Study On Separation Of Antioxidant Components From Black Cumin Seeds Based On Deep Eutectic Solvents

Nigella glandulifera is an annual herbaceous plant belonging to the Ranunculaceae family.The seeds of the plant（commonly known as black cumin seeds）,rich in oil,have been used in food and medicine for more than 3,000 years.Modern pharmacological studies have shown that black cumin seeds have great nutritional and medicinal value and pharmacological activities such as antioxidant,anti-inflammatory,anti-tumor,and immune regulation.Deep eutectic solvent（DES）,which is a new type of green solvents,has the great characteristics of low toxicity,biocompatibility,degradability,strong dissolution and penetration ability,and designability.This paper mainly focuses on the method of isolating antioxidant component based on DESs,exploring the mechanism that DES facilitates the separation of antioxidant ingredients,identifying the antioxidants and evaluating the antioxidant activities of DES extracts from black cumin seeds.The main results are as follows:（1）52 DESs were prepared by heating and stirring and the physical and chemical properties such as relative density,p H value,polarity,and viscosity of each DES were tested.The relative density of DES was greater than that of water.The p H value and polarity of DES was greatly affected by that of hydrogen bond donor.The viscosity of DESs decreased significantly as the temperature rising or adding a small amount of water.（2）The best DES for extracting antioxidant components from black cumin seed oil was selected based on the DPPH free radical scavenging ability.DESs with low viscosity（η<0.10Pa·s）,acidic p H（not less than 3.00）and low polarity(E_NR>50.00 kcal/mol)were suitable for extracting antioxidant components from black cumin seed oil,and among which,DES-B9 was the best.DPPH free radical scavenging ability of DES-B9 extracts were 2.2 times that of 70%methanol extracts at the same raw meal concentration（3.78 mg/m L）.The antioxidant components of DES-B9 extracts were further separated by rapid preparation chromatography.The antioxidant constituents of fraction DES-B9-3 were identified by Q-TOF MS,which are 5-（3,5-Dimethoxy-4-methylphenyl）pentanoic acid,sugetriol,cinnamic acid,odoratol,2-O-2-phenylethyl-β-D-glucopyranoside,thymoquinone,crocetin,carnosic acid,etc.Antioxidant activity of DES-B9 extracts and fraction DES-B9-3 was evaluated by Ferric ion reducing antioxidant power（FRAP）,oxygen radical absorbance capacity（ORAC）and cellular antioxidant assay（CAA）.The FRAP value of DES-B9 extracts and fraction DES-B9-3 was1057.6±23.1 and 483.3±7.2,respectively.The ORAC value of DES-B9 extracts and fraction DES-B9-3 was 646.5±24.1μmol TE/g and 878.3±26.8μmol TE/g,respectively.The EC₅₀ of cellular antioxidant activity of DES-B9 extracts and fraction DES-B9-3 was 36.6μg/m L and25.1μg/m L,respectively.（3）The best DES for extracting antioxidant components from black cumin seedcake was selected based on the DPPH free radical scavenging ability.DESs with low viscosity（η<0.10Pa·s）,strong acidity（p H<2.50）and greater polarity(E_NR<50.00 kcal/mol)were suitable for antioxidant components extracted from black seedcake,and among which,DES-D10 was the best.DPPH free radical scavenging ability of DES-D10 extracts were 2.4 times that of methanol extracts at the same raw meal concentration（3.33 mg/m L）.The antioxidant components of DES-D10 extracts were further separated by rapid preparation chromatography.The antioxidants of fraction DES-D10-1 are saponins,polysaccharides and organic acids,identified by physical and chemical tests.The antioxidant constituents of fraction DES-D10-2 were identified by Q-TOF MS,which are Nigelloside and Nigeglanoside.The antioxidant activity of DES-D10 extracts,fraction DES-D10-1 and fraction DES-D10-2 was evaluated by FRAP assay,ORAC assay and cellular antioxidant assay.The FRAP value of DES-D10 extracts,fraction DES-D10-1 and fraction DES-D10-2 was 1529.1±30.5,712.2±20.6 and 515.6±14.7,respectively.The ORAC value of DES-D10 extracts,fraction DES-D10-1 and fraction DES-D10-2 was 310.9±15.4μmol TE/g,490.6±11.6μmol TE/g and 239.7±6.3μmol TE/g,respectively.The EC₅₀ of cellular antioxidant activity of DES-D10 extracts,fraction DES-D10-1 and fraction DES-D10-2 was 130.3μg/m L,61.9μg/m L and 85.6μg/m L,respectively.