MAPK3-MYB36-ARF1 module regulates the tanshinone formation in Salvia miltiorrhiza.

Salvia miltiorrhiza, known as Danshen, is a traditional Chinese medicinal plant with significant cardiovascular benefits, attributed to its secondary metabolites, particularly tanshinones. Despite their medicinal value, tanshinones occur in low natural abundance, necessitating research to increase their content. This study explores the role of the ARF transcription factor (SmARF1) in tanshinone accumulation in Danshen. Overexpressing SmARF1 in hairy roots significantly increased tanshinone levels. EMSA and Dual-LUC assays revealed that SmMYB36, a transcription factor interacting with SmMAPK3, binds to and regulates the SmARF1 promoter. SmMYB36 alone inhibited the expression of SmARF1 gene, while its interaction with SmMAPK3 enhanced SmARF1 promoter activity. This MAPK3-MYB36-ARF1 module elucidates a complex regulatory mechanism for tanshinone biosynthesis, offering insights for targeted enhancement of tanshinone content through advanced biotechnological approaches.

RBOH-dependent signaling is involved in He-Ne laser-induced salt tolerance and production of rosmarinic acid and carnosol in Salvia officinalis.

BACKGROUND: In the past two decades, the impacts of Helium-Neon (He-Ne) laser on stress resistance and secondary metabolism in plants have been studied, but the signaling pathway which by laser regulates this process remains unclear. Therefore, the current study sought to explore the role of RBOH-dependent signaling in He-Ne laser-induced salt tolerance and elicitation of secondary metabolism in Salvia officinalis. Seeds were primed with He-Ne laser (6 J cm- 2) and peroxide hydrogen (H2O2, 5 mM) and 15-old-day plants were exposed to two salinity levels (0, 75 mM NaCl). RESULTS: Salt stress reduced growth parameters, chlorophyll content and relative water content (RWC) and increased malodialdehyde (MDA) and H2O2 contents in leaves of 45-old-day plants. After 48 h of salt exposure, higher transcription levels of RBOH (encoding NADPH oxidase), PAL (phenylalanine ammonia-lyase), and RAS (rosmarinic acid synthase) were recorded in leaves of plants grown from seeds primed with He-Ne laser and/or H2O2. Despite laser up-regulated RBOH gene in the early hours of exposing to salinity, H2O2 and MDA contents were lower in leaves of these plants after 30 days. Seed pretreatment with He-Ne laser and/or H2O2 augmented the accumulation of anthocyanins, total phenol, carnasol, and rosmarinic acid and increased total antioxidant capacity under non-saline and more extensively at saline conditions. Indeed, these treatments improved RWC, and K+/Na+ ratio, enhanced the activities of superoxide dismutase and ascorbate peroxidase and proline accumulation, and significantly decreased membrane injury and H2O2 content in leaves of 45-old-day plants under salt stress. However, applying diphenylene iodonium (DPI as an inhibitor of NADPH oxidase) and N, N-dimethyl thiourea (DMTU as a H2O2 scavenger) after laser priming reversed the aforementioned effects which in turn resulted in the loss of laser-induced salt tolerance and secondary metabolism. CONCLUSIONS: These findings for the first time deciphered that laser can induce a transient RBOH-dependent H2O2 burst, which might act as a downstream signal to promote secondary metabolism and salt stress alleviation in S. officinalis plants.

The methyl jasmonate-responsive transcription factor SmERF106 promotes tanshinone accumulation in Salvia miltiorrhiza.

Understanding the regulatory biosynthesis mechanisms of active compounds in herbs is vital for the preservation and sustainable use of natural medicine resources. Diterpenoids, which play a key role in plant growth and resistance, also serve as practical products for humans. Tanshinone, a class of abietane-type diterpenes unique to the Salvia genus, such as Salvia miltiorrhiza, is an excellent model for studying diterpenoids. In this study, we discovered that a transcription factor, SmERF106, responds to MeJA induction and is located in the nucleus. It exhibits a positive correlation with the expression of SmKSL1 and SmIDI1, which are associated with tanshinone biosynthesis. We performed DNA affinity purification sequencing (DAP-seq) to predict genes that may be transcriptionally regulated by SmERF106. Our cis-elements analysis suggested that SmERF106 might bind to GCC-boxes in the promoters of SmKSL1 and SmIDI1. This indicates that SmKSL1 and SmIDI1 could be potential target genes regulated by SmERF106 in the tanshinone biosynthesis pathway. Their interaction was then demonstrated through a series of in vitro and in vivo binding experiments, including Y1H, EMSA, and Dual-LUC. Overexpression of SmERF106 in the hairy root of S. miltiorrhiza led to a significant increase in tanshinone content and the transcriptional levels of SmKSL1 and SmIDI1. In summary, we found that SmERF106 can activate the transcription of SmKSL1 and SmIDI1 in response to MeJA induction, thereby promoting tanshinone biosynthesis. This discovery provides new insights into the regulatory mechanisms of tanshinones in response to JA and offers a potential gene tool for tanshinone metabolic engineering strategy.

Crosstalk between H2O2 and Ca2+ signaling is involved in root endophyte-enhanced tanshinone biosynthesis of Salvia miltiorrhiza.

Tanshinones are bioactive ingredients derived from the herbal plant Salvia miltiorrhiza and are used for treating diseases of the heart and brain, thus ensuring quality of S. miltiorrhiza is paramount. Applying the endophytic fungus Trichoderma atroviride D16 can significantly increase the content of tanshinones in S. miltiorrhiza, but the potential mechanism remains unknown. In the present study, the colonization of D16 effectively enhanced the levels of Ca2+ and H2O2 in the roots of S. miltiorrhiza, which is positively correlated with increased tanshinones accumulation. Further experiments found that the treatment of plantlets with Ca2+ channel blocker (LaCl3) or H2O2 scavenger (DMTU) blocked D16-promoted tanshinones production. LaCl3 suppressed not only the D16-induced tanshinones accumulation but also the induced Ca2+ and H2O2 generation; nevertheless, DMTU did not significantly inhibit the induced Ca2+ biosynthesis, implying that Ca2+ acted upstream in H2O2 production. These results were confirmed by observations that S. miltiorrhiza treated with D16, CaCl2, and D16+LaCl3 exhibit H2O2 accumulation and influx in the roots. Moreover, H2O2 as a downstream signal of Ca2+ is involved in D16 enhanced tanshinones synthesis by inducing the expression of genes related to the biosynthesis of tanshinones, such as DXR, HMGR, GGPPS, CPS, KSL and CYP76AH1 genes. Transcriptomic analysis further supported that D16 activated the transcriptional responses related to Ca2+ and H2O2 production and tanshinones synthesis in S. miltiorrhiza seedlings. This is the first report that Ca2+ and H2O2 play important roles in regulating fungal-plant interactions thus improving the quality in the D16-S. miltiorrhiza system.

Changes in secondary metabolites contents and stress responses in Salvia miltiorrhiza via ScWRKY35 overexpression: Insights from a wild relative Salvia castanea.

Salvia castanea Diels, a close wild relative to the medicinal plant, Salvia miltiorrhiza Bunge, primarily grows in high-altitude regions. While the two species share similar active compounds, their content varies significantly. WRKY transcription factors are key proteins, which regulate plant growth, stress response, and secondary metabolism. We identified 46 ScWRKY genes in S. castanea and found that ScWRKY35 was a highly expressed gene associated with secondary metabolites accumulation. This study aimed to explore the role of ScWRKY35 gene in regulating the accumulation of secondary metabolites and its response to UV and cadmium (Cd) exposure in S. miltiorrhiza. It was found that transgenic S. miltiorrhiza hairy roots overexpressing ScWRKY35 displayed upregulated expression of genes related to phenolic acid synthesis, resulting in increased salvianolic acid B (SAB) and rosmarinic acid (RA) contents. Conversely, tanshinone pathway gene expression decreased, leading to lower tanshinone levels. Further, overexpression of ScWRKY35 upregulated Cd transport protein HMA3 in root tissues inducing Cd sequestration. In contrast, the Cd uptake gene NRAMP1 was downregulated, reducing Cd absorption. In response to UV radiation, ScWRKY35 overexpression led to an increase in the accumulation of phenolic acid and tanshinone contents, including upregulation of genes associated with salicylic acid (SA) and jasmonic acid (JA) synthesis. Altogether, these findings highlight the role of ScWRKY35 in enhancing secondary metabolites accumulation, as well as in Cd and UV stress modulation in S. miltiorrhiza, which offers a novel insight into its phytochemistry and provides a new option for the genetic improvement of the plants.

CYP72D19 from Tripterygium wilfordii catalyzes C-2 hydroxylation of abietane-type diterpenoids.

KEY MESSAGE: CYP72D19, the first functional gene of the CYP72D subfamily, catalyzes the C-2 hydroxylation of abietane-type diterpenoids. The abietane-type diterpenoids, e.g., triptolide, tripdiolide, and 2-epitripdiolide, are the main natural products for the anti-tumor, anti-inflammatory, and immunosuppressive activities of Tripterygium wilfordii, while their biosynthetic pathways are not resolved. Here, we cloned and characterized the CYP72D19-catalyzed C-2 hydroxylation of dehydroabietic acid, a compound that has been proven to be a biosynthetic intermediate in triptolide biosynthesis. Through molecular docking and site-directed mutagenesis, L386, L387, and I493 near the active pocket were found to have an important effect on the enzyme activity, which also indicates that steric hindrance of residues plays an important role in function. In addition, CYP72D19 also catalyzed a variety of abietane-type diterpenoids with benzene ring, presumably because the benzene ring of the substrate molecule stabilized the C-ring, allowing the protein and the substrate to form a relatively stable spatial structure. This is the first demonstration of CYP72D subfamily gene function. Our research provides important genetic elements for the structural modification of active ingredients and the heterologous production of other 2-hydroxyl abietane-type natural products.

The genomes of medicinal skullcaps reveal the polyphyletic origins of clerodane diterpene biosynthesis in the family Lamiaceae.

The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into 13 pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane, and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis, and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol, and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis, which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the production of anticancer clerodanes through future metabolic engineering efforts.

A tracking work on how Sm4CL2 re-directed the biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: Overexpression and antisense expression of Sm4CL2 re-directed the biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. Danshen (Salvia miltiorrhiza Bunge) is a widely used traditional Chinese medicine and its main active ingredients are water-soluble phenolic acids and lipophilic diterpenoids which are produced through the phenylpropanoid pathway and terpenoid pathway, respectively. 4-Coumaric acid: Coenzyme A ligase (4CL) is a key enzyme in the phenylpropanoid metabolism. We had obtained Sm4CL2-overexpressing (Sm4CL2-OE) and antisense Sm4CL2-expressing (anti-Sm4CL2) danshen hairy roots over ten years ago. In the follow-up study, we found that total salvianolic acids in Sm4CL2-OE-4 hairy roots increased to 1.35 times of the control-3, and that in anti-Sm4CL2-1 hairy roots decreased to 37.32% of the control-3, but tanshinones in anti-Sm4CL2-1 was accumulated to 1.77 ± 0.16 mg/g of dry weight, compared to undetectable in Sm4CL2-OE-4 and the control-3 hairy roots. Interestingly, Sm4CL2-OE-4 hairy roots contained more lignin, 1.36 times of the control-3, and enhanced cell wall and xylem lignification. Transcriptomic analysis revealed that overexpression of Sm4CL2 caused the upregulation of other phenylpropanoid pathway genes and antisense Sm4CL2 expression resulted in the downregulation of other phenylpropanoid pathway genes but activated the expression of terpenoid pathway genes like SmCYP76AK5, SmGPPS.SSUII.1 and SmDXS2. Protein-protein interaction analysis suggested that Sm4CL2 might interact with PAL, PAL4, CSE, CCoAOMT and SmCYP84A60, and appeared to play a key role in the interaction network. The tracking work in this study proved that Sm4CL2 could redirect both salvianolic acids and tanshinones biosynthesis possibly through synergistically regulating other pathway genes. It also indicated that genetic modification of plant secondary metabolism with biosynthetic gene might cause other responses through protein-protein interactions.

The chromosome-scale assembly of the Salvia rosmarinus genome provides insight into carnosic acid biosynthesis.

Rosemary (Salvia rosmarinus) is considered a sacred plant because of its special fragrance and is commonly used in cooking and traditional medicine. Here, we report a high-quality chromosome-level assembly of the S. rosmarinus genome of 1.11 Gb in size; the genome has a scaffold N50 value of 95.5 Mb and contains 40 701 protein-coding genes. In contrast to other diploid Labiataceae, an independent whole-genome duplication event occurred in S. rosmarinus at approximately 15 million years ago. Transcriptomic comparison of two S. rosmarinus cultivars with contrasting carnosic acid (CA) content revealed 842 genes significantly positively associated with CA biosynthesis in S. rosmarinus. Many of these genes have been reported to be involved in CA biosynthesis previously, such as genes involved in the mevalonate/methylerythritol phosphate pathways and CYP71-coding genes. Based on the genomes and these genes, we propose a model of CA biosynthesis in S. rosmarinus. Further, comparative genome analysis of the congeneric species revealed the species-specific evolution of CA biosynthesis genes. The genes encoding diterpene synthase and the cytochrome P450 (CYP450) family of CA synthesis-associated genes form a biosynthetic gene cluster (CPSs-KSLs-CYP76AHs) responsible for the synthesis of leaf and root diterpenoids, which are located on S. rosmarinus chromosomes 1 and 2, respectively. Such clustering is also observed in other sage (Salvia) plants, thus suggesting that genes involved in diterpenoid synthesis are conserved in the Labiataceae family. These findings provide new insights into the synthesis of aromatic terpenoids and their regulation.

Semisynthetic Abietic and Dehydroabietic Acid Derivatives and Triptoquinone Epimers Interfere with LPS-Triggered Activation of Dendritic Cells.

Abietic acid (AA), dehydroabietic acid (DHA) and triptoquinones (TQs) are bioactive abietane-type diterpenoids, which are present in many edible vegetables and medicinal herbs with health-promoting properties. Evidence suggests that beneficial effects of diterpenes operate, at least in part, through effects on cells in the immune system. Dendritic cells (DCs) are a key type of leukocyte involved in the initiation and regulation of the immune/inflammatory response and natural or synthetic compounds that modulate DC functions could be potential anti-inflammatory/immunomodulatory agents. Herein, we report the screening of 23 known semisynthetic AA and DHA derivatives, and TQs, synthesized previously by us, in a multi-analyte DC-based assay that detects inhibition of pro-inflammatory cytokine production. Based on the magnitude of the inhibitory effect observed and the number of cytokines inhibited, a variety of activities among compounds were observed, ranging from inactive/weak to very potent inhibitors. Structurally, either alcohol or methyl ester substituents on ring A along with the introduction of aromaticity and oxidation in ring C in the abietane skeleton were found in compounds with higher inhibitory properties. Two DHA derivatives and two TQs exhibited a significant inhibition in all pro-inflammatory cytokines tested and were further investigated. The results confirmed their ability to inhibit, dose dependently, LPS-stimulated expression of the co-stimulatory molecules CD40 and/or CD86 and the production of the pro-inflammatory cytokines IL-1ß, IL-6, IL-12 and TNFα. Our results demonstrate that DC maturation process can be targeted by semisynthetic DHA derivatives and TQ epimers and indicate the potential of these compounds as optimizable anti-inflammatory/immunomodulatory agents.

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.

Flukicidal effects of abietane diterpenoid derived analogues against the food borne pathogen Fasciola hepatica.

Control of liver fluke infections remains a significant challenge in the livestock sector due to widespread distribution of drug resistant parasite populations. In particular, increasing prevalence and economic losses due to infection with Fasciola hepatica is a direct result of drug resistance to the gold standard flukicide, triclabendazole. Sustainable control of this significant zoonotic pathogen, therefore, urgently requires the identification of new anthelmintics. Plants represent a source of molecules with potential flukicidal effects and, amongst their secondary metabolites, the diterpenoid abietic acids can be isolated in large quantities. In this study, nineteen (19) chemically modified abietic acid analogues (MC_X) were first evaluated for their anthelmintic activities against F. hepatica newly excysted juveniles (NEJs, from the laboratory-derived Italian strain); from this, 6 analogues were secondly evaluated for their anthelmintic activities against adult wild strain flukes. One analogue, MC010, was progressed further against 8-week immature- and 12-week mature Italian strain flukes. Here, MC010 demonstrated moderate activity against both of these intra-mammalian fluke stages (with an adult fluke EC50 = 12.97 µM at 72 h post culture). Overt mammalian cell toxicity of MC010 was inferred from the Madin-Darby bovine kidney (MDBK) cell line (CC50 = 17.52 µM at 24 h post culture) and demonstrated that medicinal chemistry improvements are necessary before abietic acid analogues could be considered as potential anthelmintics against liver fluke pathogens.

MAPKK2/4/5/7-MAPK3-JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza.

Phenolic acids are the major bioactive metabolites produced in Salvia miltiorrhiza, a traditional Chinese medicine called Danshen. Many phytohormone elicitor treatments induce phenolic acid biosynthesis, even though the underlying mechanism remains obscure. Expression pattern analysis showed that SmMAPK3 was highly expressed in leaves, and SmMAPK3 was significantly induced by salicylic acid (SA) and methyl jasmonate (JA). Bioinformatics analysis revealed that SmMAPK3 belongs to group A and contains a TEY motif in the activation loop together with three conserved regions (P-loop, C-loop and CD-domain). A previous study speculated that SmMAPK3 is likely a positive regulator in the biosynthesis of phenolic acids in S. miltiorrhiza. In this study, overexpression of SmMAPK3 increased phenolic acid biosynthetic gene expression and enhanced the accumulation of phenolic acids in S. miltiorrhiza plantlets. Yeast two-hybrid (Y2H) analysis and firefly luciferase complementation imaging (LCI) assays revealed that SmMAPKK2/4/5/7-SmMAPK3-SmJAZs form a cascade that regulates the accumulation of phenolic acids. In summary, this work deepens our understanding of the posttranscriptional regulatory mechanisms of phenolic acid biosynthesis and sheds new light on metabolic engineering in S. miltiorrhiza.

Tanshinones: Leading the way into Lamiaceae labdane-related diterpenoid biosynthesis.

Tanshinones are the bioactive diterpenoid constituents of the traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), and are examples of the phenolic abietanes widely found within the Lamiaceae plant family. Due to the significant interest in these labdane-related diterpenoid natural products, their biosynthesis has been intensively investigated. In addition to providing the basis for metabolic engineering efforts, this work further yielded pioneering insights into labdane-related diterpenoid biosynthesis in the Lamiaceae more broadly. This includes stereochemical foreshadowing of aromatization, with novel protein domain loss in the relevant diterpene synthase, as well as broader phylogenetic conservation of the relevant enzymes. Beyond such summary of more widespread metabolism, formation of the furan ring that characterizes the tanshinones also has been recently elucidated. Nevertheless, the biocatalysts for the pair of demethylations remain unknown, and the intriguing potential connection of these reactions to the further aromatization observed in the tanshinones are speculated upon here.

HPTLC and ATR/FTIR Characterization of Antioxidants in Different Rosemary Extracts.

The effect of spontaneous fermentation by lactic acid bacteria on the extraction yield of bioactive compounds and antioxidant activity from rosemary leaf extracts was investigated using high-performance thin-layer chromatography (HPTLC). Brining and spontaneous fermentation with lactic acid bacteria more than doubled extraction of polyphenolics and antioxidants from the rosemary leaves. The results show that lactic acid fermentation enhances antioxidant activity in extracts by increasing the total phenolic content but does not increase extraction of phytosterols. Increased extraction of phenolic oxidants during fermentation assisted extraction, results from the in situ generated natural eutectic solvent from the plant sample. ATR-FTIR spectra from the bioactive bands suggests that this increased antioxidant activity is associated with increased extraction of rosmarinic acid, depolymerised lignin, abietane diterpenoids and 15-hydroxy-7-oxodehydroabietic acid.

Targeting Aurora B kinase with Tanshinone IIA suppresses tumor growth and overcomes radioresistance.

Aurora B kinase is aberrantly overexpressed in various tumors and shown to be a promising target for anti-cancer therapy. In human oral squamous cell carcinoma (OSCC), the high protein level of Aurora B is required for maintaining of malignant phenotypes, including in vitro cell growth, colony formation, and in vivo tumor development. By molecular modeling screening of 74 commercially available natural products, we identified that Tanshinone IIA (Tan IIA), as a potential Aurora B kinase inhibitor. The in silico docking study indicates that Tan IIA docks into the ATP-binding pocket of Aurora B, which is further confirmed by in vitro kinase assay, ex vivo pull-down, and ATP competitive binding assay. Tan IIA exhibited a significant anti-tumor effect on OSCC cells both in vitro and in vivo, including reduction of Aurora B and histone H3 phosphorylation, induction of G2/M cell cycle arrest, increase the population of polyploid cells, and promotion of apoptosis. The in vivo mouse model revealed that Tan IIA delayed tumor growth of OSCC cells. Tan IIA alone or in combination with radiation overcame radioresistance in OSCC xenograft tumors. Taken together, our data indicate that Tan IIA is an Aurora B kinase inhibitor with therapeutic potentials for cancer treatment.

Improving in vitro bioaccessibility and bioactivity of carnosic acid using a lecithin-based nanoemulsion system.

As a phenolic terpenoid, carnosic acid (CA) mainly exists in rosemary, which can be effectively used for the treatment of degenerative and chronic diseases by taking advantage of its health-promoting bioactivities. However, the low solubility and dissolution of CA in aqueous solutions at ambient and body temperatures result in low stability and bioaccessibility during the digestion process, which limits its application scope in the functional foods industry. In this regard, a lecithin based nanoemulsion system (CA-NE) is employed in the present work to enhance the bioaccessibility and bioactivities of CA. It is revealed that the CA-NE under investigation exhibits high loading capacity (2.80 ± 0.15%), small particle size (172.0 ± 3.5 nm) with homogeneous particle distribution (polydispersity index (PDI) of 0.231± 0.025) and high repulsive force (zeta potential = -57.2 ± 0.24 mV). More importantly, the bioaccessibility of CA-NE is improved by 2.8-fold compared to that of CA in MCT oil. In addition, the cellular antioxidant assay (CAA) and cellular uptake study of the CA-NE in HepG2 cell models demonstrate a longer endocytosis process, suggesting the well-controlled release of CA from CA-NE. Furthermore, an improved anti-inflammatory activity was evaluated via the inhibition of the pro-inflammatory cytokines, nitric oxide (NO) and TNF-α production in LPS-stimulated RAW 264.7 macrophage cells. The results clearly demonstrated a promising application of CA-NE as a functional food.

Selective stabilization of multiple promoter G-quadruplex DNA by using 2-phenyl-1H-imidazole-based tanshinone IIA derivatives and their potential suppressing function in the metastatic breast cancer.

The G-quadruplex (G4) DNA, which has been developed as a potential anticancer target in drug screening and design, plays a crucial role in the oncogene transcription and translation. Tanshinone IIA derivatives with a planar heterocycle structure may function as G4 stabilizers. We present an innovative case of imidazole-based tanshinone IIA derivatives (1-8) especially compound 4 that improve the selectivity and the binding affinity with G4 DNA and enhance the target tumor inhibition. Cellular and in vivo experiments indicate that the tanshinone IIA derivative 4 inhibits the growth, metastasis, and angiogenesis of triple-negative breast cancer cells possibly through the stabilization of multiple G4 DNAs (e.g., c-myc, K-ras, and VEGF) to induce DNA damage. Further investigation of the intermolecular interaction and the molecular docking indicates that tanshinone IIA derivatives have better selective binding capability to various G4 DNAs than to double-stranded DNA. These findings provide guidance in modifying the molecular structures of tanshinone IIA derivatives and reveal their potential to function as specific G4 stabilizers.

UPLC/MS-based untargeted metabolomics reveals the changes of metabolites profile of Salvia miltiorrhiza bunge during Sweating processing.

Salvia miltiorrhiza has numerous compounds with extensive clinical application. "Sweating", a processing method of Traditional Chinese Medicine (TCM), results in great changes in pharmacology and pharmacodynamics. Previously, chromatogram of 10 characteristic metabolites in S. miltiorrhiza showed a significant difference after "Sweating". Due to the complexity of TCM, changes in metabolites should be investigated metabolome-wide after "Sweating". An untargeted UPLC/MS-based metabolomics was performed to discover metabolites profile variation of S. miltiorrhiza after "Sweating". Multivariate analysis was conducted to screen differential metabolites. Analysis indicated distinct differences between sweated and non-sweated samples. 10,108 substance peaks had been detected altogether, and 4759 metabolites had been identified from negative and positive ion model. 287 differential metabolites were screened including 112 up-regulated and 175 down-regulated and they belong to lipids and lipid-like molecules, and phenylpropanoid and polyketides. KEGG analysis showed the pathway of linoleic acid metabolism, and glyoxylate and dicarboxylate metabolism were mainly enriched. 31 and 49 identified metabolites were exclusively detected in SSM and NSSM, respectively, which mainly belong to carboxylic acids and derivatives, polyketides and fatty acyls. By mapping tanshinones and salvianolic acids to 4759 identified metabolites library, 23 characteristic metabolites had been identified, among which 11 metabolites changed most. We conclude that "Sweating'' has significant effect on metabolites content and composition of S. miltiorrhiza.

Identifying the mechanisms underlying the protective effect of tetramethylpyrazine against cisplatin­induced in vitro ototoxicity in HEI­OC1 auditory cells using gene expression profiling.

Sensorineural hearing loss is prevalent in patients receiving cisplatin therapy. Tetramethylpyrazine (Tet) and tanshinone IIA (Tan IIA) have protective roles against hearing impairment or ototoxicity. The present study aimed to investigate the molecular mechanisms underlying cisplatin­induced ototoxicity and the protective effect of Tet and Tan IIA against it. House Ear Institute­Organ of Corti 1 auditory cells were treated with titrating doses of Tan IIA, Tet, and cisplatin. In a cell viability assay, cisplatin, Tan IIA and Tet had IC50 values of 42.89 µM, 151.80 and 1.04x103 mg/l, respectively. Tan IIA augmented cisplatin­induced cytotoxicity. However, Tet concentrations <75 mg/l attenuated cisplatin­induced cytotoxicity and apoptosis. Moreover, RNA sequencing analysis was carried out on auditory cells treated for 30 h with 30 µM cisplatin alone for 48 h or combined with 37.5 mg/l Tet for 30 h. Differentially expressed genes (DEGs) induced in these conditions were identified and examined using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Cisplatin increased the expression of genes related to the p53 and FoxO pathways, such as Fas, p21/CDKN1A, and Bcl­2 binding component 3, but decreased the expression of insulin­like growth factor 1 (IGF1), as well as genes in the histone (Hist)1 and Hist2 clusters. Treatment with Tet downregulated FOXO3 and Bcl­2 binding component 3, and increased the expression of IGF1. Moreover, Tet upregulated genes associated with Wnt signaling, but not p53­related genes. Thus, the otoprotective properties of Tet might be mediated by activation of Wnt and IGF1 signaling, and inhibition of FoxO signaling.