It is reported that salvianolic acid B, a bioactive phenolic compound contained in the root of Salvia miltiorrhiza, exhibits a much stronger activity in free radical scavenging and antioxidance than those of vitamin E. When a conventional refluxing method is adopted to extract salvianolic acid B from the root, in which the materials are subjected to higher temperature and longer time, the yield of this phenolic compound is lower due to the possibility of its hydrolysis to tanshinol. However, a higher extraction yield can be achieved over a shorter time period and lower temperature when an ultrasound-assisted extraction method is used. This paper investigated the parameters influencing the extraction of salvianolic acid B. Factors such as extraction time, frequency of the ultrasound, the ratio of solvent to material, and types of extraction solvent were examined. A comparison was also conducted between conventional refluxing and ultrasound-assisted extraction. Results showed that the optimal parameters to extract salvianolic acid B from the root of S. miltiorrhiza were as follows: ultrasonic frequency: 45 Hz; solvent: 60% aqueous ethanol; extraction temperature: 30 degrees C; extraction time duration: 25 min.; ratio of solvent to material: 20:1 (v/w, ml/g). Under these conditions, the yield of salvianolic acid B was 5.17 mg/g (33.93 mg/g) higher than those with conventional refluxing method (28.76 mg/g), indicating that the efficiency and the yield of ultrasound-assisted extraction method are higher than reflux method, and the hydrolysis of salvianolic acid B to tanshinol is effectively avoided.

Jasmonates (JAs) are important plant hormones that regulate a variety of plant development and defense processes, including secondary metabolite biosynthesis. The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the JA signaling pathways of plants. We first verified that methyl jasmonate (MeJA) enhanced the accumulation of both salvianolic acids and tanshinones in Salvia miltiorrhiza (Danshen) hairy roots by inducing the expression of their biosynthetic pathway genes. Nine JAZ genes were cloned from Danshen and their expression levels in hairy roots were all elevated by treatment with MeJA. When analyzed in detail, however, SmJAZ8 showed the strongest expression in the induced hairy roots. Overexpression or RNA interference of SmJAZ8 deregulated or up-regulated the yields of salvianolic acids and tanshinones in the MeJA-induced transgenic hairy roots, respectively, and the transcription factors and biosynthetic pathway genes showed an expression pattern that mirrored the production of the compounds. Genetic transformation of SmJAZ8 altered the expression of other SmJAZ genes, suggesting evidence of crosstalk occurring in JAZ-regulated secondary metabolism. Furthermore, the transcriptome analysis revealed a primary–secondary metabolism balance that was regulated by SmJAZ8. Altogether, we propose a novel role for SmJAZ8 as a negative feedback loop controller in the JA-induced biosynthesis of salvianolic acids and tanshinones.

Salicylic acid (SA) is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H₂O₂) plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H₂O₂in rosmarinic acid (RA) production inSalvia miltiorrhizacell cultures were investigated. The results showed that SA significantly enhanced H₂O₂production, phenylalanine ammonia-lyase (PAL) activity, and RA accumulation. Exogenous H₂O₂could also promote PAL activity and enhance RA production. If H₂O₂production was inhibited by NADPH oxidase inhibitor (IMD) or scavenged by quencher (DMTU), RA accumulation would be blocked. These results indicated that H₂O₂is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation.