Study On Extraction Technique Of Total Saponins From The Seeds Of Nigella Glandulifera And Its Immunomodulatory Activity

Objective: To investigate double water phase extraction process of total saponins from the seeds of Nigella glandulifera(NGT)as well as it?s antioxidant and immunomodulatory activities.Methods:(1)Nigegllanoside A as detection index,based on the single factor experiment,the response surface analysis method was used to optimize the extraction process of NGT,and NGT was purified by macroporous resin.(2)Active components of total saponins were isolated by reversed-phase silica gel and Sephadex LH-20,and chemical structures of the active components were identified by NMR and other spectral techniques combined with physical and chemical properties.(3)Antioxidant activity of total saponins from the seeds of Nigella glandulifera were investigated by DPPH and superoxide anion scavenging experiments and reduction ability determination.(4)Immunomodulatory activity of total saponins was evaluated in mice with low immune function induced by cyclophosphamide.Results:(1)To optimize the aqueous two-phase system extraction precess of NGT,the optimum extraction process of NGT is follows:ethanol concentration at 47%,ammonium sulfate dosage at 0.35g/mL and solvent-material ratio of 22:1.Under this condition,the extraction rate of NA was 3.15%.Further purified by macroporous resin,the content of saponin is more than 50% in NGT.(2)Three compounds were isolated from NGT,and their chemical structures were identified as follows: hederagenin 3-O-?-D-xylopyranosyl-(1?3)-?-L-pyranrhamnosyl-(1?2)-?-L-pyranarabinose-28-O-?-L-pyranrhamnosyl-(1?4)-?-D-pyranglucosyl-(1?6)-?-D-glucopyranoside(1,NA)?Hederagenin3-O-?-L-pyranrhamnosyl-(1?2)-?-L-pyranarab inose-28-O-?-L-pyranrhamnosyl-(1?4)-?-D-p-yranglucosyl-(1?6)-?-D-glucopyranoside(2)and Hederagenin-3-O-?-L-pyranrhamno-syl-(1?2)-?-L-pyranarabinose-28-O-?-Dpyranglucosyl-(1?6)--?-D-glucopyranosid-e(3).(3)NGT have better antioxidant activity,DPPH free radical and superoxide anion radical scavenging activities.(4)The medium and high doses of NGT(100 and 200 mg/kg)can significantly increase TNF-? and INF-? levels in immune deficit mice as well as the spleen and gland index(p<0.05).Conclusion: The aqueous two-phase extraction precess of NGT was a convenient,non-polluting method,and can apply to industrial production.NGT has a good antioxidant and immunomodulatory ability.The result can provide data foundation for the development and utilization of Nigella glandulifera and NGT.