Study On The Metabolism And Accumulation Of Indigo And Indirubin In Isatis Indigotica

Isatis indigotica is a kind of biennial herbaceous plant belongs to Isatis of Cruciferae,which is one of the medicinal materials in common use,and widely cultivates in China.It has many beneficial effects such as heat-clearing,detoxicating,cooling blood,dissolving purpura,and relieving sore-throat and pain,and was recorded in the Chinese Pharmacopoeia 2010.In this paper,the parameters of ultrasound assisted extraction of indigo and indirubin from Isatis indigotica were optimized according to the center combination(Box-Benhnken)experimental principles.Four factors and three levels based on single factor experiments were selected to design response surface test.The response factor was the yield of indigo and indirubin.The independent variables were concentration of methanol,ratio of water to material,ultrasound extraction temperature and ultrasound extraction time.And then the rules of metabolism and acumulation of indigo and indirubin from Isatis indigotica under the natural conditions,conditions of culture in vitro and conditions treated with different elicitors in different concentrations and times has been revealed.All these results may provide guidance to quality control,reasonable harvest,standardized production and rational exploit and utilization of Isatis indigotica.The main experimental results were as follows:(1)The HPLC method to determine indigo and indirubin were established,the mobile phase:A(methanol):B(0.2%phosphoric acid in water)= 75:25;Column temperature:25?;Flow rate:1.0 mL/min;Detection wavelength:289.Response surface analysis methodology(RSM)was used to optimize ultrasound assisted extraction of indigo and indirubin from Isatis indigotica based on the results of single factor experiments.By means of regression analysis,the optimum parameters were as follows:methanol concentration 75%,ratio of water to material 1:34,ultrasound extraction temperature 4? and ultrasound extraction time 25 min.The influence of the parameters to the yield of indigo and indirubin were:the concentration of methanol>ratio of water to material>extraction temperature>extraction time.(2)The rule of the accumulation of indigo and indirubin from Isatis indigotica under natural conditions has been studied.The results showed that,during the observed growth stages,there was similar change trend of the contents of indigo and indirubin in different organs of the plant.And the contents of indigo and indirubin from the highest to the lowest were as below:leaf>flower>fruit>stem>root.And the contents of indirubin are always higher than indigo in the all organs of Isatis indigotica except for root(3)The contents of indigo and indirubin in the calluses and adventitious buds of Isatis indigotica in different generation of subculture were determinated.The results showed that the contents of indigo were decreased with the increase of subculture generations,and that was different change trends to contents of indirubin's during the callus inducing and subculture.And during the stage of adventitious buds subcultures,the contents of indigo and indirubin increased firstly,then decreased,and the peak contents appeared(138.88,529.54 ?g/g)in the fourth generation.The contents of indigo and indirubin of the adventitious buds were higher than callus,and similar to the seedlings.(4)The effects of different elicitors on accumulation of indigo and indirubin in Isatis indigotica were disscussed.The results showed that,after being treated with the various elicitors(MJ?SA?YE?Cu2+?Ag+),the contents of indigo and indirubin suffered different influences with a 14-day period,and showed different time and density dependent effects.After being treated with the various elicitors,the contents of indigo were increased,and the highest contents reach 1.70 times,compared to the control group,treated with MJ of 750?M.But the change rules of contents of indirubin were different from indigo.The contents of indirubin were increased obviously,when treated with YE and MJ.When the plant was treated with SA,the contents of indirubin were increased quickly in the early time,but inhibited on 4th or 7th day.The plants were damaged obviously,and the contents of indirubin were decreased when treated with Cu2+ and Ag+,after treated for 1 day,but then increased in the later time.