Abstract
Main conclusion
Methyl jasmonate promotes the synthesis of rosmarinic acid in Salvia miltiorrhiza Bunge and Salvia castanea f. tomentosa Stib, and it promotes the latter more strongly.
Abstract
Salvia miltiorrhiza Bunge (SMB) is a traditional Chinese medicinal material, its water-soluble phenolic acid component rosmarinic acid has very important medicinal value. Salvia castanea f. tomentosa Stib (SCT) mainly distributed in Nyingchi, Tibet. Its pharmacological effects are similar to SMB, but its rosmarinic acid is significantly higher than the former. Methyl jasmonate (MJ) as an inducer can induce the synthesis of phenolic acids in SMB and SCT. However, the role of MJ on rosmarinic acid in SMB is controversial. Therefore, this study used SMB and SCT hair root as an experimental material and MJ as a variable. On one hand, exploring the controversial reports in SMB; on the other hand, comparing the differences in the mechanism of action of MJ on the phenolic acids in SMB and SCT. The content of related metabolites and the expression of key genes in the synthesis pathway of rosmarinic acid was analyzed by 1H-NMR combined with qRT-PCR technology. Our research has reached the following conclusions: first of all, MJ promotes the accumulation of rosmarinic acid and related phenolic acids in the metabolic pathways of SMB and SCT. After MJ treatment, the content of related components and gene expression are increased. Second, compared to SMB, SCT has a stronger response to MJ. It is speculated that the different responses of secondary metabolism-related genes to MJ may lead to different metabolic responses of salvianolic acid between the two.
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Introduction
Salvia miltiorrhiza Bunge (SMB, Danshen in Chinese), belonging to Labiate family (Kai et al. 2011; Shi et al. 2014), is an important traditional Chinese herbal plant with long history for medicine as well as healthy food (Shi et al. 2018). It has been used clinically to treat cardiovascular diseases (Wang et al. 2017a, b, 2018; Chen and Chen 2017; Fang et al. 2018) and displays anti-inflammation (Gao et al. 2018; Wu et al. 2018), neuro-protection (Di et al. 2018; Saroya and Singh 2018), cardio-protection (Hu et al. 2012; Weng et al. 2013) and anticancer activities (Wang et al. 2017a, b; Uto et al. 2018; Qiu et al. 2018; Hsieh et al. 2018). There are two major types of pharmacologically active compounds present in SMB, one is water-soluble phenolic acids (Ma et al. 2013), and the other is fat-soluble ketones (Kim et al. 2008). Salvia castanea Diels f. tomentosa Stib (SCT), formerly known as salvia villi, also known as "Nyingchi Salvia", mainly distributed in Nyingchi, Tibet. The effective ingredients of SCT roots are similar to SMB roots, and have long been used as a substitute for SMB to treat diseases in the local area (Ye et al. 2004; Sun et al. 2004). Compared with SMB, the water-soluble component rosmarinic acid is higher in SCT, but the salvianolic acid B is lower (Yang et al. 2009). Therefore, SCT has important significance as a medicine source of rosmarinic acid.
Some scholars have studied the biosynthetic pathways of phenolic acid components in SMB, and it is now generally believed that rosmarinic acid is the precursor of other more complex phenolic acid compounds. Ellis and Tower (1970) first proposed the possible source of rosmarinic acid in 1970. They fed Mentha haplocalyx Briq. with 14C radioisotope and speculated about the biogenic pathway of rosmarinic acid by separating different compounds from the young shoots. As a result, it is believed that phenylalanine and tyrosine are precursors of rosmarinic acid biosynthesis. Through the suspension culture of Plectranthus scutellarioides (L.) R.Br., Petersen and Simmonds (2003) comprehensively described the biogenic pathways of rosmarinic acid and related enzymes for the first time. They found that the biogenic pathway of rosmarinic acid is composed of two parallel branches, the phenylalanine branch and the tyrosine branch (Fig. S1). Afterwards, Di et al. (2013) fed the hairy root of SMB with phenylalanine labeled with 13C radioisotope, and corrected the metabolic flux of rosmarinic acid by liquid-mass spectrometry data analyze of different phenolic compounds in the hairy root (Fig. 1).
Methyl jasmonate (MJ) is a cyclopentanone derivative formed by the methylation of the chemical substance of jasmonic acid. It is named, because it is an important component of the flower volatiles of jasmine plants (Yan et al. 2015). It can regulate the growth and development of plants, cause cells to respond to stress, induce plants to express defense genes, synthesize defense substances, and form defense organs. It can also induce plants to produce alkaloids, terpenes, and phenols for human use (Zhu et al. 2013). MJ can induce plants to produce secondary metabolites: studying the effect of spraying MJ on pine acid, it was found that the content of coumaric acid, salicylic acid, ferulic acid, cinnamic acid and phenylacetic acid in the cones of the treatment group was higher than the control group (Wang and Yan 2012); spraying the leaves of tobacco plants with MJ can increase the nicotine content by two times (Dam et al. 2000). In addition, the effect of MJ on the active ingredient content and enzyme activity in SMB hairy root was studied (Xing et al. 2013; Xiong et al. 2018). However, the effect of MJ on rosmarinic acid is controversial. Studies have shown that MJ can significantly promote the synthesis of rosmarinic acid in SCT, but has almost no effect on SMB (Fang et al. 2017). Opposite studies have shown that MJ can promote the accumulation of rosmarinic acid (Li et al. 2013): Some evidences show that the accumulation of rosmarinic acid reaches the maximum after 24 h of MJ treatment (Li et al. 2012), and there are also studies prove that the accumulation of rosmarinic acid was the largest after treatment for 48 h by MJ (Xing et al. 2013).
Hairy root is a new technology that combines genetic engineering and cell engineering developed in the 1980s. It transforms the T-DNA contained in the Ri plasmid of agrobacterium rhizogenes into the DNA of plant cells (Yan et al. 2006), induce hairy root formation. Hairy roots are important materials for the research of root medicinal plants (Liu et al. 2015) and have important prospects in large-scale production (Wang et al. 2020). Hairy roots have a complete metabolic pathway in plant roots due to their convenient cultivation. It is an effective method for studying root medicinal materials and an effective way for large-scale production of active ingredients of Chinese medicine (Sun et al. 2014).
At present, NMR-based metabolomics and transcriptomics technologies to explore the biosynthesis of SMB phenolic acid have been applied (Liu et al. 2019). Based on the above background, this study has two purposes, one is to explore the above-mentioned controversial views, and the other is to compare the differences in the mechanism of action of MJ on SMB and SCT. We adopted 1H-NMR combined with qRT-PCR technology to study MJ treatment of SMB and SCT hairy roots, and the control group was treated with absolute ethanol. The content of cinnamic acid, caffeic acid, p-coumaric acid, l-tyrosine, l-phenylalanine, 4-hydroxyphenylpyruvic acid and rosmarinic acid (Fig. S2) were measured on 0, 1, 2, 3, 6, 9 days after treatment with MJ, combined with the expression level of key genes PAL, TAT, 4CL, C4H, RAS, HPPR, CYP98A14 in their metabolic pathways.
Materials and methods
Plant materials
Hairy root of Salvia miltiorrhiza Bunge and Salvia castanea f. tomentosa Stib were obtained from an earlier experiment in our laboratory. Both plants were obtained from Tasly Pharmaceutical Group Co. LTD (Tianjin, China). We transferred the cultures from solid medium to hormone-free MS liquid medium with 30 g L−1 sucrose and cultivated them at 25 °C in the dark. The induction experiment was conducted after stable growth reached. Specifically, induction was started 18 days after 3 g of hairy roots was inoculated into 250-mL Erlenmeyer flasks through the application of the abiotic elicitor MJ (0.1 mM) as previously described (Xiao et al. 2009). Hairy roots were harvested at 0, 1, 2, 3, 6 and 9 days post-induction.
Methods
MJ dosage and solution preparation
MJ is made up of 50% mother liquor with absolute ethanol as a co-solvent. Sterilize through a 0.22 μm microporous membrane and add to the medium to make the final MJ concentration 200 μmol L−1 (Wang et al. 2007). The control group added equal amount of absolute ethanol.
Sample preparation
SMB and SCT hairy roots with different induction time removed from the culture medium. The culture medium was washed away with tap water and blotted with absorbent paper. Take a part of the 40 °C oven to dry to constant weight, cool at room temperature, the hairy root after drying is used as the material for determining the content of phenolic acid active ingredients. Another part was wrapped in tin foil paper and then liquid nitrogen was cooled instantly and frozen in a − 80 °C refrigerator, as a material for measuring the expression of key genes.
1H-NMR determination of phenolic acids in hairy root of SMB
Grind the dried hairy roots of SMB and SCT to powder, add 70% methanol solution to soak overnight, then ultrasonically extract for 45 min, centrifuge at 3214g for 15 min, filter liquor was transferred into a clean Eppendorf tube and evaporated to dryness under nitrogen at 35 °C, then was added 600 μL D2O (contain proper amount of TMSP) to dissolve for NMR analysis. The NMR sample tube with sample was assembled for analysis at 298 K and sealed and prior to 1H-NMR measurement. Samples for NMR analysis were measured on a 600 MHz AVANCE III HD spectrometer with a TXI probe at 298 K. The solvent-suppression was used in the acquisition of NMR spectra. 1H-NMR experimental parameters were shown as follows: Lc1pnf2 pulse sequence (1D version of noesygppr using presaturation during relaxation delay and mixing time) with 32 scans of 32 K data points in a spectral width of 12,626.26 (21 ppm), acquisition time 5.0 s, relaxation delay 25 s (depending on the longest longitudinal relaxation time referring to the IS determined by the Bruker inversion recovery pulse program). All the data processing was performed by using MestReNova 11.0.
Internal standards
To test internal standards, the extracted sample M9 with 600 μL D2O, containing appropriate amount of TMSP, maleic acid and 3,4,5-trichloropyridine as IS were analyzed.
Quantitative NMR analysis
The most important fundamental relation of qNMR is signal response (integrated signal area) Ix in a spectrum that is directly proportional to the number of nuclei Nx generating the corresponding resonance line: (Malz and Jancke 2005; Gadape and Parikh 2011; Li et al. 2020):
Ks is an unknown spectrometer constant, which is a constant for all resonance lines in the same 1H single-pulse NMR spectrum. Accordingly, the determination of relative area ratios Ix/Iy is the most efficient way to obtain quantitative results using Eq. (2) when Ks cancels for the ratio:
For the purity determination of a substance an internal standard with known purity is needed. Based on Eq. (2), the component purity can be calculated from the NMR intensity via the following equations:
Wx and Px represent the mass and purity of the analyte. Mx and MStd are the molar masses of the analyte and the standard (TMSP 172.17 g mol−1). m is the weighed mass of the investigated sample. mStd and PStd are the weighed mass and the purity (99.5%) of the standard. NStd and IStd correspond to the number of protons for the standard (in this experiment is 9) and the integrated signal area of a typical NMR line (which was 9 in this experiment). Nx and Ix correspond to the number of protons for the analyte 1H.
qRT-PCR to measure gene expression of key genes
Total RNA for qRT-PCR analysis was extracted from frozen samples using polysaccharide polyphenol plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), following the manufacturer’s protocol. The yield and integrity of RNA were assessed using a NanoDrop Micro Photometer (Thermo Scientific, USA) and agarose gel electrophoresis, respectively. cDNA was synthesized from 500 ng of total RNA using a cDNA synthesis kit (Takara), following the manufacturer’s protocol. Specific primers for seven key genes to be detected were designed (Table 1) and synthesized by Zhejiang Youkang Biotechnology Co., Ltd. The internal reference gene used housekeeping gene 18S rRNA to design primers F18S (ATGATAACTCGACGGATCGC) and R18S (CTTGGATGTGGTAGCCGTTT), and the same template was used for qRT-PCR amplification of the participating samples.
PCR reactions (10 μL) included 1.5 μL cDNA, 0.4 μL of each primer, 5 μL SYBR Premix, 0.1 μL Rox Reference Dye II and 2.6 μL H2O. Reactions were performed using an ABI Quant Studio 6 Flex real-time PCR system (Applied Biosystems, USA) under the following conditions (Xing 2015): 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s, followed by 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s to obtain melt curves. The expression of each gene relative to average Ct values of the housekeeping genes was determined and analyzed using ABI 6 Flex System Sequence Detection Software (Applied Biosystems, USA). Quantification of the relative changes in gene transcript level was performed in accordance to the 2 − ΔΔCt method (Livak and Schmittgen 2001). For control samples (this refers to samples processed by MJ for 0 day), the mean relative expression level of the assayed gene was assigned a value of 1.0, and the relative expression level of all samples calculated relative to it. Results represent the mean of three biological replicates.
Results
Identification of metabolites in MJ-induced SMB hairy root cultures through 1H-NMR
Specificity and selectivity
The sample solutions were optimized to obtain the best separation and stability for all the integrated signals in 1H-NMR spectrogram. Quantification was performed by an NMR sample-tube adapter at 298 K. The result was found to give the desired signal separation of p-coumaric acid, l-phenylalanine, cinnamic acid, caffeic acid, l-tyrosine and 4-hydroxyphenylpyruvic acid (Fig. 2). As a necessary prelude to all the measurements, analyte and IS were analyzed qualitatively by routine 1H experiments to determine longest spin–lattice relaxation time, usually at least five times the T1, thus the optimized relaxation delay of 25 s was obtained. Based on the optimized NMR parameters, signals for cinnamic acid at 6.54 ppm, caffeic acid at 6.34 ppm, p-coumaric acid at 6.40 ppm, l-phenylalanine at 3.99 ppm, l-tyrosine at 7.18 ppm and 4-hydroxyphenylpyruvic acid at 6.88 ppm were selected as the quantification signals (Fig. 3). The TMSP was chosen as the IS due to its good solubility and stability.
Linearity, LOD and LOQ
The intensity of the response signal is directly proportional to the amount of nuclei, consequently, the linearity regression yielded a good correlation coefficient (r2 > 0.985). The concentration ratios of the six references ranged from 0.01 to 1.00 mg mL−1 (cinnamic acid; caffeic acid; l-tyrosine), 0.01 to 1.40 mg mL−1 (p-coumaric acid), 0.02 to 1.60 mg mL−1 (l-phenylalanine) and 0.01 to 1.20 mg mL−1 (4-hydroxyphenylpyruvic acid), respectively. The limit of detection (LOD) presents the lowest detectable analyte concentration, whilst the limit of quantitation (LOQ) represents the lowest quantifiable analyte concentration. These are two fundamental elements of method validation defining the limitations of an analytical method. In qNMR, the LOD and LOQ cannot be determined by means of SNR (signal noise ratio) as the NMR response signals are Lorentzian lines. Hence, the LOD and LOQ were determined using the standard deviation of the response σ and the slope S of a calibration curve obtained in the linearity study by the following equations:
The result of linearity, LOD and LOQ are shown in Table 2.
Real sample determination
We used 1H-NMR to quantitatively analyze six phenolic acids in SMB (Fig. 4) and SCT (Fig. 5), and calculated the content of rosmarinic acid (Hou et al. 2020).
As the picture shown, the contents of l-phenylalanine, p-coumaric acid, l-tyrosine and 4-hydroxyphenylpyruvic acid in SCT were higher than that in SMB. Before MJ treatment, the content of cinnamic acid was not much different in the both, but after MJ treatment, the content of cinnamic acid in SCT was higher than that in SMB. Especially when MJ was treated for 9 days, the content of the former was about twice that of the latter. Interestingly, after 6 days of MJ treatment, the cinnamic acid content in the SMB treatment group was significantly lower than the control group. No matter before and after MJ treatment, the content of caffeic acid in SMB was higher than that in SCT. It is speculated that this is related to the by-pass status of caffeic acid in the synthesis of rosmarinic acid, which also explains the difference in the content of rosmarinic acid between the two. After 9 days of MJ treatment, the content of p-coumaric acid in SMB and SCT and 4-hydroxyphenylpyruvic acid in SCT were significantly higher than that in the control group. In addition, when MJ was treated for 1 day, the content of l-tyrosine and 4-hydroxyphenylpyruvic acid in SCT was also significantly higher than that in the control group.
Gene expression was analyzed by qRT-PCR
qRT-PCR was performed to investigate the expression levels of seven key genes in the rosmarinic acid synthesis pathway in the hairy roots of SMB (Fig. 6) and SCT (Fig. 7) treated with MJ at 0, 1, 2, 3, 6, 9 days.
It can be seen that after MJ treatment, the expression of related genes in SMB and SCT was up-regulated within a certain range. The genes measured in SCT are all up-regulated to varying degrees. Specifically, when MJ was treated for 6, 9 days, the expression levels of TAT, RAS, and C4H were significantly higher than those of the control group. When MJ was treated for 9 days, the expression of HPPR, CYP98A14, 4CL was significantly higher than that of the control group, and when MJ was treated for 3 days, the expression of 4CL was significantly higher than that of the control group. The expression level of PAL was significantly higher than that of the control group after 1 day of MJ treatment. The response of related genes in SMB to MJ is weaker than that of SCT. When MJ was treated for 2 days, the expression of TAT and HPPR was significantly higher than that of the control group. In addition, when MJ was treated for 3 and 9 days, the expression of TAT was also significantly higher than that of the control group. However, when MJ was treated for 2 days, the expression of PAL was significantly higher than that of the control group, and 4CL also showed the same changing trend when MJ was treated for 9 days.
Discussion
1H-NMR determination of phenolic acids in SMB and SCT
Consistent with previous reports, the content of rosmarinic acid in SCT is higher than that in SMB (Cheng et al. 2005). After MJ treatment, the content of related phenolic acids in SMB and SCT increased. That is different from the previous report (Fang et al. 2017), we found that MJ can promote the accumulation of rosmarinic acid and related phenolic acids in SMB within a certain treatment time. From an overall comparison, the phenolic acids in SCT have a higher response to MJ than SMB. The promotion effect is the most significant when MJ is treated for 9 days. To better compare the differences in the mechanism of action of MJ on SMB and SCT, the qRT-PCR technology was used to analyze the expression of several key genes.
Gene expression was analyzed by qRT-PCR
After MJ treatment, the expression of related genes in SMB and SCT both showed different degrees of up-regulation within a certain processing time. In addition to the previously reported TAT, HPPR, RAS three genes (Fang et al. 2017), we also found that the expression of C4H, 4CL and CYP98A14 in SCT were up-regulated, but the SMB changes are different. However, the up-regulation of PAL gene expression previously reported (Li et al. 2012) is not very clear in our experimental results. On day 1 of MJ treatment, the expression of PAL in SCT was significantly up-regulated, but on days 2 and 3, the expression of PAL in SMB and SCT was significantly down-regulated, respectively.
MJ significantly up-regulated the expression of 4CL, TAT, HPPR, RAS, C4H, PAL, CYP98A14 in SCT within a certain treatment time, but only significantly up-regulated the expression of TAT and HPPR in SMB, and significantly down-regulated the expression levels of PAL and 4CL. That is to say, MJ plays a role in both the phenylalanine and tyrosine branches of the rosmarinic acid synthesis pathway, and promotes the accumulation of rosmarinic acid by up-regulating the expression of related genes in the two branches. MJ mainly acts on the tyrosine branch in the process of rosmarinic acid synthesis in SMB, and promotes the accumulation of rosmarinic acid by up-regulating the expression of related genes. To a certain extent, this seems to provide exploration ideas for the differential response of SMB and SCT to MJ.
To more intuitively compare the difference between SMB and SCT on MJ, we summarized the experimental results shown in Fig. 8. It can be seen that whether it is SMB or SCT, after MJ treatment, related substrates and gene expression in the rosmarinic acid synthesis pathway were increased to varying degrees. For SMB, the contents of l-phenylalanine, cinnamic acid, p-coumaric acid and caffeic acid on the phenylalanine branch and l-tyrosine, 4-hydroxyphenylpyruvic acid on the tyrosine branch and rosmarinic acid were higher compared with the control group on the 3 and 9 days of MJ treatment. Related gene expression did not show a consistent trend. It is more obvious that the expression levels of C4H, TAT RAS and CYP98A14 were higher than those of the control group during the entire time range of MJ treatment. Compared with SMB, SCT had a stronger response to MJ. Specifically, within 5 days of MJ treatment, the contents of l-phenylalanine, p-coumaric acid, caffeic acid, l-tyrosine and rosmarinic acid were higher than the control group for 4 days, the contents of cinnamic acid and 4-hydroxyphenylpyruvic acid were also higher than the control group for 3 days. Intriguingly, the expression level of HPPR in SCT also continued to be higher than that of the control group, but RAS and CYP98A14 did not show a tendency to always be higher than the control group in SCT. In summary, MJ has a significant promoting effect on the metabolism of rosmarinic acid in SMB and SCT, and its effect on SCT is greater than SMB. Based on this, we made the following conjectures: the differential response of secondary metabolism-related genes to the induction of MJ may lead to different salvianolic acid metabolic responses between the two.
Concluding remarks
To explore the mechanism of MJ on rosmarinic acid biosynthesis in SMB and SCT, we used 1H-NMR technology and qRT-PCR technology to obtain the content of phenolic acids in the hairy root of SMB and SCT under different treatment times with MJ, and combined with the expression of related genes for analysis. Compared with the control group, we found that MJ can promote both of the synthesis of rosmarinic acid and related components in its synthesis pathway in SMB and SCT. We found that MJ significantly increased the expression of TAT and HPPR in the synthesis pathway of rosmarinic acid in SMB, and significantly increased the expression of TAT, RAS, C4H, PAL, 4CL, CYP98A14 and HPPR in SCT. Compared with SMB, SCT has a stronger response to MJ. That is to say, MJ promotes the expression of related genes in the phenylalanine and tyrosine branches of rosmarinic acid synthesis in SCT, but only up-regulates the expression of TAT and HPPR genes in the tyrosine branch of SMB. This is probably due to the different reactions induced by the secondary metabolism-related genes to methyl jasmonate leading to the different metabolic reactions of salvianolic acid between the two. This discovery not only provides new ideas for the mechanism of action of MJ on rosmarinic acid, but also provides important ideas for the synthesis of other phenolic acids. Of course, there are some meaningful issues that are worthy of in-depth exploration in this research. For example, why is the expression of PAL and 4CL on the phenylalanine branch significantly down-regulated after MJ induction? Will there be changes in the response of related genes to MJ at 0–24 h increasing the sampling time point and are the changes the same? We believe that these questions can be a focus of the next research, and the analysis of this problem will provide more ideas and basis for the biosynthesis of rosmarinic acid and even phenolic acids by MJ.
Author contribution statement
Formal analysis, YL; funding acquisition, ZH and LX; project administration, ZL; resources, FS; software, JC; supervision, XZ and DY; writing—original draft, YL; writing—review and editing, ZH. All authors have read and agreed to the published version of the manuscript.
Abbreviations
- C4H:
-
Cinnamate 4-hydroxylase
- 4CL:
-
4-Coumarate: CoA ligase
- CYP98A14:
-
Cytochrome P450
- HPPR:
-
4-Hydroxyphenylpyruvate reductase
- MJ:
-
Methyl jasmonate
- PAL:
-
Phenylalanine ammonia-lyase
- PAS:
-
Rosmarinic acid synthase
- SCT:
-
Salvia castanea f. tomentosa Stib
- SMB:
-
Salvia miltiorrhiza Bunge
- TAT:
-
Tyrosine aminotransferase
References
Chen W, Chen GX (2017) Danshen (Salvia miltiorrhiza Bunge): a prospective healing sage for cardiovascular diseases. Curr Pharm Des 23:5125–5135. https://doi.org/10.2174/1381612823666170822101112
Cheng XL, Xiao XY, Cai LP, Ma SC, Lin RC (2005) Gualitative and guantitative analysis of water-soluble constituents in Salvia castanea F. tomentosa Stib. Chin J Pharm Anal 25:1041–1045
Dam NMV, Hadwich K, Baldwin IT (2000) Induced responses in Nicotiana attenuata affect behaviour and growth of the specialist herbivore Manduca sexta. Oecologia 122:371–379. https://doi.org/10.1007/s004420050043
Di P, Zhang L, Chen JF, Tan HX, Xiao Y, Dong X, Zhou X, Chen WS (2013) 13C tracer reveals phenolic acids biosynthesis in hairy root cultures of Salvia miltiorrhiza. ACS Chem Biol 8:1537–1548. https://doi.org/10.1021/cb3006962
Di CML, Piccolo M, Maione F, Ferraro MG, Irace C, De FV, Ghelardini C, Mascolo N (2018) Tanshinones from Salvia miltiorrhiza Bunge revert chemotherapy-induced neuropathic pain and reduce glioblastoma cells malignancy. Biomed Pharmacother 105:1042–1049. https://doi.org/10.1016/j.biopha.2018.06.047
Ellis BE, Tower GH (1970) Bigenesis of rosmarinic acid in Mentha. Biochem J 118:291–297. https://doi.org/10.1042/bj1180291
Fang YM, Yang DF, Liang ZS (2017) Diverse responses to methyl jasmonate in hairy roots of two Salvia species. J Zhejiang Sci Tech Univ (Nat Sci) 37:712–719. https://doi.org/10.3969/j.issn.1673-3851.2017.09.018
Fang LH, Chen YC, Yuan TY, Lyu Y, Du GH (2018) Salvianolic acid A attenuates vascular remodeling in pulmonary arterial hypertension rats induced by monocrotaline. Chin J Pharmacol Toxicol 32:47–48. https://www.cnki.com.cn/Article/CJFDTotal-YLBS201804043.htm
Gadape H, Parikh K (2011) Quantitative determination and validation of Carvedilol in pharmaceuticals using quantitative nuclear magnetic resonance spectroscopy. Anal Methods 3:2341–2347. https://doi.org/10.1039/C1AY05247K
Gao HW, Huang LT, Ding F, Yang K, Feng YL, Tang HZ, Xu QM, Feng JF, Yang SL (2018) Simultaneous purification of dihydrotanshinone, tanshinone I, cryptotanshinone, and tanshinone IIA from Salvia miltiorrhiza and their anti-inflammatory activities investigation. Sci Rep 8:8460. https://doi.org/10.1038/s41598-018-26828-0
Hsieh FS, Hung MH, Wang CY, Chen YL, Hsiao YJ, Tsai MH, Li JR, Chen LJ, Shih CT, Chao TI, Chen KF (2018) Corrigendum to “Inhibition of protein phosphatase 5 suppresses non-small cell lung cancer through AMP-activated kinase activation” [Lung Cancer 112, (October) (2017) 81–89]. Lung Cancer 119:127–128. https://doi.org/10.1016/j.lungcan.2018.03.012
Hou Z, Liang Z, Li Y, Su F, Chen J, Zhang X, Yang D (2020) Quantitative determination and validation of four phenolic acids in Salvia miltiorrhiza Bunge using 1HNMR spectroscopy. Curr Pharm Anal. https://doi.org/10.2174/1573412916666191231104909
Hu F, Koon CM, Chan JYW, Lau KM, Fung KP (2012) The cardioprotective effect of danshen and gegen decoction on rat hearts and cardiomyocytes with post-ischemia reperfusion injury. BMC Complement Altern Med 12:249. https://www.biomedcentral.com/1472-6882/12/249
Kai GY, Xu H, Zhou CC, Liao P, Xiao JB, Luo XQ, You LJ, Zhang L (2011) Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metab Eng 13:319–327. https://doi.org/10.1016/j.ymben.2011.02.003
Kim HK, Woo ER, Lee HW, Park HR, Kim HN, Jung YK, Choi JY, Chae SW, Kim HR, Chae HJ (2008) The correlation of Salvia miltiorrhiza extract—induced regulation of osteoclastogenesis with the amount of components tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone. Immunopharmacol Immunotoxicol 30:347–364. https://doi.org/10.1080/08923970801949133
Li J (2013) Effect of methyl jasmonate on the activity Salvia miltiorrhiza Bunge enzyme and the content and antioxidant activity of phenolic acid compounds. Master’s thesis, Sichuan Agricultural University, Yaan, China
Li WY, Gao W, Zhao J, Cui GH, Shao AJ, Huang LQ (2012) Research of mechanism of secondary metabolites of phenolic acids in Salvia miltiorrhiza hairy root induced by jasmonate. China J Chin Mater Med 37:13–16. https://doi.org/10.4268/cjcmm20120104
Li YY, Hou ZN, Su F, Chen JP, Zhang XD, Xu L, Yang DF, Liang ZS (2020) Quantitative determination and validation of four ketones in Salvia miltiorrhiza Bunge using quantitative proton nuclear magnetic resonance spectroscopy. Molecules 25:2043–2056. https://doi.org/10.3390/molecules25092043
Liu LW, Zhang YQ, Li XE (2015) Advances in research on hairy roots of medicinal plants. J Shandong Univ Chin Med 21:288–291. https://doi.org/10.16294/j.cnki.1007-659x.2015.03.033
Liu X, Jin MX, Zhang M, Li TQ, Sun SS, Zhang JY, Dai JG, Wang YH (2019) The application of combined 1H NMR-based metabolomics andtranscriptomics techniques to explore phenolic acid biosynthesis in Salvia miltiorrhiza Bunge. J Pharm Biomed Anal 172:126–138. https://doi.org/10.1016/j.jpba.2019.04.030
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Ma PD, Liu JJ, Zhang CL, Liang ZS (2013) Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza Bunge. Appl Biochem Biotechnol 170:1253–1262. https://doi.org/10.1007/s12010-013-0265-4
Malz F, Jancke H (2005) Validation of quantitative NMR. J Pharm Biomed 38:813–823. https://doi.org/10.1016/j.jpba.2005.01.043
Petersen M, Simmonds MS (2003) Rosmarinic acid. Phytochemistry 62:121–125. https://doi.org/10.1016/S0031-9422(02)00513-7
Qiu Y, Li CH, Wang QH, Zeng XQ, Ji P (2018) Tanshinone IIA induces cell death via Beclin-1-dependent autophagy in oral squamous cell carcinoma SCC-9 cell line. Cancer Med 7:397–407. https://doi.org/10.1002/cam4.1281
Saroya AS, Singh J (2018) Neuropharmacology of Salvia miltiorrhiza Bunge (Danshen). Pharmacotherapeutic potential of natural products in neurological disorders. Springer, Singapore, pp 153–158
Shi M, Luo XQ, Ju GH, Yu XH, Hao XL, Huang Q, Xiao JB, Cui LJ, Kai GY (2014) 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. Funct Integr Genom 14:603–615. https://doi.org/10.1007/s10142-014-0385-0
Shi M, Huang FF, Deng CP, Wang Y, Kai GY (2018) Bioactivities, biosynthesis and biotechnological production of phenolic acids in Salvia miltiorrhiza. Crit Rev Food Sci Nutr 59:953–964. https://doi.org/10.1080/10408398.2018.1474170
Sun JB, Hua R, Ou RM, Deng SG, Wu ZF, Zeng X, Wu CL, Cai LP (2004) Pharmacological test of Salvia villi (Salvia nyingchi) on resisting granuloma formation and improving hemorheology. J Chin Med Mater 27:118–120. https://doi.org/10.13863/j.issn1001-4454.2004.02.022
Sun J, Yang HY, Sui C (2014) Research progress on various influences in hairy root yield and secondary metabolites accumulation of medicinal plant. Mod Chin Med 16:945–952. https://doi.org/10.13313/j.issn.1673-4890.2014.11.017
Uto T, Tung NH, Ohta T, Juengsanguanpornsuk W, Hung LQ, Hai NT, Long DD, Thuong PT, Okubo S, Hirata S, Shoyama Y (2018) Antiproliferative activity and apoptosis induction by trijuganone C isolated from the root of Salvia miltiorrhiza Bunge (Danshen). Phytother Res 32:657–666. https://doi.org/10.1002/ptr.6013
Wang Q, Yan SC (2012) Effects of exogenous jasmonates induced systemic acquired resistance on the content of phenolic acid. For Stud China 34:98–106. https://doi.org/10.13332/j.1000-1522.2012.06.011
Wang XY, Cui GH, Huang LQ, Qiu DY (2007) Effects of methyl jasmonate on the accumulation and release of tanshinones in hairy root of Salvia miltiorrhiza. China J Chin Mater Med 32:300–302
Wang LL, Ma RF, Liu CY, Liu HX, Zhu RY, Guo SZ, Tang MK, Li Y, Niu JZ, Fu M, Gao SH, Zhang DW (2017a) Salvia miltiorrhiza: a potential Red light to the development of cardiovascular diseases. Curr Pharm Des 23:1077–1097. https://doi.org/10.2174/1381612822666161010105242
Wang XY, Gao AN, Jiao YD, Zhao Y, Yang XB (2017b) Antitumor effect and molecular mechanism of antioxidant polysaccharides from Salvia miltiorrhiza Bunge in human colorectal carcinoma LoVo cells. Int J Biol Macromol 108:625–634. https://doi.org/10.1016/j.ijbiomac.2017.12.006
Wang LL, Li YM, Deng W, Dong ZH, Li X, Liu D, Zhao LJ, Fu WG, Cho KK, Niu HY, Guo DA, Chen JL, Jiang BH (2018) Cardio-protection of ultrafine granular powder for Salvia miltiorrhiza Bunge against myocardial infarction. J Ethnopharmacol 222:99–106. https://doi.org/10.1016/j.jep.2018.04.029
Wang FY, You HQ, Du XH, You ZQ, Zhang XD, Liang ZS, Yang DF (2020) Effects of different nitrogen sources on accumulation of active components in hairy roots of Salvia miltiorrhiza and Salvia castanea f. tomentosa. Chin Tradit Herb Drugs 51:2538–2547. https://doi.org/10.7501/j.issn.0253-2670.2020.09.031
Weng YS, Kuo WW, Lin YM, Kuo CH, Tzang BS, Tsai FJ, Tsai CH, Lin JA, Hsieh DJY, Huang CY (2013) Danshen mediates through estrogen receptors to activate Akt and inhibit apoptosis effect of Leu27IGF-II-induced IGF-II receptor signaling activation in cardiomyoblasts. Food Chem Toxicol 56:28–39. https://doi.org/10.1016/j.fct.2013.01.008
Wu XX, Gao HW, Hou Y, Yu J, Sun W, Wang Y, Chen XY, Feng YL, Xu QM, Chen XP (2018) Dihydronortanshinone, a natural product, alleviates LPS-induced inflammatory response through NF-κB, mitochondrial ROS, and MAPK pathways. Toxicol Appl Pharmacol 355:1–8. https://doi.org/10.1016/j.taap.2018.06.007
Xiao Y, Gao SH, Di P, Chen JF, Chen WS, Zhang L (2009) Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures. Physiol Plant 137:1–9. https://doi.org/10.1111/j.1399-3054.2009.01257.x
Xing BC (2015) Regulation of phenolic acids and tanshinones biosynthesis by bHLH and WD40 transcription factors in Salvia miltiorrihiza Hairy Roots. Master’s Thesis, Zhejiang Sci-Tech University, HangZhou, China. https://doi.org/10.7666/d.Y2809296
Xing BY, Dang XL, Zhang JY, Wang B, Chen ZY, Dong JE (2013) Effects of methyl jasmonate on rosmarinic acid biosynthesis and related enzyme activities in cultured cells of Salvia miltiorrhiza. Plant Physiol J 49:1326–1332. https://doi.org/10.13592/j.cnki.ppj.2013.12.005
Xiong BQ, Liu DQ, Liao XJ (2018) The effect of methyl jasmonate on the content of active ingredients in Salvia miltiorrhiza hairy root. Biotechnol Bull 34:81–84. https://doi.org/10.13560/j.cnki.biotech.bull.1985.2017-1001
Yan Q, Shi M, Ng J, Wu JY (2006) Elicitor-induced rosmarinic acid accumulation and secondary metabolism enzyme activities in Salvia miltiorrhiza hairy roots. Plant Sci 170:853–885. https://doi.org/10.1016/j.plantsci.2005.12.004
Yan YY, Hu WZ, Jiang AL, Mu SY, Feng K (2015) Research progress of signal molecule of methyl jasmonate and its application in fresh-cut fruits and vegetables. Sci Technol Food Ind 36:384–387. https://doi.org/10.13386/j.issn1002-0306.2015.02.075
Yang DF, Yang SS, Zhang YJ, Liu YH, Meng XH, Liang ZS (2009) Metabolic profiles of three related Salvia species. Fitoterapia 80:274–278. https://doi.org/10.1016/j.fitote.2009.03.004
Ye HG, Liao WB, Li H, Lan YR, Wu GT (2004) Investigation of “Nyingchi Salvia” resources. J Chin Med Mater 27:809–811. https://doi.org/10.13863/j.issn1001-4454.2004.11.008
Zhu HT, Li J, Li Y, Zhang YJ, Yang CR (2013) Plant biological activity of hormone pesticides jasmonic acid and its methyl ester and its application in agricultural production. Pestic 52:552–557. https://doi.org/10.16820/j.cnki.1006-0413.2013.08.002
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We are grateful to the National Natural Science Foundation of China (nos. 31800255 and 31871694) for financial support.
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Hou, Z., Li, Y., Su, F. et al. Application of 1H-NMR combined with qRT-PCR technology in the exploration of rosmarinic acid biosynthesis in hair roots of Salvia miltiorrhiza Bunge and Salvia castanea f. tomentosa Stib. Planta 253, 2 (2021). https://doi.org/10.1007/s00425-020-03506-y
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DOI: https://doi.org/10.1007/s00425-020-03506-y