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Production of Paeoniflorin and Albiflorin From Paeonia Lactiflora. Pall Callus Tissue:Induction and

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Tutor: MeiLeHe
School: Zhejiang University
Course: Biochemical Engineering
Keywords: Paeonia lactiflora Pall,Callus,Albiflorin,Paeoniflorin,Plant cell culture,Optimi
CLC: R284.2
Type: Master's thesis
Year:  2014
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Abstract:
Paeoniflorin(PF) and albiflorin(AF) are important physiologically active substances extracted and derived from natural herbs Paeonia. Modern pharmacological experiments prove that they have anti-inflammatory, analgesic, and hepatoprotective effects and can be used to cure autoimmune disease, especially rheumatoid arthritis.This paper launched a series of studies about callus induction and culture by plant callus culture techniques for the preparation of PF and AF. Single-factor experiment and a variety of statistical methods were used to optimize callus subculture conditions and subculture medium. It offers the advantages of producing a large amount of useful secondary metabolites under controlled conditions in a shorter period of time and with relatively lower cost.Paeonia lactiflora Pall callus was successfully induced culture from different explants (root, stem, petiole, and leaf) and the optimum conditions were determined for callus induction by investigating the effects of induction conditions and explant types on callus induction efficiency. Addition, HPLC analysis for total content of PF and AF in callus and explants was established. The column packed with SinoChrom ODS-BP (4.6mm×250mm,5μm) was developed with acetonitrile and0.05%(w/v) potassium dihydrogen phosphate solution (11:89, v/v) as the flow phase, and at a flow of1.0ml/min. An aliquot of20μL of the supernatant was injected, and the total content of PF and AF were detected in UV absorption at230nm. Compared with their parent explants, RP-HPLC analysis showed that all calli had higher total levels of paeoniflorin and albiflorinSingle-factor experiment was used to study the influence of different types of basic medium, medium nutrients, carbon, trace elements, pH value, light and other culture conditions on callus growth and synthesis of secondary metabolites. Then the optimum conditions obtained were medium, WPM+0.5mg/L6-BA+1.0mg/L NAA +0.1mg/L TDZ; pH,2.5; cultural temperature,24℃and light intensity,15001x; the optimal incubation period,40days. Sucrose as carbon source is better than others and application of Na2SeO3can significantly increase the accumulation of PF and AF.The statistical method combined PB design, the steepest ascent method and CCD were used to optimize and evaluate the medium components and main external conditions for paeoniflorin and albiflorin production. A better optimization results were achieved that the yield of total glycosides was in agreement with predicted value, which increased by33%than that with initial culture conditions.
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