Effect of different fertilization treatments on yield and secondary metabolites of Codonopsis pilosula / 中国中药杂志

The research studies the effect of different fertilization treatments on yield and accumulation of secondary metabolites of Codonopsis pilosula by using single factor randomized block design, in order to ensure reasonable harvesting time and fertilization ratio, and provide the basis for standardized cultivation of C. pilosula. According to the clustering results, the nitrogen fertilizer benefitted for the improvement of root diameter and biomass of C. pilosula. The phosphate fertilizer could promote the content of C. pilosula polysaccharide. The organic fertilizers could increase the content of lobetyolin. With the time going on, C. pilosula's yield, polysaccharide and ehanol-soluble extracts increased while the content of lobetyolin decreased. According to various factors, October is a more reasonable harvest period. Organic fertilizers are more helpful to the yield and accumulation of secondary metabolites of C. pilosula.

Preparation and vitality detection of protoplast in Salvia miltiorrhiza Bunge / 生物工程学报

We prepared protoplasts from Salvia miltiorrhiza Bunge suspension culture cells. Then, the protoplasts' vitality and functions were tested by fluorescein diacetate staining method and Fluo-3/AM flourescent probe. The optimal condition of protoplast isolation was Cellulase R-10 1.5%, Pectinase Y-23 0.3%, Macerozyme R-10 0.5%, 40 r/min 12 h, 600 r/min 5 min, and the protoplasts yield was 1.1x10(6) cells/g FW, the vitality was more than 95% by using fluorescein diacetate staining method. It has been confirmed that calcium fluorescent probe Fluo-3/AM can be successfully loaded into protoplasts.

Effects of Ca2+ on salicylic-acid induced biosynthesis of salvianolic acid B in young seedlings of Salvia miltiorrhiza Bunge / 生物工程学报

In order to study the effects of Ca2+ in the biosynthesis of salvianolic acid B (Sal B) induced by salicylic acid (SA) in the young seedlings of Salvia miltiorrhiza, we used confocal laser scanning microscopy and high performance liquid chromatography to measure the change of relative fluorescence intensity of Ca2+ and the contents of Sal B induced by SA before and after the application of extracellular calcium channel inhibitors (VP and LaCl3), intracellular calcium channel inhibitor (LiCl), as well as intracellular calmodulin antagonist (TFP). SA could induce the calcium burst, and the Ca2+ peak could last to 2-3 min in the guard cells of S. miltiorrhiza, which prompted the biosynthesis of Sal B after the Ca2+ burst. Both Vp or LaCl3, and LiCl or TFP could inhibit the burst of Ca2+ and the biosynthesis of Sal B. The above results demonstrated that Ca2+ from the extracellular and the intracellular calcium store regulate the biosynthesis of Sal B elicited by salicylic acid in S. miltiorrhiz young seedlings.

Effect of calcium on medium alkalinization induced by salicylic acid in Salvia miltiorrhiza suspension cultures / 生物工程学报

We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.

Regulation effects of intracellular and extracellular Ca2+ on biosynthesis of rosmarinic acid induced by salicylic acid in young seedlings of Salvia miltiorrhiza / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of intracellular and extracellular Ca2+ on the biosynthesis of rosmarinic acid (RA) induced by salicylic acid in young seedlings of Salvia miltiorrhiza.</p><p><b>METHOD</b>Young seedlings of S. miltiorrhiza were used to select an optimal concentration of salicylic acid (SA), and then use the optimal concentration of SA to investigate the effects of extracellular Ca2+ channel inhibitors Verapamil, LaCl3, intracelluar calmodulin antagonist TFP and intracelluar Ca2+ channel inhibitors LiCl on the biosynthesis of RA and related enzymes.</p><p><b>RESULT</b>SA increased the accumulation of RA and the activities of PAL and TAT, especially the SA of 2 mmol x L(-1) after 24 h. SA improved the accumulation of RA to (40.51 +/- 2.16) mg x g(-1), which was 1.97 times than that of control, and the activities of PAL, TAT were 1.42 times and 1.29 times than those of the control. However, Vp, LaCl3, TFP, LiCl inhibited the effects of SA evidently.</p><p><b>CONCLUSION</b>Ca2+ plays a key role in the regulation of the induction process.</p>

Effects of salicylic acid on synthesis of rosmarinic acid and related enzymes in the suspension cultures of Salvia miltiorrhiza / 生物工程学报

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-a-aminooxy-beta-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914 +/- 0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 micromol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709 +/- 0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 micromol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.

Hydrogen peroxide is involved in the signal transduction of salicylic acid-induced salvianolic acid B biosynthesis in Salvia miltiorrhiza cell cultures / 生物工程学报

Hydrogen peroxide (H2O2), one of reactive oxygen species, is widely generated in many biological systems, and it mediates various physiological and biochemical process in plants. To investigate the role of H2O2 as a signaling molecule in the process of salicylic acid (SA)-induced Salvianolic acid B (Sal B) accumulation, we separately inspected the cultured cells of Salvia miltiorrhiza with SA, H2O2, catalase (CAT), 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (DMTU) and Imidazole (IMD) to investigate the influence on the activity of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) and the accumulation of Sal B. Treatment of S. miltiorrhiza cells with SA resulted in an increase of H2O2, the increase of PAL and TAT and accumulation of Sal B. Exogenous application of 10-30 mmol/L H2O2 was found to effectively increase PAL and TAT activity as well as the Sal B content. CAT, a H2O2 scavenger, eliminated the Sal B-accumulating effects of exogenous H2O2 and SA. These indicated that H2O2 may serve as an upstream signaling molecule in the SA-induced accumulation of Sal B signal transduction pathway. Disposed by DMTU, a chemical trap for H2O2, as observed to be effective in inhibiting SA-induced accumulation of Sal B. IMD strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H2O2 formation in plant cells. IMD treatment strongly inhibited the accumulation of Sal B in cultured cells of S. miltiorrhiza, but the effects of IMD, can be partially reversed by the exogenous SA. The accumulation of Sal B was blocked once the generation of H2O2 by NADPH oxidase was inhibited, and H2O2 served as signaling molecule mediated the SA-induced Sal B accumulation.

Effects of calcium on synthesis of rosmarinic acid and related enzymes in suspension cultures of Salvia miltiorrhiza / 生物工程学报

We studied the influence of the concentration of Ca2+ (0-50 mmol/L) in culture medium on the synthesis of rosmarinic acid (RA) and related enzymes in Salvia miltiorrhiza suspension cultures. Using verpamil (VP, a calcium channel antagonist) and ionophore A23187, we studied the mechanism of secondary metabolites of Salvia miltiorrhiza suspension cultures influenced by the concentration of Ca2+ in the culture medium. The synthesis of intracellular RA in 6-day incubation was significantly dependent on the medium Ca2+ concentration. At the optimal Ca2+ concentration of 10 mmol/L, a maximal RA content of 20.149 mg/g biomass dry weight was reached, which was about 37.3% and 20.4% higher than that at Ca2+ concentrations of 1 and 3 mmol/L, respectively. The variation of the activity of PAL and TAT, two key enzymes of the two branches of RA, could be affected by the concentration of Ca2+ in culture medium. The change of their activity occurred prior to the accumulation of RA, which suggested both of the key enzymes be involved in the synthesis of RA. Meanwhile, the enzymatic action of PAL was more distinct than TAT. The treatment of VP and A23187, respectively, indicated that the influence of RA affected by the concentration of Ca2+ in the culture medium was accomplished by the intracellular Ca2+, and the flow of Ca2+ from the extracellular to the intracellular environment could also participate in this process.

Effect of water stress on growth and accumulation of active components of Isatis indigotica / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of water stress on the growth and accumulation of active components of Isatis indigotica.</p><p><b>METHOD</b>By pot culture experiment,the characteristic indexes of water requirement, chlorophyll content, transpiration rate, photosynthesis, biomass and effective ingredient indirubin were studied under normal and water stress conditions, respectively.</p><p><b>RESULT</b>With the increase of the extent and duration of water stress, the quantity of water requirement, chlorophyll content, respiration rate, photosynthesis and biomass of I. indigotica were decreased, while water use efficiency increased.</p><p><b>CONCLUSION</b>It is a critical period of water requirement in July, high water content in soil and extreme water stress are not propitious to the accumulation of indirubin, appropriate water stress can stimulate indirubin accumulatio. The harvest of leaves of I. indigotica is optimal in July, while the harvest of root is appropriate in October. Yield and quantity of I. indigotica are improved when the field capacity is between 45% and 70%.</p>