(Danshen) from Cultivated F
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Zhan et al. Chin Med (2019) 14:42 https://doi.org/10.1186/s13020-019-0265-6 Chinese Medicine RESEARCH Open Access Metabolome and transcriptome analyses reveal quality change in the orange-rooted Salvia miltiorrhiza (Danshen) from cultivated feld Zhilai Zhan1†, Wentao Fang1,2†, Xiaohui Ma1,3, Tong Chen1, Guanghong Cui1, Ying Ma1, Liping Kang1, Tiegui Nan1, Huixin Lin1, Jinfu Tang1, Yan Zhang1, Changjiangsheng Lai1, Zhenli Ren4, Yanan Wang1, Yujun Zhao1, Ye Shen1, Ling Wang1, Wen Zeng1, Juan Guo1* and Luqi Huang1* Abstract Background: The dry root and rhizome of Salvia miltiorrhiza Bunge, or Danshen, is a well-known, traditional Chinese medicine. Tanshinones are active compounds that accumulate in the periderm, resulting in red-colored roots. How- ever, lines with orange roots have been observed in cultivated felds. Here, we performed metabolome and transcrip- tome analyses to investigate the changes of orange-rooted Danshen. Methods: Metabolome analysis was performed by ultra-high-performance liquid chromatography-quadrupole time-of-fight mass spectrometry (UPLC/Q-Tof–MS) to investigate the metabolites variation between orange Danshen and normal Danshen. RNA sequencing and KEGG enrichment analysis were performed to analyzing the diferentially expressed genes between orange-rooted and normal Danshen. Results: In total, 40 lipophilic components were detected in metabolome analysis, and seven compounds were signifcantly decreased in the orange Danshen, including the most abundant active compounds, tanshinone IIA and tanshinone I in normal Danshen. Systematic analysis of transcriptome profles revealed that the down-regulated genes related to catalytic dehydrogenation was not detected. However, two genes related to stress resistance, and four genes related to endoplasmic reticulum (ER)-associated degradation of proteins were up-regulated in orange Danshen. Conclusions: Decreases in the content of dehydrogenated furan ring tanshinones such as tanshinone IIA resulted in phenotypic changes and quality degradation of Danshen. Transcriptome analysis indicated that incorrect folding and ER-associated degradation of corresponding enzymes, which could catalyze C 15-C16 dehydrogenase, might be contributed to the decrease in dehydrogenated furan ring tanshinones, rather than lower expression of the relative genes. This limited dehydrogenation of cryptotanshinone and dihydrotanshinone I into tanshinones IIA and I prod- ucts, respectively, led to a reduced quality of Danshen in cultivated felds. Keywords: Salvia miltiorrhiza Bunge (Danshen), Tanshinones, Metabolome profling, mRNA expression profling, ER-associated degradation Background *Correspondence: [email protected]; [email protected] Danshen, the dry root and rhizome of Salvia miltiorrhiza †Zhilai Zhan and Wentao Fang contributed equally to this work Bunge, is one of the most versatile components in tradi- 1 State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical tional Chinese medicine. More than 636 medicinal prep- Sciences, Beijing 100700, China arations utilize Danshen as their main constituent, which Full list of author information is available at the end of the article can be found in the Chinese patent medicine prescription © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhan et al. Chin Med (2019) 14:42 Page 2 of 10 library. Among them, Danshen Tablets and Fufang Dan- Interestingly, some orange-rooted lines have appeared shen Dripping Pills have been widely used in modern during cultivation, suggesting that variations in tanshi- Chinese clinical trials and are considered efective in none content or composition may afect the phenotype treating cardiovascular and cerebrovascular diseases [1]. and quality of Danshen (Fig. 1a). Additionally, this phe- Te main components of Danshen include hydrophilic nomenon is not an isolated case and has been observed phenolic acids and lipophilic diterpenoid tanshinones, in several diferent cultivation areas, including the Shan- which are used to evaluate the quality of medicinal dong, Henan, and Shanxi Provinces. Terefore, extensive materials [1, 2]. Modern pharmacological studies and investigation of the orange lines found in cultivated felds chemical investigations suggest that both components is important for further utilization of Danshen. contribute to Danshen’s pharmacological and therapeu- Te genome sequence and transcriptome analysis of tic efects. Phenolic acid compounds are distributed in Danshen from various development periods [7–9], difer- the roots and leaves of S. miltiorrhiza, while tanshinones ent tissues [10], and tissue cultures from diferent induc- mainly accumulate in the roots and rhizomes. More than tion times were taken [11], which provide a foundation 40 structurally diverse tanshinones have been isolated for the biological analysis of this useful medicinal plant. and identifed since their discovery in the 1930s. Among Based on these analyses, the biosynthesis and regula- them, tanshinone IIA, cryptotanshinone, tanshinone I, tion of tanshinones and phenolic acids have made great and dihydrotanshinone I are the main active ingredients strides [12–14]. However, there are still many aspects [3]. Most tanshinones have colors ranging from orange to that need to be studied based on comparative omics data red (Fig. 1), which elicit the reddish phenotype of Dan- and genome sequencing. shen. Tese compounds have been extensively investi- In this study, we utilized ultra-high-performance liquid gated for their well-known cardiovascular activities, as chromatography-quadrupole time-of-fight mass spec- well as for their anti-cancer activities in vitro and in vivo trometry (UPLC/Q-TOF–MS) to analyze the component [4]. Tanshinone IIA has also been reported to have poten- variations in both normal and orange Danshen to assess tial for treating human infammation [5], as well as anti- their diferences in quality. In order to determine a pos- adipogenic efects on 3T3-L1 cells and in zebrafsh [6]. sible explanation for these phenotypic changes, high- In China, more than 70,000 tons of Danshen is con- throughput RNA sequencing was performed to detect sumed every year. Before the 1970s, wild Danshen was diferences in gene expression between the two types of the main ingredient in traditional Chinese medicine. Danshen. However, with increased demand, wild resources have greatly decreased. Now, Danshen is cultivated in north- Materials and methods ern and middle China, providing almost all the material Plant materials for traditional patent prescriptions and for clinical use in Danshen were collected in July 2015 from the Chinese Chinese medicine. Tus, the quality and consistency of herbal medicine cultivation base located in Changqing, cultivated Danshen are essential for medicinal security. Shandong Province, China. Tree normal Danshen a b c normal 1: TOFMSES+ 100 297.1518 297.1553 295.1381 BPI 297.1518 295.1346 1.49e6 283.1724 Tanshinone IIA % 279.1044281.1176 705.1882 274.2759 277.0882 521.1052 261.0898 150.1274 219.1866 318.2997 0 Crypototashinone orange 100 297.1483 1: TOFMSES+ BPI 1.43e6 Tanshinone I % 283.1724 279.1044 281.1176 223.9979 274.2759 150.1299 521.1052 0 Time Dihydrotanshinone I 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.00 12.0013.00 Fig. 1 a Color phenotypes of normal Danshen (left) and orange Danshen (right). b Phenotypes of tanshinone IIA, cryptotashinone, tanshinone I, and dihydrotanshinone I. c The chromatogram of total ions found in normal Danshen (top) and orange Danshen (bottom) Zhan et al. Chin Med (2019) 14:42 Page 3 of 10 samples and three orange Danshen samples represent- 12.3–14.0 min, 98% B; 14.0–14.5 min, 98–30% B; 14.5– ing three replicates were collected for metabolite profl- 16.0 min, 30% B. Te injection volume was 1 µL. Te tar- ing and transcriptome analysis during the forescence get column temperature was 40 °C. period in July. At this time, growth conditions transfer Chemical profle analysis was performed on a Xevo from vegetative to reproductive growth and secondary G2-S Q-Tof–MS analyzer (Waters Micromass, Manches- metabolites are accumulated during this period. Te root ter, UK) operated in both positive electrospray ionization of these Danshen were sampled and frozen with liquid modes. In the ESI + mode, parameters were set as follows: nitrogen and stored at − 80 °C respectively for subse- mass range, 50–1500 Da; collision energy, 35–60 V; capil- quent metabolome and RNA-sequencing analyses. lary voltage, 0.5 kV; cone voltage, 40 V; source tempera- ture, 100 °C; desolvation gas temperature, 450 °C; cone Chemicals and reagents gas fow rate, 50 L/h; desolvation fow rate, 900 L/h. Reference standards of tanshinone IIA, cryptotanshi- none, tanshinone I, dihydrotanshinone I, tanshinone IIB, Data pretreatment and statistical analyses dihydroisotanshinone I, danshenxinkun B, methylenetan- Te centroid MS raw data were analyzed using the Pro- shinquinone, tetrahydrotanshinone