Study On Extraction And Detection Of Sinigrin And Establishment Of Brassica Campestris L. Cell Line With High Sinigrin Content

Glucosinolates are the generic name of a kind of glucose derivatives and hydrophilic secondary metabolites, which are contained in fifteen families and most important in crucifer. Sinigrin is the flavor ingredient and has some medical value, which is a kind of glucosinolates. Allyl isothiocynate is one of sinigrin degradation products. It has antitumous effect and is used for food additive and antibacterial agent.Chemical extraction and HPLC detection system were set up to detect sinigrin contents of twenty rape varieties, in which a high sinigrin content and stable variety was picked out to establish rape cell line with high sinigrin content. It was researched into the influences of some factors to sinigrin content in rape cell primarily by tissue culture. The main conclusions of this study were showed as follow:1. The best extraction conditions of sinigrin were chosen by unifactor experiment. Extraction agent: 70% methanol. Extraction temperature: 70℃. Extraction time: 30min. Extraction times: 3 times. Material to solvent ratio: 30:1.2. The best HPLC detection conditions of sinigrin were: mobile phase:methanol:H2O (0.005mol/LKCl)=9:91, flow rate: 0.5ml/min,column temperature: 30℃,wave length: 230nm.3. Contents of sinigrin in 20 kinds of rapeseed were determined. Concentration of sinigrin in Longyou No.5 was as high as19.38mg/g. Results of cross-breed were generally higher than commercial varieties.4. The best inducing condition of rape callus: The induced callus was much better in dark than in light. Leaves: MS+1.0mg/L2,4-D+2.0mg/L6-BA, stems: MS+ 0.2mg/L 2,4-D +3.0mg/L 6-BA, roots:MS+0.2mg/L2,4-D+0.5mg/L6-BA, embryos and endosperms: MS+0.5 mg/L 2,4-D+2.5 mg/L6-BA.5. Root and embryo were the best organs to induce rape callus with higher sinigrin content.6. The best media of rape secondary culture: MS+3.0 mg/L6-BA+0.5 mg/L2,4-D.7. The additions of five kinds amino acids didn't promote the rape callus multiplication. When the concentrations of Val were 2 mmol/L and 4 mmol/L, the sinigrin contents of rape callus were promoted obviously. The other four amino acids had no promotion to sinigrin content.8. Sucrose was the best carbon source of rape callus multiplication and the best addition concentration was 30g/L. The sinigrin content was the highest when the addition was 10g/L. With the increase of carbon source concentration, the sinigrin content descended. Lactose couldn't be used for callus growth.9. In the growth cycle of rape callus, the callus multiplication firstly increased and then decreased, which maximized in 34d. The sinigrin content came to the highest in 6d, and then decreased slowly. According to the gross production, the best harvest time was 34d to 36d.