Active Components Extraction From Inonotus Obliquus By PEF And Its Chemical Modification & Anticancer Activities

Inonotus Obliquus is an anticancer epiphyte. It can strengthen human's immune system and is effective in preventing cancer, (e.g. gastric cancer, liver cancer, intestinal cancer), heart disease, diabetes, etc. It is highly recommended by Japanese researchers as being effective in preventing and curing liver cancer, AIDS, colon bacillus intoxication. It has been used as an omnipotent medicine for 400 years. It is a natural, health-care food. It is of significance to make full use of it.PEF non-thermal pasteurization technique was known as one of the most popular and advanced technology in the world. It is a revolution in traditional pasteurization technique and definitely an innovative, scientific and advanced technology. It is significant to develop PEF technology further and extend its application in order to meet the demand for low-cost and high-efficiency extraction method. It will become an ideal way to extract effective ingredients from Chinese herbal medicine, which will be an impetus for the medicine's popularity and availability in the world.In the study, PEF is applied to extracting useful ingredients from Inonotus Obliquus. It focuses on the process to increase the extraction efficiency of polysaccharide and triterpenoids of Inonotus Obliquus under normal temperature with less time. In addition, the indissoluble triterpenoid was chemical modification. In the same time, we investigate extracts of Inonotus Obliquus of against cancer cell, using MTT assay. Through the study, it provides a new idea and new method for China herbal medicine development. Therefore, the study has significance not only in application but also in theory.The study includes:⑴theoretical analysis of mechanism of reinforced extraction during PEF extraction, the findings are:Under PEF, the polar solution moves in fixed way, accelerating its infiltrating through the pores of herbal medicine. The greater the electric field strength, the less the extraction time.PEF will lead to violent eddy diffusion inside the cells, causing sharp difference of concentration in and outside the cells, thus increase solute's rate of extraction. PEF will cause cell membrane polarization, puncture, perforation, and thus fasten the extraction of effective ingredients inside the cells. Through dynamics extraction analysis, the factors that affect PEF extraction process are: electric field strength, pulse number, solvent-to-solid ratio, appropriate solvent.⑵To understand the reinforced extraction by PEF, and to study the feasibility of polysaccharide of Inonotus Obliquus, the paper studied the polysaccharide extraction by PEF. Nowadays, no such studies has been reported neither at home nor abroad.Though the above studies, four factors that affect the extraction were found out and proved. The following optimized process conditions were finalized: electric field =30kV/cm, pulse number=6, solvent-to-solid rate=25mL/g, and pH=10, under this condition, the extraction yield of polysacchride is 49.8%, the purity is 25.6%. PEF extraction is higher than those of hot alkali extraction for 41%, and microwave extraction for 13%, and the purity is higher than ultrasonic extraction for 5%.It indicates that PEF can improve the extract efficiency, a significant reduction of extracting time, and excellent than any other motheds. When using PEF extract polysaccharide, with increasing the electric field strength, pulse number and time, the extraction rate decreases. The possible reason is lengthened time and greater electric field strength caused polysaccharide, and therefore affects the extraction rate.⑶To further testify PEF's effect on improved extraction efficiency, and the feasibility of non-polar substance extraction, the process of extracting triterpenoids of Inonotus Obliquus by PEF was studied. In the meanwhile, GC-MS was used to test the effect of PEF on the ingredients of triterpenoids.Though the above studies, four factors that affect the extraction was found out and proved. Orthogonal experiment is used to optimize the test. The following optimized process conditions was finalized: electric field =50kV/cm, pulse number=5, solvent-to-solid rate=30mL/g, and 80% ethanol solution, under this condition, the extraction yield of triterpenoids is 11.8g/kg. The extraction rate is higher when using 75% ethanol than that of pure ethanol, which shows PEF can accelerate polar molecule's movement, thus accelerate solvent diffusion, as a result, increase efficient. With the increase of electric field strength from 0 to 60kV/cm, inotodiol and betulin of triterpenoids ingredients increase gradually, while lanosterol has a tendency of decrease when the electric field strength higher than 60kV/cm. PEF extraction is higher than those of ultrasonic extraction for 5%, and maceration extraction for 8%, with 20μs. It indicates that PEF can improve the extract efficiency, a significant reduction of extracting time.⑷After separating, purifying and identifying the extracts of triterpenoids, the findings are:Using silica gel separating, 1H-NMR, 13C-NMR combining mass spectrum structure analysis, the compoundⅠ,Ⅱ,Ⅲ,Ⅳare inotodiol, betulin, ergosterol lanosterol. Inotodiol is indissoluble and therefore hard to absorb by the body, which limits its application. Though sulphatising, inotodiol-3-sulphate sodium(Ⅴ)and inotodiol- 3,24-disulphate sodium(Ⅵ)are generated, identified through IR, 13C-NMR. The output are 26.4% and 7.1% respectively. The solubility of the medicineⅤ,Ⅵare 47.2 times and 72.7 times separately, which indicate sulphatze ester has satisfactory improved solubility of inotodiol.Betulin is indissoluble,we synthesize betulinic acic(Ⅶ), the product is identified by IR,13C-NMR, with an output of 32.5%. This method has the following advantages: less time, low cost, high efficient and less pollution.⑸After MTT test for investigating Inonotus Obliquus extracts'effect on killing hela cells, the following conclusions are reached:Through compared the kill rate of hela cell, it shows that the concentration is 100μg/mL, the 8 medicines are proved to kill hela cells. Given 100μg/mL after 72h, medicine 4 can kill hela cell 11.9% more than medicine 3, which indicates that the anticancer activity of inotodiol increases after sulphate. Given 100μg/mL after 72h, medicine 6 can kill hela cell 20% more than medicine 5, which shows that the betulinic acid enhances the activity of the medicine than betulin, it is the same with literature.To sum up, the study explores the extraction of Chinese herbal medicine Inonotus Obliquus, as well as its extracts'separation, chemical modification and anticancer activity. PEF has the following advantages: high extraction rate, less time, high purity, retaining biologic activity, normal temperature, easy-to-operate, low cost and high efficient, which make it possible to extract the effective elements incide herbal cells under normal temperature. It is a new, green method. After systematic study of the activity ingredients of Inonotus Obliquus, and comparing the efficacy of medicines, the findings lays a theoretical foundation for developing anticancer medicine.