide canonical structure,and the XRD analysis showed that PGP-a is a semicrystalline material.Thermogravimetric analysis indicates ti has a good thermal stability,and Congo Red experiments indicate no triple-helical structure.To exam the effects of different doses of PGP-a on body weight,immune organ index,phagocytic capacity of macrophages,delayed anaphylaxis,serum lysozyme and cytokines in immunosuppressed mice.The results showed that PGP-a could not only significantly improve the weight loss,thymus index and spleen index of immunosuppressed mice caused by cyclophosphamide,but also could improve the liver damage,significantly enhance the the phagocytosis of macrophage cells and the degree of ear swelling in mice caused by DNFB,then significantly increased the lysozyme content and cytokines such as IL-2,IL-4 and TNFa.In summary,PGP-a was shown to enhance immune function in immunosuppressed mice.