黑果枸杞黄酮提取工艺优化及配方液活性检测

Detection of Activity of Liquid Formula of Flavonoid from Lycium ruthenicum and Optimization of Extraction Process

目的探究黑果枸杞配方液对肿瘤细胞的生物学活性检测。方法以无菌幼苗茎段为外植体,不同浓度的6-呋喃氨基嘌呤(KT)、2,4-二氯苯氧乙酸(2.4-D)及吲哚丁酸(IBA)考察组织培养条件,比较组培生根苗抗氧化能力差异,超声法提取黑果枸杞叶中黄酮,噻唑兰(MTT)法检测黑果枸杞黄酮多种配方液在体外对肿瘤细胞的生物学活性。结果愈伤组织最佳诱导培养基为MS+2.4-D 0.50 mg·mL^(-1)+KT 0.20 mg·mL^(-1)+蔗糖30 g·L^(-1)+琼脂8.0 g·L^(-1),分化培养最佳培养基为植物组织培养基(MS)和激动素(KT)0.15 mg·mL^(-1)+IBA 0.01 mg·mL^(-1),根诱导最适培养基为1/2MS;黑果枸杞组培苗提取物的过氧化物酶(POD)、过氧化氢酶(CAT)及超氧化物歧化酶(SOD)活性明显优于移栽苗;当超声功率270 W、超声时间35 min、乙醇浓度体积分数70%和料液比1∶25时粗黄酮得率为5.54%,当黑果枸杞组培叶黄酮-锁阳多糖-螺旋藻多糖为3∶1∶1,终浓度为160μg·mL^(-1),作用48 h后,体外对人黑色素瘤B16-F0细胞的抑制率最大为52.53%。结论黑果枸杞黄酮与锁阳多糖配方液在体外对多种肿瘤细胞均有抑制作用,为充分利用黑果枸杞及锁阳在抗肿瘤方面的开发和应用提供数据基础及参考。

OBJECTIVE To investigate the biological activity of Lycium ruthenicum Murr on tumor cells.METHODS Aseptic plantlet stem segments were utilized as explants,different concentrations of KT,2.4-D and IBA were used to investigate the tissue culture conditions,the antioxidant capacity of rooting plantlets were compared,flavones were extracted from leaves of Lycium ruthenicum Murr by ultrasonic method,and the biological activities of formula liquid of Lycium ruthenicum and Songaria cynomorium polysaccharide on the tumor cells were tested by MTT method in vitro.RESULTS The best medium for callus induction was MS+KT 0.20 mg·mL^(-1)+2.4-D 0.50 mg·mL^(-1)+sucrose 30 g·L^(-1)+agar 8.0 g·L^(-1),the best medium for differentiation was MS+KT 0.15 mg·mL^(-1)+IBA 0.01 mg·mL^(-1),and the best medium for root induction was 1/2MS.The biological activities of POD,CAT and SOD of the extract from Lycium ruthenicum tissue culture plantlets were significantly stronger than that from transplant plantlets.Under the extracting condition of ethanol concentration 70%,solid-liquid ratio 1∶25,ultrasonic time 35 min,and ultrasonic power 270 W,the extraction rate of crude flavone was 5.54%.When the ratio of flavone extracted from Lycium ruthenicum tissue culture leaves:Songgaria cynomorium polysaccharide:spirulina polysaccharide was 3∶1∶1 and the total concentration was 160μg·mL^(-1),the inhibition rate of human melanoma B16-F0 cells was 52.53%at the maximum after 48 h in vitro.CONCLUSION The flavone of Lycium ruthenicum Murr and the formulation of Songgaria cynomorium have inhibitory effect on many kinds of tumor cells in vitro,which provides data basis and reference for the development and application of Lycium ruthenicum Murr and Songgaria cynomorium for anti-tumor therapy.