Effects and action mechanisms of sodium fluoride (NaF) on the growth and cephalotaxine production of Cephalotaxus mannii suspension cells.

To explore the effects of NaF on the growth and cephalotaxine production of Cephalotaxus mannii suspension cells, NaF was added into the C mannii cell suspension cultures at day 15 of culture time. It is documented that NaF suppressed cell growth but enhanced cephalotaxine production. The largest yield obtained of cephalotaxine reached to 9.57mg/L when the cultures were treated with an appropriate dosage of 15mg/L NaF, which was 3.7 times that of the control cultures (2.58mg/L). Additionally, NaF markedly enhanced the activity of glucose 6-phosphate dehydrogenase (G6PDH) and reduced the level of hydrogen peroxide (H2O2) of cells. It was also found that NaF weakened the oxidative damage of cell membrane and led to lower content of malonyl dialdehyde (MDA) in NaF-treated cells compared with the control cells. The MDA content of NaF-treated cells decreased 91% compared to the controls. Although lower membrane lipid peroxidation in NaF-treated cells, its membrane permeability was higher than the control cells and showed a high product secret rate. What is more, NaF boosted the activity of phenylalanine ammonium-lyse (PAL), but did not burst a peak of PAL activity in the time curve of PAL activity. These results indicated NaF acted as an inhibitor of the Enbden Parnas (EMP) pathway, not as an elicitor to promote cephalotaxine production.

[Quantitative analysis of hinokiol from cell suspension cultures of Cephalotaxus fortunei].

A high performance liquid chromatography (HPLC) method was developed for the separation of secondary metabolites and quantitative analysis of hinokiol from cell suspension cultures of Cephalotaxus fortunei. The samples were prepared by extraction using methanol followed by partitioning between ammonium hydroxide and chloroform. The HPLC separation was achieved on an Apollo C18 column (250 mm x 4.6 mm, 5 microm) with gradient elution using methanol and water at 1 mL/min and 30 degrees C. The detection was carried out at 290 nm. A good linear correlation between the hinokiol peak area and mass concentration was observed over the mass concentration range of 0.012 5 - 0.2 g/L. The proposed method was applied to the determination of hinokiol in the actual samples with recoveries of 87.2% - 94.7% and with the relative standard deviations of 0.9% - 4.2%. This method is reliable and reproducible and is suitable for the analysis of hinokiol in plant cell cultures.

[Biotransformation of artemisinic acid by cell suspension cultures of Cephalotaxus fortunei and Artemisia annua].

OBJECTIVE: To investigate the biotransformation of artemisinic acid by cell suspension cultures of Cephalotaxus fortunei and Artemisia annua. METHODS: Artemisinic acid was added into to the media of the suspension cells of Cephalotaxus fortunei and Artemisia annua in their logarithmic growth phase. The biotransfromed product was detected with HPLC and isolated by silica gel column, Sephadex LH20 and ODS chromatography methods. The chemical structure of biotransformed product was elucidated on the basis of physical-chemical properties and spectroscopic data. Otherwise, the influence of co-cultured time on conversion ratio was investigated with HPLC. RESULTS: One biotransformed product, 3-alpha-hydroxyartemisinic acid, was obtained after two days of artemisinic acid administration to the suspension cells of Cephalotaxus fortunei and Artemisia annua. The optimal co-cultured time in suspension cells of Cephalotaxus fortunei was 2 days with the highest biotransformation rate of 8.42%, and in the case of Artemisia annua, it was 3 days and 3.95% respectively. CONCLUSION: It was the first time for the biotransformation of artemisinic acid to 3-alpha-hydroxyartemisinic acid by using cell suspension cultures of Cephalotaxus fortunei and Artemisia annua.