Study On Tissue Culture And Quality Of High Effective Components Of Fritillaria Cirrhosa D.Don

Fritillaria Cirrhosa D.Don is a famous and precious medicine in Sichuan,which mainly distributed in the altitude of 2800 ~ 4700 m in alpine shrub and grassland,and mostly located in the southwest of China in mountains.Fritillaria Cirrhosa D.Don has a remarkable effect when used as a cure for coughing,expelling phlegm and relieving asthma because it contain alkaloids such as Peimine,Peiminin and Sipeimine.However Fritillaria Cirrhosa D.Don only harvest every five years when growth under natural conditions and in recent years people dug it without any constraint that make its resourc shortage and has a low content of alkaloids.To resolve this problem,our study use tissue culture technique to establish the rapid propagation system of Fritillaria Cirrhosa D.Don with high effective components.Furthermore,in order to obtain more effective components we also have a research on extraction process of effective components by orthogonal design,and we strictly accordance with pharmacopoeia of the people’s republec of China(Edition 2010) to research its quality for having a better control. The results were as follows:1 Study on the in vitro rapid propagation of tissue culture seedlings of Fritillaria Cirrhosa D.Don:Using scale leaf as the study subject,have a research on the influence of the different plant hormones to the callus induction, bulb differentiation, seedling and rooting induction of Fritillaria Cirrhosa D.Don.The result showed that the best culture medium of callus induction is MS+6-BA 1.0 mg/L+2,4-D0.5mg/L, after 45 d the induction rate is 84.33%;the best culture medium of bulb differentiation is MS+KT 2.0 mg/L+NAA 0.3mg/L, after 45 d the induction rate is 82.77%;the best culture medium of seedling induction is MS+6-BA 3.0 mg/L+IBA0.3mg/L, after 45 d the induction rate is 85.39%;the best culture medium of rooting induction is MS+NAA 0.3mg/L, after 60 d the induction rate is 83.20%.2 Have a research on the influence of the concentration of colchcine,the time of being soaked in colchcine,the volume fraction of dimethyl sulfoxide and temperature for inducing the polyploid of Fritillaria Cirrhosa D.Don.The result showed that the optimal combination was the concentration of colchcine 900 mg/L, the time of being soaked in colchcine is 48 h, the volume fraction of dimethyl sulfoxide is 4% and the temperature is 20℃,under this condition cultured 30 d,the induction rate is 94.3%.Have a determination with wild,tissue culture of diploid and polyploid bulbs of Fritillaria Cirrhosa D.Don.The result showed that their content of total alkaloids is 0.237%、0.249% and 0.657% respectively,so it can be proved that no matter diploid or polyploid bulbs of Fritillaria Cirrhosa D.Don by tissue cultured, their content are higher than wild, further more the polyploid bulbs is 2.8 times of the wild.3 Using orthogonal design to investigate the effect of the concentration and amount of ethanol,extraction time and frequance on extracting process with the issue culture of Fritillaria Cirrhosa D.Don.The result show that the four factors between each other effect on the extraction is concentration of ethanol>amount of ethanol>extraction time>extraction frequance,and when the extraction process is extracted twice with 6 times the amount of 85%ethanol,1.5 h for each time can obtain the most effective components.Have a determination with tissue culture of Fritillaria Cirrhosa D.Don which including the total ash,acid-insoluble ash,extract,alkaloid content and TLC for quality standard,The result show that the total ash is not more than2%,the acid-insoluble ash should not exceed 1%,the extract should be not less than 20% and the content of total alkaloids should be between 0.2%~0.3%.4 Have a research on quality difference between human intervention of Fritillaria Cirrhosa D.Don,The result show that different cultivation methods and growth years of Fritillaria Cirrhosa D.Don have an impact on total ash and acid insoluble ash content,extract,alkaloid content determination,but different treatments of it have no significant impact on them.