Study On The Anthocyanin With Qualitative,Quantitative,Purification And The Induced Optimization Conditions From Lycium Ruthenicum's Leaves

Lycium ruthenicum Murr is a Lycium plant belonging to Solanaceae.It is widely distributed in arid areas of northwest China.Its fruit is atropurpureus and rich in anthocyanin.It has antioxidant,anti-aging and anti-radiation effects.Lycium ruthenicum Murr anthocyanin is a phenolic hydroxyl-containing flavonoid compound,mainly petunia anthocyanin,which is a functional component of Lycium ruthenicum Murr.However,Lycium ruthenicum Murr has low yield and the supply of its fruit is short of demand.It is unrealistic to obtain anthocyanin from Lycium ruthenicum Murr fruit.In addition,due to the variety and similar nature of Lycium ruthenicum Murr anthocyanin,the process of separating and obtaining high purity petunia anthocyanins from Lycium ruthenicum Murr fruits is complicated and difficult,which restricts the experimental research on physiological and biochemical activities of petunia anthocyanins.Based on this,this paper used the detached leaves of Lycium ruthenicum Murr as materials,so as to induce the detached leaves of Lycium ruthenicum Murr to compound anthocyanin by utilizing the principle of environmental stress induction and the tissue culture method.This provides a way to obtain major anthocyanins from Lycium ruthenicum Murr and prepare high purity petunia anthocyanins,and also provides an experimental reference for purification and preparation of anthocyanin compounds.In this paper,the optimal environmental conditions for the synthesis and accumulation of anthocyanin from detached leaves of Lycium ruthenicum Murr were optimized by orthogonal test.And using ultra-high performance liquid chromatography-mass spectrometry,the qualitative and quantitative analysis of anthocyanin induced by this method was carried out,and the separation and purification of anthocyanin induced by tissue culture were studied in this paper.The main research results are as follows:1.Studied on the optimum of conditions in inducing anthocyanin from the detached leaves of Lycium ruthenicum Murr.The seeds of Lycium ruthenicum Murr were pretreated at low temperature,sterilized conventionally and germinated aseptically.And after 30 days of seedling growth,the leaves were picked and cultured aseptically.L₉?3⁴?orthogonal test was used to optimize the induction conditions.Its results showed that the leaves were cultured for20 days under the conditions of temperature 20?and blue light irradiation in a culture medium containing 150mM/L sucrose and 1.0 mg/L ABA,and the leaves changed from green to red,and the highest color value was measured at 530nm.2.Qualitative analysis of anthocyanin synthesis induced from the leaves of Lycium ruthenicum Murr.According to UPLC-DAD-TQD analysis,combining the chromatography,mass spectrometry,ultraviolet analysis data from experiments and references obtained,there were mainly 4 kinds of anthocyanins in induced synthesis.Their were Pt-3-O-[rut-?p-c?-glu]-5-O-diglu,Dp-3-O-[rut-?p-c?-glu]-5-O-glu,Pt-3-O-[rut-?p-c?-glu]-5-O-glu and Mv-3-O-[rut?p-c?-glu]-5-O-glu respectively.The Pt-3-O-[rut-?p-c?-glu]-5-O-glu was accounting for 75%of the total induced synthesis.3.Studied on purification of anthocyanin induced from Lycium ruthenicum Murr leaves.The anthocyanin in leaves was passed through AB-8 macroporous resin,polyamide and dextran gel filling chromatography columns respectively,and the purity was monitored by high performance liquid chromatography.The results showed that the main anthocyanin Pt-3-O-[ru?p-cou?-glu]-5-O-glu in Lycium ruthenicum Murr fruit could be easily obtained after purification of anthocyanin induced by leaves,and its purity could reach 95%.4.Studied on quantitative analysis of anthocyanin generated by inducing.Vanillic acid-HCl colorimetry was used to measure the content of proanthocyanidins in Lycium ruthenicum Murr fruit and induced leaves,and the total anthocyanin content in induced leaves was 0.72±0.21g/100g,which was about 40%of the fruit.The total anthocyanin content in Lycium ruthenicum Murr fruit and induced leaves was measured by pH differential method,and the anthocyanin content in induced leaves was 0.96±0.28g/100g,which was about 30%of the fruit.UPLC-DAD-TQD method was used to measure the content of Pt-3-O-[ru?p-cou?-glu]-5-O-glu in anthocyanin induced from leaves,which was 6.56±0.25 mg/100g,that was about25%of fruit.Through the research of this subject,it is determined that the anthocyanin obtained by inducing the detached leaves of Lycium ruthenicum Murr is consistent with the anthocyanin of Lycium ruthenicum Murr fruit,mainly Pt-3-O-[rut-?p-cou?-glu]-5-O-glu.This study provides a new way to obtain lycium barbarum anthocyanin and a new way to prepare lycium barbarum anthocyanin,which can provide a reference for the comprehensive development and utilization of lycium barbarum.