Micropropagation Techniques And Identification Of Some Activity Substances From Clinacanthus Nutans

Clinacanthus nutans is an important herbal medicine that has been widely used in pharmacology and has good efficacy in antiinflammatory,antioxidation,antivirus,antibacterial and antitumor activities.It is one of the effective ways to promote the sustainable development of Clinacanthus nutans by optimizing the culture conditions and breeding it rapidly through plant tissue culture.The purpose of this study is to achieve the micropropagation of Clinacanthus nutans and to isolate and identify some of its active substances.The main contents include:（1）Observed the growth conditions of the plantlets and measured stem height and induction rate of axillary buds by changing hormone ratio,type of medium,concentration of sucrose,NO₃^-/NH₄⁺and phosphorus concentration in solid culture conditions.Finally,determined the most suitable culture conditions for the growth of Clinacanthus nutans.（2）Induced clustered shoots and roots using the stem segments with axillary buds of Clinacanthus nutans,and explored the effects of different media types,hormone ratio on the induction of clustered shoots and roots,to determine the best culture conditions for the induction of the clustered shoots and roots.（3）By changing the liquid culture conditions,the microscopic soilless fast propagation of Clinacanthus nutans was established.（4）Extracted and purified the total flavonoids of Clinacanthus nutans,and then conducted antibacterial and antioxidative tests to compare the differences of the content of total flavonoids,the antibacterial activity and the antioxidation between tissue culture seedlings and seedlings.The main findings are as follows:1.The optimal culture conditions for the growth and the induction of axillary buds of Clinacanthus nutans were as follows:Modified MS medium(40 mmol·L^-1 KNO₃+10mmol·L^-1 NH₄NO₃+3 mmol·L^-1 KH₂PO₄,other conditions were unchanged)+Sucrose 30g·L^-1+agarose 6 g·L^-1+6-BA 1.0 mg·L^-1+NAA 0.8 mg·L^-1,pH 6.0.2.The optimal culture conditions for the induction of the clustered shoots were as follows:MS+sucrose 30 g·L^-1+agarose 6 g·L^-1+6-BA 0.8 mg·L^-1+NAA 0.5 mg·L^-1+KT 0.2 mg·L^-1,pH 6.0.3.The optimal conditions for the induction of roots were as follows:1/2 MS+sucrose 30g·L^-1+agarose 6 g·L^-1+IBA 1.0 mg·L^-1+NAA 0.5 mg·L^-1,pH 6.0.4.The optimal conditions for the micropropagation were as follows:modified 1/2 MS medium(20 mmol·L^-1 KNO₃+5 mmol·L^-1 NH₄NO₃+1.5 mmol·L^-1 KH₂PO₄,other conditions were unchanged)+Sucrose 20 g·L^-1+agarose 6 g·L^-1+IBA 1.0 mg·L^-1+6-BA 0.5 mg·L^-1+NAA 0.8 mg·L^-1+KT 0.2 mg·L^-1,pH 6.0.5.The crude extraction of total flavonoids of Clinacanthus nutans was extracted with75%ethanol,and the content of total flavonoids of the seedlings was 13.08±0.15 mg·g^-1.The content of total flavonoids of the tissue culture seedlings was 1.3 times that of the seedlings,which was 17.22±0.31 mg·g^-1.And then purified by AB-8 macroporous resin,the content of total flavonoids I reached 60.56%±0.70%.The content of total flavonoids II reached 65.62%±0.53%.By antibacterial and antioxidant tests,total flavonoids I and total flavonoids II inhibited the growth of E.coli and Staphyloccocus aureus Rosenbach,and were able to scavenge DPPH free radicals and hydroxyl free radicals.The total flavonoids II was better in all aspects than the total flavonoids II.Therefore,cultivating Clinacanthus nutans by means of tissue culture not only provides the seedlings basis for the industrialized cultivation,but also has further developmental value in the pharmacological research.