RESUMEN
Tetrandrine, a bioactive active compound mainly found in the roots of Stephania tetrandra, exhibits various pharmacological properties. In vitro hairy root (HR) culture may serve as a promising solution for the extraction of tetrandrine, overcoming the limitations of natural cultivation. The present study describes the consistent production of tetrandrine from S. tetrandra hairy roots induced by different strains of Agrobacterium rhizogenes. Cultivation in woody plant medium (WPM) resulted in the highest HR biomass (0.056â¯g/petri-dish) and tetrandrine content (7.28â¯mg/L) as compared to other media. The maximum HR biomass (6.95â¯g dw/L) and tetrandrine production (68.69â¯mg/L) were obtained in the fifth week of cultivation. The presence of ammonium nitrate (800â¯mg/L), calcium nitrate (1156â¯mg/L), sucrose (20â¯g/L) and casein (2â¯g/L) enhanced the tetrandrine production. Moreover, the fed-batch cultivation demonstrated that the NH4NO3 (1200â¯mg/L) was an important growth limiting factor that yielded the highest tetrandrine amount (119.59â¯mg/L). The cultivation of hairy roots in a mist trickling bioreactor for eight weeks was less (26.24â¯mg/L) than in the flask. Despite a lower tetrandrine yield observed in bioreactors compared to flask cultures, refining the growth medium and fine-tuning bioreactor operations hold promise for boosting tetrandrine yield.
Asunto(s)
Agrobacterium , Bencilisoquinolinas , Medios de Cultivo , Raíces de Plantas , Stephania tetrandra , Bencilisoquinolinas/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Raíces de Plantas/crecimiento & desarrollo , Agrobacterium/genética , Stephania tetrandra/metabolismo , Medios de Cultivo/química , BiomasaRESUMEN
Tetrandrine is the most effective small molecule that has been found to inhibit the Ebola virus. It is a typical bisbenzylisoquinoline alkaloid and is the main active ingredient in Stephania tetrandra. Metabolic engineering and synthetic biology are potential methods for efficient and rapid acquisition of tetrandrine. S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting step involved in the biosynthesis of tetrandrine. In this study, we identify S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase from S. tetrandra, which catalyzes the conversion of (S)-norcoclaurine to (S)-coclaurine. Four 6OMT-like genes were cloned from S. tetrandra. An in vitro enzyme assay showed that St6OMT1 could catalyze the conversion of (S)-norcoclaurine to produce (S)-coclaurine. St6OMT2 can catalyze the production of very few (S)-coclaurine molecules, accompanied by more by-products with m/z 300, compared to St6OMT1. The newly discovered 6OMTs will provide an optional genetic component for benzylisoquinoline alkaloid (BIA) synthetic biology research. This work will lay the foundation for the analysis of the biosynthetic pathway of tetrandrine in S. tetrandra.
Asunto(s)
Antivirales/metabolismo , Bencilisoquinolinas/metabolismo , Metiltransferasas/genética , Proteínas de Plantas/genética , Stephania tetrandra/genética , Secuencia de Aminoácidos , Metiltransferasas/química , Metiltransferasas/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Stephania tetrandra/enzimología , Stephania tetrandra/metabolismoRESUMEN
BACKGROUND/AIMS: Tetrandrine and Fangchinoline (Fcn) are two natural products that are found in Stephania tetrandra. Tetrandrine is a known anti-bladder cancer compound, but the effects of Fcn on bladder cancer have been previously unclear. In the present study, we focused on the anti-tumor effects of Fcn on bladder cancer. METHODS AND RESULTS: We treated T24 and 5637 bladder cancer cell lines with Fcn in vitro. We observed that Fcn inhibited the viability of bladder cancer cells in a concentration-dependent manner. The expression of PCNA, a biomarker of proliferation, was down-regulated. Fcn treatment induced both apoptosis and autophagy in bladder cancer cells, as shown by the increased cleavage of caspase-3, an up-regulated LC3-II/LC3-I ratio and the down-regulated p62 level. Blocking autophagy with 3-MA (3-Methyladenine) enhanced Fcn-induced apoptosis, indicating that Fcn-induced autophagy was adaptive. Additionally, we observed that Fcn treatment inhibited mTOR and reduced the intracellular ATP levels. The exogenous addition of methyl pyruvate (MP) to compensate metabolic substrates alleviated Fcn-induced apoptosis and autophagy. CONCLUSIONS: Our data indicated that Fcn caused an impairment in energy generation, which led to apoptosis and adaptive autophagy in bladder cancer. These results demonstrated that Fcn may be a potential candidate for use in the prevention and treatment of bladder cancer.