2.1 TGSO extraction and fatty acids analysis
Thorn grapes(Vitis davidii Foex.)，authorized by Cultivar Variety Examination and Approving Committee of Hunan Province(No：XPD010-2005)，were picked from Mayang county，Huaihua city，Hunan province，P.R.China and deposited in nursery garden of Hunan Agricultural University.The seeds were collected and dried at 50℃for 24 h and then smashed into the powder of 40 meshes.TGSO was extracted by using supercritical CO2 fluid extraction machine(HA231-50-06，Nantong Hua'An Supercritical Extraction Co.Ltd.)at CO2 flow of 20～30L/h，extraction pressure of 34.2Mpa，extraction temperature of 39.3℃，separation pressure of 12 Mpa and separation temperature of 55℃for 95minutes.(https://www.daowen.com)
Fatty acid composition of TGSO were analyzed by GC/MS with a QP2010 gas chromatograph-mass spectrometer(Shimadzu，Japan)，equipped with flame ionization detector(FID)，Rtx-WAX capillary column(30.00m×0.25mm×0.25μm).Samples(1μL)were prepared following the protocols：TGSO(100 mg)were dissolved in 5mL hexane，5mL sodium hydroxide-carbinol solution(0.5mol/L)，then vortex-mixed for 30s and incubated in a water bath at 80℃for 30min.After cooled to room temperature，1mL hexane and 1mL deionized water were added，vortex mixed for 30s and centrifuged for 10min at 8000 revolutions per minute(RPM).The supernatant were transferred into a GC vial after filtered with 0.45μm membrane(Shanghai Xingya purification material factory，Shanghai，China)and injected in split mode(1∶10)with injector temperature of 250℃for GC/MS analysis.Helium was used as carrier gas at a flow rate of 1.02m L/min.The oven temperature was 100℃for 3min，and then increased from 100℃to 200℃at 5℃/min and at 200℃for 17min.The detector operated in scan mode from m/z 40 to 500.Initial pretreatment of the raw chromatographic data，such as baseline correction，chromatogram alignment，noise reduction，and normalization，were performed by using GC/MS solution software.The peaks were then tentatively identified from their retention characteristics and mass fragmentation patterns by using NIST mass spectrum database.