Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis / 中国天然药物

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Accumulation of biomass and four triterpenoids in two-stage cultured Poria cocos mycelia and diuretic activity in rats / 中国天然药物

Poria cocos (Schw.) Wolf, an important medicinal and food fungus, is well known in East Asia. Due to growing market demand, long cultivation period, and consumption of pine trunk during cultivation, developing alternative methods for producing P. cocos and/or its active components is of interest. In the present study, the effects of different culture methods on biomass and accumulation of four triterpenoids were investigated. The ethanol extract of fermented mycelium (EFM) was orally administered to rats. Urine output and concentrations of electrolytes (Na, K, and Cl) were measured. Our results showed that mycelia grew better under continuous shaking culture condition (7.5 g DW·L), and higher triterpenoid levels were accumulated in two-stage culture (112 mg·L, 2.03%). The optimal starting time of static culture for triterpenoid yield was 4 d after shaking culture. Single administration of middle and high dose of EFM significantly increased urine output, Na and Cl excretion, and Na/K ratio. These results suggested that ethanol extract of cultured mycelia showed significant diuretic activity in rats and two-stage culture of P. cocos could be an alternative way to produce mycelia and triterpenoids.

Effects of feeding tyrosine and phenylalanine on the accumulation of phenylethanoid glycosides to Cistanche deserticola cell suspension culture / 中国天然药物

AIM@#To investigate the effects of feeding phenylalanine (Phe) and tyrosine (Tyr) on the accumulation of total phenolic compounds and four phenylethanoid glycosides (PeGs) to a cell suspension culture of the parasitic plant Cistanche deserticola.@*METHOD@#A cell suspension culture of C. deserticola was established and precursors of different concentrations were fed. In each group, the cell was sampled at the 24(th) day after inoculation. The content of total phenolic compounds and four PeGs compounds were determined using the Folin-Ciocalteu method and an HPLC method, respectively.@*RESULTS@#In the Phe fed cells, the maximum PeGs yield was achieved when Phe was fed at 1.5 mmol·L(-1) and the yield reached 1.13 times the control cell concentration. In the Tyr fed cells, the maximum yield of PeGs was 1.60 times of control when 0.75 mmol·L(-1) Tyr was fed to the cells. Furthermore, it was found that the salidroside yield was 4.01 times of control group when 5 mmol·L(-1) Tyr was fed.@*CONCLUSION@#Tyr is a better precursor for PeGs accumulation compared with Phe, and the rate limiting enzymes might be involved in the Tyr branch.

Effects of lovastatin, clomazone and methyl jasmonate treatment on the accumulation of purpurin and mollugin in cell suspension cultures of Rubia cordifolia / 中国天然药物

AIM@#To determine the IPP origin of the naphthoquinones (NQs) in Rubia cordifolia, and to evaluate the effects of methyl jasmonate (MeJA) treatment, MEP, and MVA pathway inhibitor treatment on the accumulation of anthraquinones (AQs) and NQs in cell suspension cultures of R. cordifolia.@*METHODS@#Cell suspension cultures of R. cordifolia were established. Specific inhibitors (lovastatin and clomazone) and MeJA were supplied to the media, respectively. Treated cells were sampled every three days. Content determination of purpurin (AQs) and mollugin (NQs) were carried out using RP-HPLC. The yield of the two compounds was compared with the DMSO-supplied group and the possible mechanism was discussed.@*RESULTS@#Lovastatin treatment increased the yield of purpurin and mollugin significantly. Clomazone treatment resulted in a remarkable decrease of both compounds. In the MeJA-treated cells, the purpurin yield increased, meanwhile, the mollugin yield decreased compared with control.@*CONCLUSION@#The IPP origin of mollugin in R. cordifolia cell suspension cultures was likely from the MEP pathway. To explain the different effects of MeJA on AQs and NQs accumulation, studies on the regulation and expression of the genes, especially after prenylation of 1,4-dihydroxy-2-naphthoic acid should be conducted.

Dynamic accumulation of three main triterpenic acids in submerged cultivation mycelium of Poria cocos / 中国中药杂志

<p><b>OBJECTIVE</b>To study the dymamic accumulation of triterpenic acids production in submerged cultivation mycelium of Poria cocos.</p><p><b>METHOD</b>Liquid culture method of P. cocos was established and RP-HPLC was applied to determine the contents of three main triterpenic acids dehydrotumulosic acid (DTA), 3-epi-dehydrotumulosic acid (eDTA) and polyporenic acid C (PAC) in submerged cultivation mycelium P. cocos at different culture stages and the contents were compared with cultivated P. cocos. The HPLC method is as follows, column: Plastisil ODS (4.6 mm x 250 mm, 5 microm); mobile phase: ACN/0.5% phosphate (80:20); flow rate: 1.0 mL . min-1; detective wavelength: 242 nm.</p><p><b>RESULT</b>The maximum biomass occurred at the 8th d after inoluctation, however, the contents and yield of three compounds increased till the 17th day. The contents of three compounds were 1. 2% (DTA), 0. 42% (eDTA) and 1.0% (PAC) at the 17th day after inoculation, which were significantly higher than that in cultivated material [0.2% (DTA), 0. 12(eDTA) and 0. 16% (PAC) ]. Furthermore, a correlation analysis between the content ratios of three independent compounds was carried out. The results showed that DTA negatively correlated with eDTA and PAC, with R2 of 0. 857 6 and 0. 971 7, respectively, which suggested the role of DTA as an important intermediate in the biosynthesis of triterpenic acids in P. cocos.</p><p><b>CONCLUSION</b>The sum content of three main terpenoids in submerged cultivation mycelium P. cocos was 5. 55 times as that in cultivated material, which strongly suggested the possibility of fermentation in the production of medicinally important triterpenic acids in the future.</p>