Separation And Purification Of Inoculum By Preparative HPLC

Inonotus obliquus,also known as Chaga,is mainly distributed in parts of Eastern Europe,Russia and North America,and also distributed in Heilongjiang and Jilin.Its sclerotia are used to treat cancer,heart disease and diabetes.Inotodiol is one of its main active ingredients.The substance is a triterpenoid compound of the lanostane type,which inhibits the activity of transplanted tumors and also has certain inhibitory effects on various cancer cells such as lung cancer and gastric cancer in vitro.There are few commercially available high-purity standards on the market,which not only limits its further pharmacological and pharmacodynamic studies,but also makes it difficult to quantify the quality of existing oblong inotodiol products.Therefore,in this study,a high-purity preparation of inotodiol was prepared by preparative HPLC method that was efficient,rapid,and in line with the requirements of the pharmaceutical process.Raw material Inonotus obliquus sclerotium was extracted with 95%ethanol at room temperature to obtain 28 g of ethanol extract.Extracts were extracted successively with 1050 mL of petroleum ether and then 2050 mL of ethyl acetate.The ethyl acetate extract was crudely separated on a silica gel column(2.6 cm×30 cm),chloroform:methanol= 10:90 was eluted to remove polar impurities,and C18 was passed.The column chromatography(lcmxlcm)eluted with methanol and adsorbed non-polar impurities.The pre-HPLC sample(3.37g)was obtained.The inotodiol content was determined to be approximately 15%.Preparation of separation conditions was performed on an analytical column(Hypersil GOLD,C18 reversed-phase column,250 × 4.6 mm,5 μm,)and 75%acetonitrile and 70%to 90%methanol-water solution against inotodiol were tested.Separation effect of impurities and impurities;the influence of flow rate(0.6-1.0 ml/min)and injection volume(20-1100 μL)on preparation and separation was tested.After balancing the sample volume and the purity,I selected 90%methanol as the mobile phase and linearly amplify at a flow rate of 0.8 mL/min.In the preparative column(SinoChrom ODS-BP,15μm,300mm×40.0mm),With a flow rate of 32 mL/min and an injection volume of 4 mL,separate 160 mL of the fraction with a retention time of 40-45 min.A sample with a purity of 57.09%was obtained.The fraction was again subjected to secondary separation on a preparative column and detected by an evaporative light scattering detector(drift tube temperature 32℃.,gas flow rate 0.5 mL/min)and a photodiode array detector(190-360 nm),respectively.The purity was 99%.and 97%..The identification of the purified product of Inonotus obliquus was performed using high and low resolution ESI mass spectrometry,1H-NMR,and 13C-NMR.The molecular ion peak m/z for this isolate was 425.4(M+H-H20),407.4(M+H-2H20),and 443.3(M+H)as measured by Agilent 1100 LC-MS low resolution mass spectrometry;Thermo Fisher Scientific LTQ FTICR ESI-HRMS(DART Positive mode)Analysis of molecular ion peaks with an m/z of 425.4 in a low resolution mass spectrum confirmed that the obtained m/z was 425.3772(M+H-H2O).After database comparison,the structure was found to be C30H49O,consistent with inotodiol.1H-NMR(500MHz CDCl3)can be seen in the high-field area of triterpenoids typical five corners of the methyl peak δ0.75(3H,s),0.81(3H,s),0.87(3H,s),0.98(3H,s)and 1.00(3H,s),13C-NMR(500MHz CDCl3),DEPT,and COSY results show that the number of carbon atoms and the chemical shifts are in good agreement with reported inotodiol.Therefore,we confirmate the preparation of high purity is Inotodiol.In this study,high-purity inotodiol was obtained by preparative HPLC,which provides a standard for the quality analysis and detection of Inonotus obliquus related products.The methanol mobile phase used for preparation can be recovered,which overcomes the disadvantages of low efficiency,large pollution,and long time consumption of the traditional silica gel column chromatography,and provides a theoretical and experimental basis for further industrial preparation of inotodiol.