Effects of different drying methods on Rubus chingii Hu fruit during processing.

In this study, the dried fruits of Rubus chingii Hu (Chinese name: Fu-Pen-Zi; FPZ) were processed and dried by three methods-in the shade, the sun, and the oven. The composition regarding the standard ingredient, color, and antioxidant capacities were investigated pro- and post-processing. The technique of headspace-solid-phase-microextraction-gas-chromatography-mass spectrometry (HS-SPME-GC-MS) and flavoromics were used to analyze the flavor-conferring metabolites of FPZ. The results obtained revealed that the highest use value and antioxidant capacities were detected in the FPZ fruits processed and dried in the shade. A total of 358 metabolites were detected from them mainly consisting of terpenoids, heterocyclic compounds, and esters. In differential analysis, the down-regulation of the metabolites was much greater than their up-regulation after all three drying methods. In an evaluation of the characteristic compounds and flavors produced after the three methods, there were variations mainly regarding the green and fruity odors. Therefore, considerable insights may be obtained for the development of novel agricultural methods and applications in the pharmaceutical and cosmetic industries by analyzing and comparing the variations in the chemical composition detected pre- and post-processing of the FPZ fruits. This paper provides a scientific basis for quality control in fruits and their clinical applications.

Metabolomic and antioxidant analyses of Salvia miltiorrhiza Bunge and Salvia prattii Hemsl. seeds.

Salvia miltiorrhiza and Salvia prattii seeds are rich in metabolites that are beneficial to human health and can be utilised as nutritional supplements. In this study, UPLC-MS and GC-MS based on extensively focused metabolomics were used to compare the seed metabolomics of the two species. LC-MS detected 118 metabolites, primarily Lipids and phenylpropanoids. GC- MS detected a total of 188 metabolites, mainly organic acids and their derivatives, of which Salvia prattii seeds contain high levels of nutrients. In addition, we experimentally determined antioxidant activity of two Salvia species, and the results showed that the antioxidant activity of Salvia prattii seeds was about twice as high as that of Salvia miltiorrhiza seeds. We used WGCNA to group the metabolites, and found the central metabolites in the focal modules including flavonoids and terpenoids. Our study contributes valuable knowledge for future research on the chemical makeup of Salvia prattii seeds.

Soil applied silicon and manganese combined with foliar application of 5-aminolevulinic acid mediate photosynthetic recovery in Cd-stressed Salvia miltiorrhiza by regulating Cd-transporter genes.

Salvia miltiorrhiza is an important medicinal plant that experiences significant growth and biomass losses when cultivated on cadmium (Cd) contaminated soils. High Cd accumulation in plant tissues also increases the risk of metal entry into the food chain. In this study, we proposed that Cd accumulation in S. miltiorrhiza can be restricted through plant growth regulators and nutrient management. Therefore, S. miltiorrhiza seedlings were transplanted into mixed nutrient soil for two weeks, then treated with 30 mg kg-1 CdCl2, 200 mg kg-1 Na2SiO3·9H2O, and 100 mg kg-1 MnSO4, and simultaneously sprayed with 10 mg L-1 ALA on the leaves one week later. This study showed that elevated Cd accumulation significantly reduced plant growth and biomass. This growth inhibition damaged photosynthetic machinery and impaired carbon assimilation. In contrast, 5-aminolevulinic acid (ALA) significantly promoted the biomass of S. miltiorrhiza, and the dry weight of plants treated with ALA combined with manganese (Mn)/silicon (Si) increased by 42% and 55% as compared with Cd+Mn and Cd+Si treatments. Exogenously applied ALA and Si/Mn significantly activated antioxidant enzymes and promoted the growth recovery of S. miltiorrhiza. Further, exogenous ALA also reduced the Cd concentration in S. miltiorrhiza, especially when combined with Si. Compared with the Cd+Si treatment, the Cd+Si+ALA treatment reduced the Cd concentration in roots and leaves by 59% and 60%, respectively. Gene expression analysis suggested that ALA and Si significantly up-regulated genes associated with Cd transport. Other genes related to heavy metal tolerance mechanisms are also regulated to cope with heavy metal stress. These results indicated that the combined action of ALA and Si/Mn could reduce Cd-toxicity by increasing chlorophyll content and changing oxidative stress and can also affect Cd accumulation by regulating gene expression.

Insights into the plateau adaptation of Salvia castanea by comparative genomic and WGCNA analyses.

INTRODUCTION: Salvia castanea, a wild plant species is adapted to extreme Qinghai-Tibetan plateau (QTP) environments. It is also used for medicinal purposes due to high ingredient of tanshinone IIA (T-IIA). Despite its importance to Chinese medicinal industry, the mechanisms associated with secondary metabolites accumulation (i.e. T-IIA and rosmarinic acid (RA)) in this species have not been characterized. Also, the role of special underground tissues in QTP adaptation of S. castanea is still unknown. OBJECTIVES: We explored the phenomenon of periderm-like structure in underground stem center of S. castanea with an aim to unravel the molecular evolutionary mechanisms of QTP adaptation in this species. METHODS: Morphologic observation and full-length transcriptome of S. castanea plants were conducted. Comparative genomic analyses of S. castanea with other 14 representative species were used to reveal its phylogenetic position and molecular evolutionary mechanisms. RNA-seq and WGCNA analyses were applied to understand the mechanisms of high accumulations of T-IIA and RA in S. castanea tissues. RESULTS: Based on anatomical observations, we proposed a "trunk-branches" developmental model to explain periderm-like structure in the center of underground stem of S. castanea. Our study suggested that S. castanea branched off from cultivated Danshen around 16 million years ago. During the evolutionary process, significantly expanded orthologous gene groups, 24 species-specific and 64 positively selected genes contributed to morphogenesis and QTP adaptation in S. castanea. RNA-seq and WGCNA analyses unraveled underlying mechanisms of high accumulations of T-IIA and RA in S. castanea and identified NAC29 and TGA22 as key transcription factors. CONCLUSION: We proposed a "trunk-branches" developmental model for the underground stem in S. castanea. Adaptations to extreme QTP environment in S. castanea are associated with accumulations of high secondary metabolites in this species.

Genome-Wide Characterization and Expression Analysis of bZIP Gene Family Under Abiotic Stress in Glycyrrhiza uralensis.

bZIP gene family is one of the largest transcription factor families. It plays an important role in plant growth, metabolic, and environmental response. However, complete genome-wide investigation of bZIP gene family in Glycyrrhiza uralensis remains unexplained. In this study, 66 putative bZIP genes in the genome of G. uralensis were identified. And their evolutionary classification, physicochemical properties, conserved domain, functional differentiation, and the expression level under different stress conditions were further analyzed. All the members were clustered into 13 subfamilies (A-K, M, and S). A total of 10 conserved motifs were found in GubZIP proteins. Members from the same subfamily shared highly similar gene structures and conserved domains. Tandem duplication events acted as a major driving force for the evolution of bZIP gene family in G. uralensis. Cis-acting elements and protein-protein interaction networks showed that GubZIPs in one subfamily are involved in multiple functions, while some GubZIPs from different subfamilies may share the same functional category. The miRNA network targeting GubZIPs showed that the regulation at the transcriptional level may affect protein-protein interaction networks. We suspected that domain-mediated interactions may categorize a protein family into subfamilies in G. uralensis. Furthermore, the tissue-specific gene expression patterns of GubZIPs were analyzed using the public RNA-seq data. Moreover, gene expression level of 66 bZIP family members under abiotic stress treatments was quantified by using qRT-PCR. The results of this study may serve as potential candidates for functional characterization in the future.

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.

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. 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.

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

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.

The ethylene response factor SmERF8 regulates the expression of SmKSL1 and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots.

Saliva miltiorrhiza ethylene response factor (SmERF), predicted to be expressed genome-wide, is the potential regulator of tanshinone biosynthesis. However, few studies have investigated its transcriptional regulation pathways in tanshinone biosynthesis. Here, we report an ethylene response factor (SmERF8), which was screened by the SmKSL1 (a key gene in tanshinone biosynthesis) promoter from the S. miltiorrhiza cDNA library. The SmERF8, highly expressed in S. miltiorrhiza root head, is sensitive to Eth stress, and its protein was enriched in the nucleus. The SmERF8 recognizes the GCC-box in the SmKSL1 promoter. Overexpression and RNAi of SmERF8 in S. miltiorrhiza transgenic hairy roots showed that the tanshinone contents were significantly increased in the overexpression transgenic lines and decreased in RNAi lines. These results suggest that the SmERF8 may be a central activator that regulates the expression of SmKSL1 by binding the GCC-box and then promoting tanshinone biosynthesis. Thus, the SmERF8 may functionally accelerate tanshinone biosynthesis by the transcriptional regulation of its key gene.

Overexpression of SmbHLH148 induced biosynthesis of tanshinones as well as phenolic acids in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: SmbHLH148 activated the whole biosynthetic pathways of phenolic acids and tanshinones, thus upregulated the production of both the two groups of pharmaceutical ingredients in Salvia miltiorrhiza. Phenolic acids and tanshinones are the two important groups of pharmaceutical ingredients presented in Salvia miltiorrhiza Bunge. The bHLH transcription factors could regulate secondary metabolism efficiently in plants. However, there are only some MYCs have been studied on regulation of either phenolic acids or tanshinones biosynthesis. In this study, a bHLH TF named SmbHLH148, which is homologous to AtbHLH148, AtbHLH147 and CubHLH1, was isolated and functionally characterized from S. miltiorrhiza. Transcription of SmbHLH148 could be intensely induced by ABA and also be moderately induced by MeJA and GA. SmbHLH148 is present in all the six tissues and mostly expressed in fibrous root and flowers. Subcellular localization analysis found that SmbHLH148 was localized in the nucleus. Overexpression of SmbHLH148 significantly increased not only three phenolic acids components accumulation but also three tanshinones content. Content of caffeic acid, rosmarinic acid and salvianolic acid B were reached to 2.87-, 4.00- and 5.99-fold of the control in the ObHLH148-3, respectively. Content of dihydrotanshinone I, cryptotanshinone, and tanshinone I were also present highest in ObHLH148-3, reached 2.5-, 5.04- and 3.97-fold of the control, respectively. Expression analysis of pathway genes of phenolic acids and tanshinones in transgenic lines showed that most of them were obviously upregulated. Moreover, transcription of AREB and JAZs were also induced in SmbHLH148 overexpression lines. These results suggested that SmbHLH148 might be taken part in ABA and MeJA signaling and activated almost the whole biosynthetic pathways of phenolic acids and tanshinones, thus the production of phenolic acids and tanshinones were upregulated.

Transcriptional Profiles of SmWRKY Family Genes and Their Putative Roles in the Biosynthesis of Tanshinone and Phenolic Acids in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge is a Chinese traditional herb for treating cardiovascular and cerebrovascular diseases, and tanshinones and phenolic acids are the dominated medicinal and secondary metabolism constituents of this plant. WRKY transcription factors (TFs) can function as regulators of secondary metabolites biosynthesis in many plants. However, studies on the WRKY that regulate tanshinones and phenolics biosynthesis are limited. In this study, 69 SmWRKYs were identified in the transcriptome database of S. miltiorrhiza, and phylogenetic analysis indicated that some SmWRKYs had closer genetic relationships with other plant WRKYs, which were involved in secondary metabolism. Hairy roots of S. miltiorrhiza were treated by methyl jasmonate (MeJA) to detect the dynamic change trend of SmWRKY, biosynthetic genes, and medicinal ingredients accumulation. Base on those date, a correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite network and identify 9 SmWRKYs (SmWRKY1, 7, 19, 29, 45, 52, 56, 58, and 68), which were most likely to be involved in tanshinones and phenolic acids biosynthesis. Taken together, this study has provided a significant resource that could be used for further research on SmWRKY in S. miltiorrhiza and especially could be used as a cue for further investigating SmWRKY functions in secondary metabolite accumulation.

Bivariate Correlation Analysis of the Chemometric Profiles of Chinese Wild Salvia miltiorrhiza Based on UPLC-Qqq-MS and Antioxidant Activities.

To better understand the mechanisms underlying the pharmacological actions of Salvia miltiorrhiza, correlation between the chemical profiles and in vitro antioxidant activities in 50 batches of wild S. miltiorrhiza samples was analyzed. Our ultra-performance liquid chromatography-tandem mass spectrometry analysis detected twelve phenolic acids and five tanshinones and obtained various chemical profiles from different origins. In a principal component analysis (PCA) and cluster analysis, the tanshinones cryptotanshinone, tanshinone IIA and dihydrotanshinone I exhibited higher weights in PC1, whereas the phenolic acids danshensu, salvianolic acids A and B and lithospermic acid were highly loaded in PC2. All components could be optimized as markers of different locations and might be suitable for S. miltiorrhiza quality analyses. Additionally, the DPPH and ABTS assays used to comprehensively evaluate antioxidant activities indicated large variations, with mean DPPH and ABTS scavenging potencies of 32.24 and 23.39 µg/mL, respectively, among S. miltiorrhiza extract solutions. Notably, samples that exceeded the mean IC50 values had higher phenolic acid contents. A correlation analysis indicated a strong correlation between the antioxidant activities and phenolic acid contents. Caffeic acid, danshensu, rosmarinic acid, lithospermic acid and salvianolic acid B were major contributors to antioxidant activity. In conclusion, phenolic compounds were the predominant antioxidant components in the investigated plant species. These plants may be sources of potent natural antioxidants and beneficial chemopreventive agents.

Bergamotane Sesquiterpenes with Alpha-Glucosidase Inhibitory Activity from the Plant Pathogenic Fungus Penicillium expansum.

Two new bergamotane sesquiterpene lactones, named expansolides C and D (1 and 2), together with two known compounds expansolides A and B (3 and 4), were isolated from the plant pathogenic fungus Penicillium expansum ACCC37275. The structures of the new compounds were established by detailed analyses of the spectroscopic data, especially 1D-, 2D-NMR, and HR-ESI-MS. In an in vitro bioassay, the epimeric mixture of expansolides C and D (1 and 2) (in a ratio of 2:1 at the temprature of the bioassay) exhibited more potent α-glucosidase inhibitory activity (IC50 =0.50 ± 0.02 mm) as compared with the positive control acarbose (IC50 = 1.90 ± 0.05 mm). To the best of our knowledge, it was the first report on the α-glucosidase inhibitory activity of bergamotane sesquiterpenes.

Quantitative determination and validation of avermectin B1a in commercial products using quantitative nuclear magnetic resonance spectroscopy.

Nuclear magnetic resonance is defined as a quantitative spectroscopic tool that enables a precise determination of the number of substances in liquids as well as in solids. There is few report demonstrating the application of NMR in the quantification of avermectin B1a (AVB1a ); here, a proton nuclear magnetic resonance spectroscopy ((1) H NMR) using benzene [1-methoxy-4-(2-nitroethyl) (PMN)] as an internal standard and deuterochloroform as an NMR solvent was tested for the quantitative determination of AVB1a . The integrated signal of AVB1a at 5.56 ppm and the signal of PMN at 8.14 ppm in the (1) H NMR spectrum were used for quantification purposes. Parameters of specificity, linearity, accuracy, precision, intermediate precision, range, limit of detection (LOD), limit of quantification (LOQ), stability and robustness were validated. The established method was accurate and precise with good recovery (98.86%) and relative standard deviation (RSD) of assay (0.34%) within the linearity of the calibration curve ranging from 5.08 to 13.58 mg/ml (R(2) = 0.9999). The LOD and LOQ were 0.009 and 0.029 mg/ml, which indicated the excellent sensitivity of the method. The stability of the method was testified by a calculated RSD of 0.11%. The robustness was testified by modification of four different parameters, and the differences among each parameter were all less than 0.1%. Comparing with the assay described by the manufacturer of avermectin tablets, there was no significant difference between the assay obtained by HPLC and quantitative NMR (qNMR), which indicated qNMR was a simple and efficient method for the determination of AVB1a in commercial formulation products.