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Determination of Triterpene Increment in Calli of Poacynum Hendersonii (hook. F.) Woodson

Author: XingSiLei
Tutor: JiQiaoLing
School: Xinjiang University
Course: Cell Biology
Keywords: Large leaf white linen Callus Triterpenoids HPLC Oleanolic acid Ursolic Antibacterial activity
CLC: R284
Type: Master's thesis
Year: 2010
Downloads: 57
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Abstract

Large leaf white linen (Poacynum hendersonii (Hook. f.) Woodson) is Apocynaceae (Apocynaceae) white linen genus (Poacynum Baill) a perennial herb. Purpose of this study is how to use large leaf white linen to produce callus triterpenoids. First, large-leaved white linen hypocotyls explants, callus induction explored the basic conditions and different levels of callus growth hormone status; HPLC method was established big-leaf white linen callus bearberry acid, oleanolic acid, a method using L9 (34) orthogonal experimental design optimization of large-leaved white linen callus ursolic acid, oleanolic acid extraction process; to MS 6-BA 0.2 mg / L NAA0.6 mg / L as the basic medium of different explants cultured days, subculture, photoperiod, medium carbon and additives for large leaf white linen callus growth factor and triterpenoids compound accumulation; filter paper patch method and tube MIC assay were big white hemp leaf callus triterpenoids against E. coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis ( Bacillus subtilis), Aspergillus niger (Aspergillus niger) and Penicillium (Penicillium chrysogenum) inhibitory effect. Specific results are as follows: 1. White linen with large leaves hypocotyls of material, in MB NAA0.6 mg / L 6-BA0.6 mg / L, MB NAA0.6 mg / L 6-BA0.2 mg / L and MB NAA0.2 mg / L 6-BA0.6 mg / L three media on callus induction rate reached 90%. The medium MB NAA0.6 mg / L 6-BA0.2 mg / L compared for the amplification of callus 2 by HPLC analysis triterpenoids extract column was C18-ODS column ( 150 mmx4.6mm), column temperature was 25 ℃, the detection wavelength was 215 nm, mobile phase was methanol -0.1% phosphoric acid (85:15, v / v), flow rate of 0.6 mL / min at a better separation efficiency, Bear acid concentration of 0.08-0.40 mg / mL and the peak area within the linear regression equation was Y = 584410X 230003 (r = 0.9998); oleanolic acid concentration of 0.12-0.60 mg / mL range and the peak area was linear regression equation Y = 9870608X 165283 (r = 0.9997). Oleanolic acid and ursolic acid retention time was 24.245 and 25.316 minutes, a resolution of 1.52, to meet the qualitative identification and quantitative analysis. Large leaf white linen calli triterpenoids optimum extraction conditions were: 70 ℃ reflux extraction with methanol 1.0 h, solid-liquid ratio of 1:16. The greatest impact on the extraction solvent extraction, followed by the temperature, the amount of extraction, the extraction time with minimal impact. Under the optimum conditions, the measured ursolic acid and oleanolic acid average content of 1.2 mg / g and 3.4 mg / g. 3. Investigation of culture conditions on a single large leaf white linen callus growth and accumulation of triterpenoids as follows: (a) different explants on callus callus growth and production of triterpenoids impact quite different. Of which the following hypocotyl callus derived growth factor and triterpenoids largest output, respectively, 7.4 times and 5.2 mg / g; (2) in cultured callus growth factor and the first 20 d triterpenoids production are maximum, respectively, 7.2 times and 5.6 mg / g; (3) callus growth in the first three generations multiples largest triterpenoids production reached its maximum, respectively, 7.3 times and 5.4 mg / g; ( 4) four kinds of photoperiod treatment, 16 h light / d lower the amount of callus growth and triterpenoids highest yield, respectively, 7.2-fold and 5.2 mg / g; (5) than sucrose, glucose, fructose more callus growth and biomass accumulation of triterpenoids to additional 5% sucrose best, were 8.3-fold and 7.2 mg / g; (6) of the three medium containing additives to add 2 mg / L salicylic acid on callus growth medium largest amount of triterpenoids also the highest yields were 7.2 times and 6.8 mg / g. Taking all these medium optimal culture conditions, callus growth factor and triterpenoids production was 8.3-fold and 7.6 mg / g. 4 large white hemp leaf callus extract triterpenoid inhibitory effect of each species are as follows: Staphylococcus aureus gt; E. gt; Bacillus subtilis gt; Penicillium gt; Aspergillus niger. In our experimental conditions, the three terpene substances Escherichia coli, Staphylococcus aureus and Bacillus subtilis minimum inhibitory concentrations were 0.91,1.82 and 3.65mg/mL, Aspergillus and Penicillium on the minimum inhibitory concentration was 7.31 mg / mL.

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