PEG and ABA Trigger the Burst of Reactive Oxygen Species to Increase Tanshinone Production in Salvia miltiorrhiza Hairy Roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Tanshinones, a group of active compounds in S. miltiorrhiza, are derived from two biosynthetic pathways: the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastids. Water stress is well known to stimulate the accumulation of secondary metabolites in plants. Reactive oxygen species (ROS) serve as important secondary messengers in water stress-induced signal transduction pathways. In this study, the effects of polyethylene glycol (PEG) and abscisic acid (ABA) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of ROS in PEG- and ABA-induced tanshinone production were further elucidated. The results showed that contents and yields of four tanshinones in S. miltiorrhiza hairy roots were significantly enhanced by 2 % PEG and 200?μM ABA. Simultaneously, the mRNA levels and activities of two key enzymes (3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose 5-phosphate synthase) involved in tanshinone biosynthesis were upregulated. Both PEG and ABA were able to trigger the burst of H₂O₂ and O₂ ^?. The PEG- and ABA-induced increases of tanshinone production, gene expression, and enzyme activity were all dramatically suppressed by two ROS scavengers, catalase and superoxide dismutase. In addition, ROS treatments resulted in a significant increase in tanshinone production. These results demonstrated that the MVA and MEP pathways were activated by PEG and ABA to stimulate tanshinone biosynthesis, and the increase of tanshinone production was probably via ROS signaling.     

PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots

Tanshinones, a group of active ingredients in Salvia miltiorrhiza, are derived from at least two biosynthetic pathways, which are the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the plastids. Abscisic acid (ABA) and methyl jasmonate (MJ) are two well-known plant hormones induced by water stress. In this study, effects of polyethylene glycol (PEG), ABA and MJ on tanshinone production in S. miltiorrhiza hairy roots were investigated, and the role of MJ in PEG- and ABA-induced tanshinone production was further elucidated. The results showed that tanshinone production was significantly enhanced by treatments with PEG, ABA and MJ. The mRNA levels of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS), as well as the enzyme activities of HMGR and DXS were stimulated by all three treatments. PEG and ABA triggered MJ accumulation. Effects of PEG and ABA on tanshinone production were completely abolished by the ABA biosynthesis inhibitor [tungstate (TUN)] and the MJ biosynthesis inhibitor [ibuprofen (IBU)], while effects of MJ were almost unaffected by TUN. In addition, MJ-induced tanshinone production was completely abolished by the MEP pathway inhibitor [fosmidomycin (FOS)], but was just partially arrested by the MVA pathway inhibitor [mevinolin (MEV)]. In conclusion, a signal transduction model was proposed that exogenous applications of PEG and ABA triggered endogenous MJ accumulation by activating ABA signaling pathway to stimulate tanshinone production, while exogenous MJ could directly induce tanshinone production mainly via the MEP pathway in S. miltiorrhiza hairy roots.     

Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures

 Salvia miltiorrhiza contains two groups of biologically active secondary metabolites termed phenolic compounds (e.g. rosmarinic acid) and tanshinones (e.g. cryptotanshinone). Their roles in plant defense responses were examined using a simplified system consisting of a yeast elicitor and a Ti C58 transformed S. miltiorrhiza cell line. Both dosage and time course studies were carried out on the effects of yeast elicitor on the formation of rosmarinic acid and cryptotanshinone. It was found that the yeast elicitor reduced the constituent level of rosmarinic acid (from ca. 5% to ca. 3.0% of dry cell weight) whereas the level of cryptotanshinone was enhanced greatly (from a negligible amount to ca. 20 mg/l). These results suggest that in S. miltiorrhiza, rosmarinic acid and cryptotanshinone may take part in plant passive and active defense responses, respectively, against pathogen attack. Cryptotanshinone was identified as a phytoalexin in S. miltiorrhiza for the first time. Results of the treatment of cell cultures with 2-aminoindan-2-phosphonic acid, a highly specific and potent inhibitor of phenylalanine ammonia-lyase (EC 4.3.1.5), indicated that this compound did not inhibit yeast elicitor induced tanshinone formation, but did inhibit rosmarinic acid biosynthesis. Received: 22 May 1999 / Revision received: 27 August / Accepted: 1 September 1999     

Molecular characterization and expression of 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose 5-phosphate reductoisomerase (DXR) gene from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plants. The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255). Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. Phylogenetic tree analysis indicated that SmDXR belongs to the plant DXR superfamily and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression pattern analysis revealed that SmDXR expressed strongly in leaves, followed by roots and stems, implying that SmDXR was a constitutively expressed gene. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in tanshinone biosynthetic pathway in Salvia plants. The expression profiles revealed by RT-PCR under different elicitor treatments such as methyl jasmonate (MJ) and salicylic acid (SA) were compared for the first time, and the results revealed that SmDXR was an elicitor-responsive gene, which could be induced by SA in leaves and inhibited by exogenous MJ in three tested tissues. The functional color assay in Escherichia coli showed that SmDXR could accelerate the biosynthesis of lycopene, indicating that SmDXR encoded a functional protein. The characterization, expression profile and functional analysis of SmDXR gene will be helpful for further study in the role of SmDXR in tanshinones biosynthetic pathway and metabolic engineering to increase tanshinones production in S. miltiorrhiza.     

Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate reductoisomerase

Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g^?1 DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag⁺ than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g^?1 DW by YE, Ag⁺, and YE-Ag⁺ treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.     

Effect of yeast elicitor and salicylic acid on the fluctuation of phytohormone contents in Ti-transformed Salvia miltiorrhiza cell cultures

When Ti transformed Salvia miltiorrhiza cells werecultured in a MS-NH₄ medium (MS without ammonium nitrate, containing30 g/L sucrose) at 25 °C in darkness for 18d, the total tanshinone (cryptotanshinone and tashinone IIA)contents in cultures were 12.23 mg/L and 15.07 mg/Lfor yeast elicitor (4 g/L), and yeast elicitor plus 200mol/L salicylic acid (SA) treated cultures, respectively,whereas only trace amounts of tanshinone were detected in the control or SAtreated cells. To explore the hormonal background concerning these phenomena,endogenous phytohormones were determined using ELISA kits. We found that ABA andiPAs contents in yeast elicitor plus SA treated cell cultures were increased 2.8to 9.8-fold and 3.6 to 5.8-fold respectively, while contents of GA₁and IAA were decreased by 13.2%–56.9% and 34.8%–74.6% respectively.This suggests that higher levels of ABA and iPAs combined with lower levels ofGA₁ and IAA inhibit the growth of cells, then probably stimulate thetanshinone production.     

Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures

This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co²⁺, Ag⁺, Cd²⁺), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g^-1 versus 0.2 mg g^-1 in control). The stimulating effect was concentration-dependent, most significant at 25 μM of Ag and Cd and 100 mg l^-1 (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1–5.5 g l^-1 versus 8.9 g l^-1 in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.     

Effect of nutritional factors on cryptotanshinone and ferruginol production by cell suspension cultures of Salvia miltiorrhiza

The effects of the components of Murashige-Skoog (MS) medium on the production of the diterpenes cryptotanshinone and ferruginol in cell suspension cultures of Salvia miltiorrhiza were examined. Sucrose, a nitrogen source and thiamine were needed for the production of these compounds, and phosphate, MnSO₄ and kinetin showed slight beneficial effects. All the other components of MS medium were found to be either unnecessary or inhibitory for the production of these compounds, when tested separately. A revised medium for the production of cryptotanshinone, a clinically active principle in this plant, was designed.     

Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae

Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans.     

In vitro inhibitory effects of ethanol extract of Danshen (Salvia miltiorrhiza) and its components on the catalytic activity of soluble epoxide hydrolase

Background: Soluble epoxide hydrolase (sEH) has been demonstrated to be a key enzyme involved in the pathologic development of several cardiovascular diseases and inflammation, and inhibition of sEH is therefore very helpful or crucial for the treatment of ischemia-reperfusion injury, cardiac hypertrophy, hypertension and inflammation. Danshen, the dried root of Salvia miltiorrhiza (Fam. Labiatae), has been used for the treatment of cardiovascular and cerebrovascular diseases in China and other countries for hundreds of years. Recent studies indicated that Danshen and its preparations also have potential for the management of inflammation. However, little information is available about the possibility of Danshen and its components on sEH inhibition.Purpose and methods: Danshen extracts and its constituents were tested for sEH inhibition using its physiological substrate, 8,9-EET, based on a LC–MS/MS assay in this study.Results: Among the tested 15 compounds, tanshinone IIA and cryptotanshinone were found to be the potent (K_i = 0.87 μM) and medium (K_i = 6.7 μM) mixed-type inhibitors of sEH, respectively. Salvianolic acid C (K_i = 8.6 μM) was proved to be a moderate noncompetitive sEH inhibitor. In consistent with the inhibition results of the pure compounds, the 75% ethanol extract of Danshen (EE, IC₅₀ = 86.5 μg/ml) which contained more tanshinone IIA and cryptotanshinone exhibited more potent inhibition on sEH than the water extract (WE, IC₅₀ > 200 μg/ml) or 1 M NaHCO₃ (BE, IC₅₀ > 200 μg/ml) extract.Conclusion: These data indicated that using the ethanol fraction of Danshen and increasing the amounts of tanshinone IIA, cryptotanshinone and salvianolic acid C, especially the contents of tanshinone IIA in Danshen extract or preparations to enhance the inhibitory effects on sEH might be efficient ways to improve its cardiovascular protective and anti-inflammatory effects, and that herbal medicines could be an untapped reservoir for sEH-inhibition agents and developing sEH inhibitors from the cardiovascular protective and anti-inflammatory herbs is a promising approach.     

Overexpression of a chrysanthemum transcription factor gene <Emphasis Type="Italic">DgNAC1</Emphasis> improves drought tolerance in chrysanthemum

Phenolic acids and tanshinones are two groups of pharmaceutical components present in Salvia miltiorrhiza Bunge. Methyl jasmonate (MeJA) has been reported to influence the accumulation of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. However, there is currently a lack of information regarding the comparison of how these two groups of bioactive compounds in S. miltiorrhiza respond to MeJA under the same conditions. In the present study, the effect of 100 µM MeJA on the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza hairy roots was investigated. The results showed that MeJA dramatically induced the accumulation of five different phenolic acids, especially rosmarinic acid and salvianolic acid B, which reached their highest contents at day 3 (20.3 mg/g DW, 1.5-fold of control) and day 6 (47.49 mg/g DW, 2.5-fold of control), respectively. The total production of phenolic acids was induced by as much as 3.3-fold of the control (day 9 after treatment), reaching 357.5 mg/L at day 6. However, tanshinone I was almost unaffected by MeJA treatment, and the accumulation of tanshinone IIA was inhibited. Furthermore, cryptotanshinone and dihydrotanshinone I were moderately induced by MeJA. The gene expression results indicated that MeJA probably induced the whole pathways, especially the tyrosine-derived pathway and the methylerythritol phosphate pathway, and finally resulted in the increased production of these metabolites. This study will help us to further understand how the different biosynthetic mechanisms of phenolic acids and tanshinones respond to MeJA and provide a reference for the future selection of elicitors for application to improving the production of targeted compounds.     

Determination of Tanshinones in Danshen (Salvia miltiorrhiza) by High‐Performance Liquid Chromatography with Fluorescence Detection after pre‐Column Derivatisation

Introduction

Tanshinones are a major class of bioactive ingredients in the traditional herbal medicines, Danshen (Salvia miltiorrhiza). A sensitive and reliable determination method for tanshinones is useful to ensure the quality of Danshen.

Objective

To develop a sensitive and selective analytical method for tanshinones by high‐performance liquid chromatography (HPLC) with fluorescence detection after pre‐column derivatisation.

Methodology

The proposed method depends on derivatisation reaction of tanshinones with 4‐carbomethoxybenzaldehyde and ammonium acetate forming intensely fluorescent imidazole derivative.

Results

The proposed method provided excellent sensitivity with the detection limits of 3.3 nM (66 fmol/injection), 3.2 nM (64 fmol/injection) and 2.0 nM (40 fmol/injection) for cryptotanshinone, tanshinone I and tanshinone IIA, respectively, without the necessity of complicated instrumentations. The developed method is successfully applied to quantify the contents of tanshinones in Danshen.

Conclusion

The developed method is the first analytical method for tanshinones by fluorescence detection. Since the derivatisation reaction is selective for the o‐quinone structure of tanshinone, the developed method will become a suitable mean for the discovering of tanshinone type diterpenoids from herbal samples. Copyright © 2017 John Wiley & Sons, Ltd.     

Enhanced secondary metabolite (tanshinone) production of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots in a novel root–bacteria coculture process

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g⁻¹ dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l⁻¹). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.     

Induction and potentiation of diterpenoid tanshinone accumulation in Salvia miltiorrhiza hairy roots by β-aminobutyric acid

The non-protein amino acid -aminobutyric acid (BABA) is a proven inducer of plant defense against pathogens. This work examines its effect on the production of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures, both separately and in combination with a yeast elicitor (YE, the carbohydrate fraction of yeast extract). In the absence of YE, BABA at 0.1, 1 and 2 mM caused a dose-dependent enhancement of tanshinone accumulation, with up to a 4.5-fold increase (from 0.24 to 1.09 mg/g DW) in total content of three major tanshinones (cryptotanshinone, tanshinone I and tanshinone IIA) in the hairy roots. The combination of BABA with YE treatment further enhanced tanshinone production, but only when the BABA treatment was applied to the culture a few days before the YE treatment. Compared with methyl jasmonate, BABA was more effective in enhancing tanshinone production. A 3-day pretreatment with 1 mM BABA followed by YE-treatment, increased the total tanshinone content of roots by 9.4 times to 2.26 mg/g cells, and the volumetric tanshinone yield of culture by 6.3 times (from 3.2 to 20.1 mg/l). The results suggest that BABA can strongly potentiate elicitor-induced secondary metabolism in plant tissue cultures.     

Plant density-dependent variations in bioactive markers and root yield in Australian-grown Salvia miltiorrhiza Bunge

The plant density‐dependent variations in the root yield and content, and the yield of biomarkers in Australian grown Salvia miltiorrhiza Bunge , a commonly used Chinese medicinal herb for the treatment of cardiovascular diseases, were investigated in a field trial involving six different plant densities. The key biomarker compounds cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B were quantified by a validated RP‐HPLC method, and the root yields were determined per plant pair or unit area. There were significant variations (p<0.05) in the root yields and contents and the yields of the biomarkers between the different plant densities. Positive linear correlations were observed between the contents of the three tanshinones, whereas negative linear correlations were revealed between the contents of the tanshinones and salvianolic acid B. The highest root yield per plant pair was achieved when the plants were grown at 45×30 cm or 45×40 cm, whereas the highest root production par unit area was obtained for a plant density of 30×30 cm. The highest contents of the three tanshinones and the most abundant production of these tanshinones per unit area were achieved when the plants were grown at 30×30 cm. However, the highest content of salvianolic acid B was found for a density of 45×40 cm, while its highest yield per unit area was obtained for densities of 30×40 cm or 45×30 cm. The findings suggest that the plant density distinctly affects the root yield and content and the yield of tanshinones and salvianolic acid B in Australian grown S. miltiorrhiza, which may be used as a guide for developing optimal agricultural procedures for cultivating this herb.     

Production of cryptotanshinone and ferruginol by immobilized cultured cells of Salvia miltiorrhiza

The production of the diterpenes cryptotanshinone and ferruginol by immobilized cultured cells of Salvia miltiorrhiza was examined. Cryptotanshinone and ferruginol were produced continuously by the immobilized cells. Much of the cryptotanshinone was released into the medium, while most of the ferruginol was retained in the cells. The production of cryptotanshinone and ferruginol by the immobilized cells was about 39% and 61% of those by cell suspensions. Re-use of the immobilized cells for the production of these compounds was also examined.