Study On Extraction, Separation, Purification And Bioactivity Of Total Flavonoids From Nigella Sativa Seed

In this paper, the determination method of total flavonoids from Nigella sativa seed（Pakistan, NSS-P） was studied. Then, the extraction condition was optimized. The total flavonoids extract was further separated and purified. The antioxidant activity of purified product from each purification step was investigated. The main contents are as follows:（1） An AlCl₃-HClColourimetry method was established for determination of total flavonoids from Nigella genus seeds using rutin as standard control. The results of methodological study showed that the precision and repeatability RSD were 0.70 % and 0.80 %, respectively, and the average recovery was 95.1 %（n=9, RSD 1.61 %）. The total flavonoids content were derminated by the established method from Nigella seeds of 4 different origin. The results showed that the method has good applicability.（2） The basal components of NSS-P was analysed. There were 5.41 % of water, 4.49 % of ash, 38.63 % of oil, 18.44% of protein, and 2.68 % of polysaccharides. The extraction condition of total flavonoids was optimized by Single-factor Experiment and Reponse Surface Methodology. The optimized condition were methanol concentration of 63 %, liquid-solid ratio of 24:1（m L:g）, extraction temperature of 62 ℃, extraction time of 50 min, and extraction times of 2. Under this condition, the average yield of total flavonoids was 1.95 ‰.（3） The total flavonoids extract was successively separated by n-butanol liquid extraction, polyamide chromatography, and sephadex gel LH-20 chromatography. The purity of flavonoids increased from 0.82 % to 85.54 %. NTF-D1 was obtained by sephadex gel LH-20 chromatography and analyzed by LC-MS. Results showed that 4 compounds were detected and 3 compounds could be quercetin-O-α-rhamnosyl-triglucoside, Nigelloside, and Nigeglanoside.（4） The antioxidant activities of NTF, NTF-D, NTF-D1 were analysed by 4 in-vitro methods which included the total recovery power and the free radical scavenging rate of DPPH·, ·OH, H2O₂. The results showed that the antioxidant activity of NTF was the weakest; NTF-D1 and NTF-D had good antioxidant activity; NTF-D1 had similar activity with rutin.