Studies On The Active Ingredient From Callus And Suspension Cell Of Carpesium Macrocephalum Franch. Et Sav.

Carpesium.macrocephalum Franch.et Sav is perennial herb.It has long been used as Chinese folk medicine for hemostatic,vermifuge,antipyretic anti-inflammatory,pain-relief,and detoxication properties in traditional Chinese medicine.The flavonoid,lactone,sterid etc many compounds were isolated from the plant. At the same time,the extract of the plant has insecticidal,fungicidal and weeded activity.Due to its long develop period,limited clime,so it has much localization to distill its insecticidal,fungicidal and weeded product.It is one of important research to industrially produce flavone,lactone using cell cultivation. We have studied on the active ingredient from callus and suspension cell of Carpesium.macrocephalum since 2002.To further exploit and utilize the insecticidal,fungicidal and weeded plant Carpesium macrocephalum Franch. et Sav., this dissertation carried out a systematic research on seven fields of its cell culture. The results showed as follows:1. The callus were induced from the radicle of Carpesium macrocephalum. There are 0.72% flavonoid content and 0.056% lactone content in the callus(cultivated 40d). The optimum media and culture conditions were determined through the researches of induction and proliferation of callus derived from root,hypocotyl and cotyledon of asepsis seedling of Carpesium.macrocephalum . The results showed that:⑴the radicle of asepsis seedling were good explant for callus inducing,B5 was the optimum media than MS and NT;⑵the optimum combination of callus inducing conditions was found to be a solid medium of B5+2.0～3.0 mg/L NAA+0.2～0.4 mg/L 6-BA containing sucrose 20 g/L and pH 5.8 at the light of 12 h/d,the linduction rate was 100%;⑶the flavonoid content and yield of root-drived callus was the highest;⑷B5+ 3.0 mg/L NAA +0.2 mg/L 6-BA (sucrose 45g/L,pH 5.7,25℃,12h/d) was propitious to increase callus multiplication;⑸the flavonoid and lactone content of root-drived callus was steady-going after 3～12 subculture frequency;⑹the optimum ultraviolet radiation time was propitious to increase callus flavonoids and lactone content was 10 min;⑺the callus growth,flavonoids and lactone cumulation curve were as"S"in different culture medium,the optimal culture medium to produce flavonoids was NT,the optimal culture medium to produce lactone was MS+NT.2.The cell suspension culture line was established using the callus There are 1.30% flavonoid content and 0.086% lactone content in the suspension cell(20 d).The dynamical model of the suspension cell line was also established. The optimum media for suspension cell growth is NTB. The components of NTB is mainly composed of NT,mixed with 1.0 mg/L B3,pH 5.0～6.0,and then 1.0 mg/L NAA,0.2 mg/L 6-BA,40 g/L sucrose,1.0～2.0 mg/L B1 were added.The inoculation weight was 40-50 FWg/L in the 250 m1 flask which contained 100 mL culture liquid. At 25±2℃, 1500～2000 Lux,12/d light treatment,the rotation speed is 120 r/min.The dynamical model of the suspension cell . calculated result coincided well with experimental data. The model could be used for good description of culture process.3.The primary medium(NTF)to produce flavonoid was established.There are 1.89%. flavonoid content and 476.97 mg/L flavonoid yield in the suspension cell cultured in NTF(cultivated 20d). The components of NTF is mainly composed of NT,mixed with 1237.5 mg/L NH4NO3,1425 mg/L KNO3,0.75 mg/L Na2MoO4?2H2O,ZnSO4?7H2O,H3BO3,KI,MnSO4?4H2O,CoSO4?5H2O,CuSO4?5H2O were increased one time,1.0 mg/L NAA+0.2 mg/L 6-BA,1.0 mg/L B6 were added.. The other culture condition was same as NTB.4.The results of physiology and biochemical modulation on cell suspension culture line show that the total flavonoid content and yield were increased 2.05～2.27% and 516.06～551.76 mg/L,when SA,KT,GA3,Phe,AgNO3 etc were added separately in the primary medium(NTF).5.In this experiment, the active ingredient in the tissue and suspension cell of Carpesium.macrocephalum were extracted. The active ingredient of suspension cell were mainly existed inside cell. The active ingredient of suspension cell were not affected by freeze-drying or drying. The dried tissue and suspension cell were pulverized in 40-60 orders, and were extracted in acetone solution at the rate of material and solution 1:10～1:15, extracted 18h by surging,then extracted 60 min by ultrasonic. The extraction rate of active ingredient can reach 100%.6.In this experiment, combination techniques to detect the active ingredient of cultivation cell and nutriment consumed in liquid medium were established using UV and bioactivity detectng by Rhizoctonia cerealis and Amaranthus retroflexus.7.The chemical components in the tissue and suspension cell of C.macrocephalum were qualitatively analysed.The results showed that the fruit of C. macrocephalum contained tannic acids,coumarin,phenols,steroidal,terpenoids,flavone,carbohydrates,amino acids,polypeptide,protein,volatile oils and oilsand fats. Compared with fruit,There were little chemical components in the tissue and suspension cell ,it were composed of terpenoids, flavone,carbohydrates,amino acids,polypeptide.The little lactone content in the tissue and suspension cell were determined by GC-MS.It showed that the flavone and lactone had been biosynthesized in the tissue and suspension cell of Carpesium.macrocephalum.So the objective of further increasing the active ingredient in the tissue and suspension cell of Carpesium.macrocephalum by physiology and biochemical modulation was to set a foundation for efficient large scale cultivation .The fungicidal and weeded activity of cultivation cell were tested by 16 pathogens,6 kinds of crop and 3 kinds weed seeds . The results showed that the ethanol extract and petroleum ether,ethyl acetate and n-butyl alcohol extraction from ethanol extract were active to all tested pathogens,crop and weed seeds. The ethyl acetate extraction has the hightest inhibiting effect on weed,the EC50 of A. retroflexus and M.sativa were 0.2945 and 0.1673 mg/mL, respectively,were 0.29 and 0.13 times of carabrone's.The extraction of ethyl acetate from ethanol extract leached by petroleum ether had the hightest fungicidal activities against different pathogens.The fungicidal activities against Rhizoctonia cerealis,Alternaria solani,Phytophthora capsici and Fusarium bulbigenum were above 95% under concentration 10 mg/mL, the EC50 were between 0.24～3.56 mg/mL.