Suspension Culture System Establishment Of Licorice Cell And Analysis Of Active Ingredients In Culture Cell

Glycyrrhiza inflata Batal (licorice) is called"king of herbs", which takes up the first position in hundreds of traditional Chinese medicine in"Compendium of Materia Medica". As a result of the increasing need of Glycyrrhiza inflata Batal in recent years, wild Glycyrrhiza inflata Batal resources was over-exploited, facing a severe situation of exhaustion. However, plant cell culture was developed as an ideal solution for the current crisis in the exploitation of medicinal plant resources. The present study was mainly focused on the establishment of Glycyrrhiza inflata Batal cell suspension culture system, the kinetic analysis of Glycyrrhiza inflata Batal cell growth and nutrient consumption, and the establishment of the fingerprint detection of active ingredients in Glycyrrhiza inflata Batal. The results were mainly shown as follows:(1) Glycyrrhiza inflata Batal cell suspension culture system were established through explants screening,medium selection and culture condition optimization.The result showed the hypocotyledonary axis in Glycyrrhiza inflata Batal was the most suitable explant material for embryogenic callus induction, with MS+2, 4-D (1.0 mg L^-1)+6-BA (1.0 mg L^-1) +NAA (1.0 mg L^-1) as the induction medium and the induction rate reaching over 90%. However, in medium containing MS+NAA (0.5 mg L^-1)+2, 4-D (0.5 mg L^-1)+6-BA (0.5 mg L^-1), the callus displayed its fastest growth rate and best growth state. Therefore, a stable Glycyrrhiza inflata Batal cell suspension culture system were established in which the MS medium, 10% inoculum concentration, pH 5.8 as the initial culture pH were selected.(2) The kinetic analysis of cell growth and nutrient consumption during Glycyrrhiza inflata Batal cell suspension culture was performed. The pH value of the liquid culture medium was found to be maintained between 4.5～6.0. The S-shaped cell growth curve was demonstrated in accordance with the consumption of carbon, nitrogen and phosphorus and changes in electrical conductivity. The growth cycle for Glycyrrhiza inflata Batal cell suspension culture was 21 days, in 18th of which the biomass achieved its maximum value.(3) The fingerprint detection method of active ingredients in Glycyrrhiza inflata Batal was also established. The results showed the glycyrrhiza acid content in Glycyrrhiza inflata Batal suspension cultured cells was 1.138 mg g^-1. 86% similarity of glycyrrhizic acid in HPLC profiles were revealed between cultured cells and wild Glycyrrhiza inflata Batal plants. Total glycyrrhiza flavonoids were extracted from the cultured cells, the maximum content of which reached 6.7 mg g^-1, almost 0.67% of the weight of dried cells. The total flavonoid content in the Glycyrrhiza inflata Batal plants was 3.53%. 75.2% similarity were revealed according to the similarity evaluation by HPLC profiles.