Study On The Ioslation, Separation, Identification And Biological Activity Of Triterpenoids From Inonotus Obliquus

Inonotus obliquus is a kind of precious medicinal fungi belonging to the family Hymenochaetaceae Donk, which is mainly distributed in the northeast of China, Finland, Poland, Russia and Japan, et al. It has been used as a folk medicine which has beneficial effects in the treatment of breast cancer, liver cancer, uterine cancer, and gastric cancer, as well as hypertension and diabetes. Triterpenoids is an important chemical composition in Inonotus obliquus which has good biological activities. Inonotus obliquus was used as the research object in the study. The extraction, isolation, identification and biological activities in vitro of triterpenoids from inonotus obliquus were studied in this paper to provide theoretical basis for research and application of triterpenoids from inonotus obliquus. The results were as follows:1. Optimization of ultrasonic extraction conditions of triterpenoids from inonotus obliquus by RSM. Based on the ethanol concentration, ratio of liquid to raw material, ultrasonic power and ultrasonic time as single-factors experiment, Box-Behnken design was used to optimize ratio of liquid to raw material, ultrasonic power and ultrasonic time three factors. The results showed that the optimum conditions were ethanol concentration at 60%; ratio of liquid to raw material 30:1(mL/g); ultrasonic power 380 W and extraction time 32 min, and the highest triterpenoids from Inonotus obliquus extracting yield reached 3.78% under the conditions.2. The purification process parameters of triterpenoids from inonotus obliquus with macroporous resin were studied. According to the static adsorption and desorption experiments, AB-8 macroporous resin was confirmed as the more suitable resin for enriching triterpenoids of Inonotus obliquus. Through the dynamic adsorption and dynamic experiments, the optimum process parameters as followed:the sample concentration 1.5 mg/mL, the volume of sample 1.5 times of column volume, sample flow velocity 1.5 mL/min, sample solution pH value 7, elution concentration 95% ethanol and eluent volume 5 times of column volume. Under this condition, the purity of triterpenoids from Inonotus obliquus reached 69.96%.3. By employing silica gel column chromatography and semipreparative high performance liquid chromatography, two compounds were isolated from Inonotus obliquus and the purity reached 95.99% and 97.27% by TLC and HPLC determination, respectively. On the basis of UV, IR, EI-MS and 1H NMR spectral data analysis and related literature, the structures of two compounds were identified as Lup-20(29)-ene-3β,28-diol and lanosta-8,24-dien-3β-ol.4. The antioxidant activities of crude triterpenoids from Inonotus obliquus were evaluated by in vitro assays including DPPH, ABTS, hydroxyl radical and reducing power. The inhibiting effects of crude triterpenoids and two compounds isolated against the growths of A549 cells and a-glucosidase activity in vitro were also studied, respectively. The results showed, in the ranges of the experiment, there was a linear relationship between the reducing power and concentration of crude triterpenoids from inonotus obliquus, the EC50 of DPPH, ABTS and hydroxyl radical were 0.39,0.61,0.51 mg/mL,respectively. In addition, IC5o of inhibiting the growths of A549 cells and a-glucosidase activity were 167 μg/mL,150 μg/mL and 163 μg/mL, respectively.