The application of combined 1H NMR-based metabolomics and transcriptomics techniques to explore phenolic acid biosynthesis in Salvia miltiorrhiza Bunge

Background: Although chromatography and spectrometry based methods have been used to analyse phenolic acids in Chinese traditional medicine Salvia miltiorrhiza Bunge (SMB), quantitative nuclear magnetic resonance (qNMR) has never previously been used to analyse fresh SMB root extracts. Objective: To establish a fast and simple method of quantitating danshensu, lithospermic acid, rosmarinic acid and salvianolic acid B content in fresh SMB root using 1H-NMR spectroscopy. Method: Fresh SMB root were extracted using a 70% methanol aqueous solution and quantitatively analysed for danshensu, lithospermic acid, rosmarinic acid and salvianolic acid B using 1H-NMR spectroscopy. Different internal standards were compared and the results were validated using high performance liquid chromatography. Results: The established method was accurate and precise with good recovery. The LOD and LOQ indicated the excellent sensitivity of the method. The robustness was testified by modification of four different parameters, and the differences among each parameter were all less than 2%. Conclusion: qNMR offers a fast, reliable and accurate method of identifying and quantifying danshensu, lithospermic acid, rosmarinic acid and salvianolic acid B in fresh SMB root extracts.

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Quantitative Determination and Validation of Four Ketones in Salvia miltiorrhiza Bunge Using Quantitative Proton Nuclear Magnetic Resonance Spectroscopy

Molecules ◽

10.3390/molecules25092043 ◽

2020 ◽

Vol 25 (9) ◽

pp. 2043

Author(s):

Yuanyuan Li ◽

Zhuoni Hou ◽

Feng Su ◽

Jipeng Chen ◽

Xiaodan Zhang ◽

...

Keyword(s):

Nmr Spectroscopy ◽

1H Nmr ◽

1H Nmr Spectroscopy ◽

Salvia Miltiorrhiza ◽

Tanshinone Iia ◽

Proton Nuclear Magnetic Resonance ◽

Resonance Spectroscopy ◽

Salvia Miltiorrhiza Bunge ◽

Simple Method ◽

Tanshinone I

Salvia mltiorrhiza Bunge (SMB) is native to China, whose dried root has been used as medicine. A few chromatographic- or spectrometric-based methods have already been used to analyze the lipid-soluble components in SMB. However, the methodology of qNMR on the extracts of fresh SMB root has not been verified so far. The purpose of this study was to establish a fast and simple method to quantify the tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone in fresh Salvia Miltiorrhiza Bunge root without any pre-purification steps using 1H-NMR spectroscopy. The process is as follows: first, 70% methanol aqueous extracts of fresh Salvia Miltiorrhiza Bunge roots were quantitatively analyzed for tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone using 1H-NMR spectroscopy. Different internal standards were tested and the validated method was compared with HPLC. 3,4,5-trichloropyridine was chosen as the internal standard. Twelve samples of Salvia Miltiorrhiza Bunge were quantitatively analyzed by qNMR and HPLC respectively. Then, the results were analyzed by chemometric approaches. This NMR method offers a fast, stable, and accurate analysis of four ketones: tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone in fresh roots of Salvia Miltiorrhiza Bunge.

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SmMYB111 Is a Key Factor to Phenolic Acid Biosynthesis and Interacts with Both SmTTG1 and SmbHLH51 in Salvia miltiorrhiza

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.8b02548 ◽

2018 ◽

Vol 66 (30) ◽

pp. 8069-8078 ◽

Cited By ~ 16

Author(s):

Shasha Li ◽

Yucui Wu ◽

Jing Kuang ◽

Huaiqin Wang ◽

Tangzhi Du ◽

...

Keyword(s):

Phenolic Acid ◽

Salvia Miltiorrhiza ◽

Acid Biosynthesis ◽

Key Factor

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Comparative RNA-Sequence Transcriptome Analysis of Phenolic Acid Metabolism in Salvia miltiorrhiza, a Traditional Chinese Medicine Model Plant

International Journal of Genomics ◽

10.1155/2017/9364594 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Zhenqiao Song ◽

Linlin Guo ◽

Tian Liu ◽

Caicai Lin ◽

Jianhua Wang ◽

...

Keyword(s):

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Phenolic Acid ◽

Expression Profiles ◽

Salvia Miltiorrhiza ◽

Lignin Biosynthesis ◽

Biosynthesis Pathway ◽

Acid Biosynthesis ◽

The Core ◽

Phenylpropanoid Biosynthesis

Salvia miltiorrhiza Bunge is an important traditional Chinese medicine (TCM). In this study, two S. miltiorrhiza genotypes (BH18 and ZH23) with different phenolic acid concentrations were used for de novo RNA sequencing (RNA-seq). A total of 170,787 transcripts and 56,216 unigenes were obtained. There were 670 differentially expressed genes (DEGs) identified between BH18 and ZH23, 250 of which were upregulated in ZH23, with genes involved in the phenylpropanoid biosynthesis pathway being the most upregulated genes. Nine genes involved in the lignin biosynthesis pathway were upregulated in BH18 and thus result in higher lignin content in BH18. However, expression profiles of most genes involved in the core common upstream phenylpropanoid biosynthesis pathway were higher in ZH23 than that in BH18. These results indicated that genes involved in the core common upstream phenylpropanoid biosynthesis pathway might play an important role in downstream secondary metabolism and demonstrated that lignin biosynthesis was a putative partially competing pathway with phenolic acid biosynthesis. The results of this study expanded our understanding of the regulation of phenolic acid biosynthesis in S. miltiorrhiza.

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R2R3-MYB Transcription Factor SmMYB52 Positively Regulates Biosynthesis of Salvianolic Acid B and Inhibits Root Growth in Salvia miltiorrhiza

International Journal of Molecular Sciences ◽

10.3390/ijms22179538 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9538

Author(s):

Rao Yang ◽

Shengsong Wang ◽

Haolan Zou ◽

Lin Li ◽

Yonghui Li ◽

...

Keyword(s):

Transcription Factor ◽

Root Growth ◽

Phenolic Acid ◽

Salvia Miltiorrhiza ◽

Salvianolic Acid B ◽

Myb Transcription Factor ◽

Acid Biosynthesis ◽

Key Enzyme ◽

R2r3 Myb ◽

Encoding Genes

The dried root of Salvia miltiorrhiza is a renowned traditional Chinese medicine that was used for over 1000 years in China. Salvianolic acid B (SalB) is the main natural bioactive product of S. miltiorrhiza. Although many publications described the regulation mechanism of SalB biosynthesis, few reports simultaneously focused on S. miltiorrhiza root development. For this study, an R2R3-MYB transcription factor gene (SmMYB52) was overexpressed and silenced, respectively, in S. miltiorrhiza sterile seedlings. We found that SmMYB52 significantly inhibited root growth and indole-3-acetic acid (IAA) accumulation, whereas it activated phenolic acid biosynthesis and the jasmonate acid (JA) signaling pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that SmMYB52 suppressed the transcription levels of key enzyme-encoding genes involved in the IAA biosynthetic pathway and activated key enzyme-encoding genes involved in the JA and phenolic acid biosynthesis pathways. In addition, yeast one-hybrid (Y1H) and dual-luciferase assay showed that SmMYB52 directly binds to and activates the promoters of several key enzyme genes for SalB biosynthesis, including SmTAT1, Sm4CL9, SmC4H1, and SmHPPR1, to promote the accumulation of SalB. This is the first report of a regulator that simultaneously affects root growth and the production of phenolic acids in S. miltiorrhiza.

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The Protein Kinase SmSnRK2.6 Positively Regulates Phenolic Acid Biosynthesis in Salvia miltiorrhiza by Interacting with SmAREB1

Frontiers in Plant Science ◽

10.3389/fpls.2017.01384 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 7

Author(s):

Yanyan Jia ◽

Zhenqing Bai ◽

Tianlin Pei ◽

Kai Ding ◽

Zongsuo Liang ◽

...

Keyword(s):

Protein Kinase ◽

Phenolic Acid ◽

Salvia Miltiorrhiza ◽

Acid Biosynthesis

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CRISPR/Cas9-mediated targeted mutagenesis of bZIP2 in Salvia miltiorrhiza leads to promoted phenolic acid biosynthesis

Industrial Crops and Products ◽

10.1016/j.indcrop.2021.113560 ◽

2021 ◽

Vol 167 ◽

pp. 113560

Author(s):

Min Shi ◽

Zhiyan Du ◽

Qiang Hua ◽

Guoyin Kai

Keyword(s):

Phenolic Acid ◽

Salvia Miltiorrhiza ◽

Targeted Mutagenesis ◽

Acid Biosynthesis

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The methyl jasmonate-responsive transcription factor SmMYB1 promotes phenolic acid biosynthesis in Salvia miltiorrhiza

Horticulture Research ◽

10.1038/s41438-020-00443-5 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Wei Zhou ◽

Min Shi ◽

Changping Deng ◽

Sunjie Lu ◽

Fenfen Huang ◽

...

Keyword(s):

Transcription Factor ◽

Methyl Jasmonate ◽

Phenolic Acids ◽

Phenolic Acid ◽

Salvia Miltiorrhiza ◽

Water Soluble ◽

Biosynthesis Pathway ◽

Acid Biosynthesis ◽

Expression Of Genes ◽

Genes Encoding

AbstractWater-soluble phenolic acids are major bioactive compounds in the medicinal plant species Salvia miltiorrhiza. Phenolic acid biosynthesis is induced by methyl jasmonate (MeJA) in this important Chinese herb. Here, we investigated the mechanism underlying this induction by analyzing a transcriptome library of S. miltiorrhiza in response to MeJA. Global transcriptome analysis identified the MeJA-responsive R2R3-MYB transcription factor-encoding gene SmMYB1. Overexpressing SmMYB1 significantly promoted phenolic acid accumulation and upregulated the expression of genes encoding key enzymes in the phenolic acid biosynthesis pathway, including cytochrome P450-dependent monooxygenase (CYP98A14). Dual-luciferase (dual-LUC) assays and/or an electrophoretic mobility shift assays (EMSAs) indicated that SmMYB1 activated the expression of CYP98A14, as well as the expression of genes encoding anthocyanin biosynthesis pathway enzymes, including chalcone isomerase (CHI) and anthocyanidin synthase (ANS). In addition, SmMYB1 was shown to interact with SmMYC2 to additively promote CYP98A14 expression compared to the action of SmMYB1 alone. Taken together, these results demonstrate that SmMYB1 is an activator that improves the accumulation of phenolic acids and anthocyanins in S. miltiorrhiza. These findings lay the foundation for in-depth studies of the molecular mechanism underlying MeJA-mediated phenolic acid biosynthesis and for the metabolic engineering of bioactive ingredients in S. miltiorrhiza.

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