High-efficiency Extraction And Separation Process Of Main Triterpenoids From Inonotus Obliquus

Inonotus obliquus,a natural medicinal fungus,has been extensively researched and applied in the medicine and food production.It is rich in a variety of chemical components,such as triterpenoids,terpenoids,polyphenols,polysaccharides,etc.,among which triterpenoids have extremely significant anti-tumor and other pharmacological activities.At present,there are few reports on the extraction and separation of triterpenoids from I.obliquus at home and abroad.Most of the separation of triterpenoids from l.obliquus rely on traditional organic solvent extraction and silica gel column chromatography,which has the disadvantages of cumbersome operation,time-consuming and laborious.In this experiment,the enzyme-induced combined negative pressure cavitation extraction technique was used to extract the triterpenoids from I.obliquus firstly and the macroporous adsorption resin was used to effectively enrich the triterpenoids.Finally,the monomer components of the triterpenoids were separated and purified by high-speed countercurrent chromatography to obtain four triterpenoid compounds.The experiment systematically established a comprehensive extraction and separation process of triterpenoids from I.obliquus to provide a scientific basis for the high development and utilization of I.obliquus resources.The results of this paper were as follows:1.The process of enzyme-induced negative pressure cavitation extraction was optimized by single-factor,Box-Behnken(BBD)and response surface methodology.The optimal extraction process parameters were determined:Enzyme type:cellulase Enzyme concentration:1 mg/mL Induction temperature:40?Induction time:24 hThe experiment was carried out under the optimal enzyme-inducing conditions,the content of triterpenoids in the enzyme-induced I.obliquus was 16.35 mg/g,which increased 28.31%by comparing to untreated ones.Extraction solvent:75%ethanol solution Extraction pressure:-0.08 Mpa Liquid to solid ratio:16 Extraction temperature:43?Extraction time:30 minUnder the above optimized conditions,the extraction yield of triterpenoids from enzyme-induced 1.obliquus was 17.41 mg/g(n=3)and the relative standard deviation was 0.85%.2.The macroporous adsorption resin was used to purify the triterpenoids from I.obliquus extract and the optimum technological conditions were determined:Resin type:AB-8 Loading concentration:1 mg/mL Loading volume:8/3 BV Loading flow rate:1 BV/h Desorption solution concentration for impurities:60%ethanol Desorption solution volume for impurities:3 BV Desorption solution concentration for target compounds:90%ethanol solution Desorption solution volume for target compounds:4.5 BVUnder the above optimized conditions,the purity of the triterpenoids was 53.26%,increased 4.67-fold.The recovery rate of triterpenoids was 85.92%.3.High-speed countercurrent chromatography was used to separate the triterpenoids from I.obliquus.The optimal conditions were optimized as follows:Solvent system:n-hexane-acetonitrile(1:1;v/v)Stationary phase:upper phase Mobile phase:lower phase Temperature:25 0C Host speed:800 rpm Mobile phase flow rate:1 mL/minUnder the above optimized conditions,The four triterpenoid compounds,namely 3?-hydroxy-lanosta-8,24-diene-21-al,inoterpene F,inotodiol and lanosterol,were separated in one run HSCCC within 5 hours successfully and the yields of compounds were 0.10 mg/g,0.13 mg/g,0.35 mg/g,0.17 mg/g and the recovery rates were 93.1%,95.2%,97.8%,98.1%,respectively.And the purities of four compounds were over 95%.In summary,the enzyme-induced negative pressure cavitation extraction was used to obtain the 17.41 mg/g triterpenoids from l.obliquus,the purity of the triterpenoids enriched by macroporous adsorption resin is 53.26%and the four triterpenoid compounds,namely 3?-hydroxy-lanosta-8,24-diene-21-al,inoterpene F,inotodiol and lanosterol,were separated in one run HSCCC within 5 hours successfully.The study established a high-efficiency,rapid and reliable technical route of extraction and separation for triterpenoids from I.obliquus successfully.Meanwhile,it provided effective technical support for the preparation and utilization of secondary metabolites in other plant resources.