Studies On The Culture Of Hairy Roots And Callus Of Sophora Flavescents Ait

Sophora flavescens Ait., a type of bush belonging to Sophora Linn.,Leguminosae, is one kind of the traditional Chinese medicine in our country,mainly distributing in Shanxi, Shaanxi, Beijing, Heilongjiang, Gansu, Hebei,Hunan, Guangxi, Sichuan, etc. Sophora flavescens Ait. has many functions,such as clearing heat, killing pesticides, dieresis, etc. Modern pharmacologicalstudies indicate that Sophora flavescens Ait. has effect of anti-cancer.Forexample, it can control melanoma, bladder, stomach cancer, etc. in differentlevels. At present, many preparations have been developed with Sophoraflavescens Ait.and its ingredients for raw material are used in clinical widely.market demand of Sophora flavescens Ait. is increasing because its growthcycle is2-3year and preparations are deeply developed. The price rises fromabout2.5yuan in2003to about14yuan in2011， which showed the issue ofsustainable development of Sophora flavescens Ait. resources.This paperresearches training system of hairy roots and callus to solve the shortageproblem of Sophora flavescens Ait. resource exploration by new ways.In this paper, the inducing conditions and growing conditions of callus andhairy roots of Sophora flavescens Ait., the matrine and oxymatrine devive fromthe callus and hairy roots were studied. The content of Matrine and oxymatrinewas detected by High Performance Liquid Chromatography （HPLC）.The resultswere as follows:1. The determination of matrine and oxymatrine in callus and hairy rootsby HPLC was performed on NH₂column（4.6mm×250mm，5μm） at roomtemperature with a mobile phase of acetonitrile： anhydrous alcohol:3%phosphate （84:8:8）. The detective wave length was220nm, flow rate was1mL/min, sampling volume was15μL.1) Under that condition, the retention time of matrine was10.4min.When the concentration of matrine was in the range of10.0²40.0μg/mL, it kept agood linearity（r=0.9990）, and the average recovery was1.75%, RSD was97.21%.2) Under that condition, the retention time of oxymatrine was15.9min.When the concentration of oxymatrine was in the range of10.0²40.0μg/mL, itkept a good linearity （r=0.9995）, and the average recovery was1.90%, RSD was99.12%, the accuracy test RSD was1.92%.2. The best conditions of inducing hairy roots was as follows: young budsas explant, infected by R1601with100μmol/L acetosyringone for20min，andinduced at25±1℃for2d in the dark. Results showed that, in this conditions,the inducting rate was28.6%.3．Research on the culture conditions of hairy roots indicated that the hairyroots grew better in the improved1/2MS medium. The accumulation ofoxymatrine was1.79mg/g （dry weight of hairy roots） and the matrine contentdid not be detected.4. The best induction conditions of callus：young buds were the bestexplant in the studies. The best inducing culture medium of callus was MS+1.0mg/L2,4-D+1.0mg/L6-BA+1.0mg/L NAA under the conditions of25±1℃,dark.The young bud, roots, stems, leaves of Sophora flavescens Ait. werechosen to induced the callus, and the inducting rates was94.12%,74.19%,35.71%,23.08%respectively.5. The best proliferating culture medium of callus: MS+0.5mg/L2,4-D+0.5mg/L6-BA+0.5mg/L NAA, and the increment rate was25.3%in thisconditions.Different combinations of hormone had a small impact on the induct ofcallus, but it had a notable effect on the increment rate of callus, especiallyNAA.Researches on the matrine and oxymatrine in the callus showed that, thecontent of matrine was0.40%, the content of oxymatrine was4.34%.