Research On Puriifcation, Characterization And Biological Activity Of Torulene In Sporidiobolus Pararoseus

Torulene (TL) shares potential functional activities with carotenoids, such asanti-oxidation, immunity-regulation, inhibiting tumor and cancer etc. Microbial fermentationsynthesis has become a major means of the natural carotenoids production. SP possesses quitea lot of advantages, such as simple production process and high yield of carotenoids includingtorulene, which provide a new strain resource for microbiological biosythesis of torulene andthe experimental basis for further study of torulene. Latest studies are mainly concentrated onthe microbial fermentation of torulene, but so far, scarce data have been reported in relation toits biological activities. In this paper, the spectral, chromatographic coupled with massspectrographic analysis were used to separate, purify and indentify torulene, and the TLstorage stability and biological effects on oxidative stress were also studied, which is aim tolay theoretical foundation for reasonable and effective exploitation of torulene.(1) The major carotenoids were determined as β-, γ-carotene, torulene and torularhodinby high performance liquid chromatography coupled with diode array detection(HPLC-DAD), with torulene present in the largest amount (167μg/g dry mass cell). Torulene,preparaed by silica gel column chromatography was further characterized by HPLC, massspectrometry and Raman spectroscopy, which showed that it is the polyene compound whichhas the basic structure of carotenoids and the molecular weight of TL is534, consistent withits chemical structure of C40H54. The analysis method of HPLC(C30)-DAD-APCI-MS wasfirstly established to separate torulene geometrical isomers and obtained a total of8cis/transisomers and all-trans(65.43%) and5’-cis-torulene(15.71%) were tentatively determined, othercis-isomers can not be confirmed due to the lack of relevant references to verify.(2) The torulene stability separated from Sporidiobolus pararoseus was then studied andthe retention rate of total TL(%) and the percent of all-trans TL among its geometricalisomers were determined under different conditions, such as light, oxygen, temperature andantioxidant. The results demonstrated that TL was sensitive to light, heat and oxygen, and theeffects of the above factors on its retention rate and all-trans configuration percent showed thesame trend. Light and heat had a marked effect on the geometrical isomerization of TL.Torulene was extremely unstable to the temperature higher than80℃and at the same time50%~90%all-trans TL isomer transformed to its cis configuration; The influences of light onthe stability of TL were as follows: sunlight<ultraviolet light<daylight<dark light; The TLretention rate descended from100%to9.4%after60days’storage exposed to oxygen, butexposed to the oxygen had no significant effect on the ratio of all-trans TL configuration.Filled with nitrogen or carbon dioxide can not only prevent TL oxidative degradation, but alsoinhibit the conversion from its cis to trans configuration, and preserved with CO2was moreeffective than treated with N2; In addition, the stability of TL partly depended on the antioxidants. The retention rate of TL adding BHTwas slightly higher (85.9%) than that ofadding TBHQ (82.4%), and was significantly higher than that without adding antioxidants.Moreover, the all-trans ratio of TL containing BHT and TBHQ were5.4%and4.1%higherthan the control group, respectively.(3) HepG2cells were used as an oxidative stress model to investigate the protectiveeffects of torulene against H2O2-induced cytotoxicity and apoptosis. The results demonstratedthat the HepG2cell viability decreased to59.18%when cells were incubated for8h by200μmol/L H2O2. The survival rate of HepG2cells treated with torulene at differentconcentrations (4~8μmol/L) increased gradually compared to the viability of theH2O2-stressed cells, and a6μmol/L TL played the most important role in improving the cellviability by35.68%(P<0.01), which was higher than the positive control group of8μmol/Lβ-carotene (19.73%) and lycopene (32.13%); The cytotoxicity was evaluated bymorphological observation: TL can obviously reduce the number of injuried HepG2cellsinduced by H2O2; A dose-dependent increase of4~8μmol/L torulene in inhibiting theaccumulation of reactive oxygen species（ROS）of HepG2cells was showed. What’more, TLprotection groups promoted oxidative-stress-related antioxidase system. As illustrated, TLsignificantly enhanced the activities of superoxide dismutase (SOD), total antioxidantcapacity (T-AOC), catalase (CAT) and glutathione peroxidase (GSH-Px), and the content ofmalonaldehyde (MDA) was decreased as well (P<0.05).