Diterpenoid Tanshinone Research Articles

Exploring the Potential Mechanisms of Guanxinshutong Capsules in Treating Pathological Cardiac Hypertrophy based on Network Pharmacology, Computer-Aided Drug Design, and Animal Experiments.

Cardiovascular diseases (CVDs) are significant causes of morbidity and mortality worldwide, and pathological cardiac hypertrophy (PCH) is an essential predictor of many heart diseases. Guanxinshutong capsule (GXST) is a Chinese patent medicine widely used in the clinical treatment of CVD, In our previous research, we identified 111 compounds of GXST. In order to reveal the potential molecular mechanisms by which GXST treats PCH, this study employed network pharmacology methods to screen for the active ingredients of GXST in treating PCH and predicted the potential targets. The results identified 26 active ingredients of GXST and 110 potential targets for PCH. Through a protein-protein interaction (PPI) network, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we confirmed AKT1, MAPK1, and MAPK3 as the core proteins in GXST treatment of PCH, thus establishing the PI3K/AKT and MAPK signaling pathways as the significant mechanisms of GXST in treating PCH. The results of molecular docking (MD) demonstrate that flavonoid naringenin and diterpenoid tanshinone iia have the highest binding affinity with the core protein. Before performing molecular dynamics simulations (MDSs), the geometric structure of naringenin and tanshinone iia was optimized using density functional theory (DFT) at the B97-3c level, and RESP2 atomic charge calculations were carried out at the B3LYP-D3(BJ)/def2-TZVP level. Further MDS results demonstrated that in the human body environment, the complex of naringenin and tanshinone iii with core proteins exhibited high stability, flexibility, and low binding free energy. Additionally, naringenin and tanshinone iia showed favorable absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics and passed the drug similarity (DS) assessment. Ultrasound cardiograms and cardiac morphometric measurements in animal experiments demonstrate that GXST can improve the PCH induced by isoproterenol (ISO). Protein immunoblotting results indicate that GXST increases the expression of P-eNOS and eNOS by activating the PI3K/AKT signaling pathway and the MAPK signaling pathway, further elucidating the mechanism of action of GXST in treating PCH. This study contributes to the elucidation of the key ingredients and molecular mechanisms of GXST in treating PCH.

Diterpenoid tanshinone attenuates the metastasis of non-small-cell lung cancer (NSCLC) cells by inhibiting the Cavin-1-mediated ERK/Smad2 signaling pathway.

The objective of this study is to explore the potential therapeutic role of diterpenoid tanshinone (DT) against non-small cell lung cancer (NSCLC) in vitro and elucidate the molecular mechanism involved in tumor metastasis. Human NSCLC lines (A549 and NCI-H1299) were transfected with pcDNA3.1-Cavin-1 plasmids and corresponding controls. We tested the effects of DT on migration and invasion of lung cancer cells using transwell filters coated with fibronectin and Matrigel. Next, Quantitative Real-Time PCR and western blot were used to determine the transcriptional and protein levels of epithelial-mesenchymal transition (EMT) markers, transcription factors (Snail, Slug), and matrix metalloproteinases. As expected, Cavin-1 related to the enhanced ability of cell migration and invasion. DT not only inhibited the migratory and invasive capacity of Cavin-1-transfected NSCLC cells but also significantly increased the expression of ZEB1 and E-cadherin and decreased the level of N-cadherin, Vimentin, Snail, and Slug. Moreover, DT treatment obviously alleviated Cavin-1 overexpression-induced high levels of MMP2, MMP7, and MMP9 at both the protein and transcriptional levels. Furthermore, overexpressed Cavin-1 upregulated ERK and Smad2 signaling pathways in NSCLC cells, which were also strongly weakened by DT administration. Our results suggested that DT effectively attenuates Cavin-1-mediated NSCLC metastasis via the ERK/Smad2 signaling pathway.

The AP2/ERF transcription factor SmERF128 positively regulates diterpenoid biosynthesis in Salvia miltiorrhiza.

The novel AP2/ERF transcription factor SmERF128 positively regulates diterpenoid tanshinone biosynthesis by activating the expression of SmCPS1, SmKSL1, and SmCYP76AH1 in Salvia miltiorrhiza. Certain members of the APETALA2/ethylene-responsive factor (AP2/ERF) family regulate plant secondary metabolism. Although it is clearly documented that AP2/ERF transcription factors (TFs) are involved in sesquiterpenoid biosynthesis, the regulation of diterpenoid biosynthesis by AP2/ERF TFs remains elusive. Here, we report that the novel AP2/ERF TF SmERF128 positively regulates diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza. Overexpression of SmERF128 increased the expression levels of copalyl diphosphate synthase 1 (SmCPS1), kaurene synthase-like 1 (SmKSL1) and cytochrome P450 monooxygenase 76AH1 (SmCYP76AH1), whereas their expression levels were decreased when SmERF128 was silenced. Accordingly, the content of tanshinone was reduced in SmERF128 RNA interference (RNAi) hairy roots and dramatically increased in SmERF128 overexpression hairy roots, as demonstrated through Ultra Performance Liquid Chromatography (UPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. Furthermore, SmERF128 activated the expression of SmCPS1, SmKSL1, and SmCYP76AH1 by binding to the GCC box, and to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs within their promoters during in vivo and in vitro assays. Our findings not only reveal the molecular basis of how the AP2/ERF transcription factor SmERF128 regulates diterpenoid biosynthesis, but also provide useful information for improving tanshinone production through genetic engineering.

Anti-lung cancer mechanisms of diterpenoid tanshinone via endoplasmic reticulum stress-mediated apoptosis signal pathway

At present, lung cancer ranks second and first respectively in the incidence and the mortality among malignant tumors. It is urgent to find new effective anti-lung cancer drugs with less side effects and relatively defined mechanisms. Endoplasmic reticulum stress (ERS)-mediated apoptosis pathway is an effective way to promote tumor cell apoptosis; diterpenoid tanshinone (DT), an effective part separated from Salviae Miltiorrhizae Radix et Rhizoma, was found to have an anti-lung cancer effect in previous studies via ERS-induced PERK-EIF2α pathway. In this paper, human lung adenocarcinoma PC9 cell line and nude mouse transplantation tumor model were applied to verify the anti-lung cancer effect of DT in vivo and in vitro, and illuminate the potential mechanism via ERS induced IRE1α/caspase 12 apoptosis pathway. The results showed that in vivo, DT could promote PC9 cell apoptosis in a concentration-dependent manner, up-regulate Bip, IRE1 and TRAF2 protein expressions in tumor tissue, reduce tumor weight and alleviate bodyweight loss. In vitro, DT inhibited the proliferation of PC9 cell line in a concentration-dependent manner, and destroyed the structure of mitochondria in PC9 cell, promoted Bax, IRE1α, Bip, TRAF2 and caspase 12 protein expressions, lower Bcl-2 protein expression in a time-dependent manner. DT shows a good effect on anti-lung cancer both in vivo and in vitro. The mechanism is related to the activation of ERS-induced IRE1α/caspase 12 apoptosis pathway and the promotion of cell apoptosis. ERS-mediated apoptosis pathway may be an important target of DT on anti-lung cancer.

Diterpenoid Tanshinones, the extract from Danshen (Radix Salviae Miltiorrhizae) induced apoptosis in nine human cancer cell lines

ObjectiveTo identify the active anti-tumor constituents in the extract from Danshen (Radix Salviae Miltiorrhizae) and investigate the mechanisms underlying the actions. MethodsFirst, we introduced a two-step counter-current chromatography to extract the therapeutically active diterpenoid, tanshinone from Danshen (Radix Salviae Miltiorrhizae). The cholecystokinin (CCK-8) method was used to evaluate the inhibitory effect of diterpenoid tanshinone in liver cancer QGY-7703, lung cancer PC9, lung cancer A549, gastric cancer MKN-45, gastric cancer HGC-27, colon cancer HCT116, myeloma cellU266/RPMI8226, and human breast cancer MCF-7 in vitro. Fluorescence staining was used to observe the cytotoxicity ofditerpenoid tanshinone on PC9 cells. The Western blot was used to detect apoptosis-related protein poly ADP-ribose polymerase (PARP), cysteinyl aspartate specific proteinase3/9 (caspase3/9), and cleaved-cysteinyl aspartate specific proteinase3/9 (cleaved-caspase3/9). The endoplasmic reticulum stress-related activating transcription factor 4 (ATF4), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and phosphorylated jun amino-terminal kinase (p-JNK), and caspase-12 were also analyzed using the Western blot. ResultsDiterpenoid tanshinone inhibited the nine human tumor cell lines, with an IC50 of 4.37–29 μg/mL, with the PC9 and MCF-7 displaying the lowest values. Fluorescence staining showed a lethal effect of diterpenoid tanshinone on PC9 cells. The Western blot showed that the expression of caspase3/9 protein and ATF-4 protein decreased gradually. However, the PARP, cleaved-caspase 3/9 and the expression of p-eIF2 α, P-JNK, and caspase-12 increased gradually, in a dose-dependent fashion. ConclusionWe successfully introduced a two-step counter-current chromatography method to extract diterpenoid tanshinone, and demonstrated its antitumor activity. Diterpenoid tanshinone can induce apoptosis in nine human cancer cell lines.

Activation tagging in Salvia miltiorrhiza can cause increased leaf size and accumulation of tanshinone I and IIA in its roots.

BackgroundSalvia miltiorrhiza Bunge (Danshen), an important herb in traditional Chinese medicine, is commonly used for treatment of cardiovascular diseases. One of the major bioactive constituents of Danshen, diterpenoid tanshinone, has been proved with pharmacological properties and have the potential to be a new drug candidate against various diseases. In our previous study, we have established an activation tagging mutagenesis (ATM) population of callus lines of S. miltiorrhiza Bunge by Agrobacterium- mediated transformation.ResultsIn the present study, we have identified ATM transgenic Salvia plant (SH41) with different leaf morphology and more tanshinones in its roots. The transgenic background of SH41 was identified by PCR (using hpt II primers) and Southern blots. PCR analysis showed a single band of hpt II gene and Southern blot analysis showed single insertion in SH41. External appearance of ATM transgenic SH41 was observed with broader leaves comparing to non-transformed plants. More healthy trichomes as well as bigger and wobbly guard cells and stomata were observed in SH41 by scanning electron microscopy (SEM). Quantitative analysis of active compounds in SH41 roots revealed a significant increase in tanshinone I (3.7 fold) and tanshinone IIA (2 fold) contents as compared to the wild plant.ConclusionsWe have generated an activation tagged transgenic Salvia plant (SH41) with different leaf morphology and high diterpenes content in its roots. The increased amount of tanshinones in SH41 will definitely offer a route for maximizing the benefits of this plant in traditional Chinese herbal medicines. The present report may also facilitate the application of ATM for genetic manipulation of other medicinal crops and subsequent improved metabolite contents.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-37) contains supplementary material, which is available to authorized users.

Investigation on equilibrium solubility and apparent oil/water partition coefficient of diterpenoid tanshinone component by similarity analysis methods

To study the equilibrium solubility and apparent oil/water partition coefficient of diterpenoid tanshinone component by similarity analysis methods, so as to lay the foundation for Salvia diterpenoid quinone component of the overall solubility characterization. Taking Salvia diterpenoid quinone component as model drug, determined the equilibrium solubility and partition coefficients of the components in different buffer, evaluated the degree of similarity of each component based on the cosine and Grubbs. The representative composition of Salvia diterpenoid quinone component, namely dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone II(A) had similar properties of equilibrium solubility and partition coefficients in different pH buffer. This similarity was not only manifested in the trends, but also reflected the value. This similarity assessment reflected the degree of deviation and dispersion of components, which was applicable to the study of the components; the similarity assessment could increase the science and rationality of component evaluation, and at the same time could optimize the structure of the component.

Cloning, molecular characterization and functional analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza Bge. f. alba

The enzyme 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) is a terminal-acting enzyme in the plastid MEP pathway, which produce isoprenoid precursors. The full-length cDNA of HDR, designated SmHDR1 (Genbank Accession No. JX516088), was isolated for the first time from Salvia miltiorrhiza Bge. f. alba. SmHDR1 contains a 1389-bp open reading frame encoding 463 amino acids. The deduced SmHDR1 protein, which shows high identity to HDRs of other plant species, is predicted to possess a chloroplast transit peptide at the N-terminus and four conserved cysteine residues. Transcription pattern analysis revealed that SmHDR1 has high levels of transcription in leaves and low levels of transcription in roots and stems. The expression of SmHDR1 was induced by 0.1mM methyl-jasmonate (MeJA) and salicylic acid (SA), but not by 0.1mM abscisic acid (ABA), in the hairy roots of S.miltiorrhiza Bge. f. alba. Complementation of SmHDR1 in the Escherichia coli HDR mutant MG1655 ara < > ispH demonstrated the function of this enzyme. A functional color assay in E.coli showed that SmHDR1 accelerates the biosynthesis of β-carotene, indicating that SmHDR1 encodes a functional protein. Overexpression of SmHDR1 enhanced the production of tanshinones in cultured hairy roots of S.miltiorrhiza Bge. f. alba. These results indicate that SmHDR1 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S.miltiorrhiza Bge. f. alba.

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.

Cloning and characterization of the 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza (Chinese sage) hairy roots

Salvia miltiorrhiza Bunge (Chinese sage; Lamiaceae) is a valuable Chinese herbal plant, and its rhizome, known as Danshen in Chinese because of its characteristic red pigment, is the part of the plant used in herbal medicine. The red pigment in S. miltiorrhiza roots is mainly composed of numerous diterpenoid tanshinones, as the major bioactive ingredients of the herb. In plants, diterpenes are synthesized through the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway in the plastids, and DXR [DXP (1-deoxy-D-xylulose 5-phosphate) reductoisomerase] is an enzyme catalysing the first step of the MEP pathway. In the present study, a full-length cDNA encoding DXR (GenBank Nucleotide Sequence Database accession no. DQ991431) was cloned from the hairy roots of S. miltiorrhiza in culture. The enzyme activity of DXR protein was verified by complementation of an Escherichia coli mutant deficient in dxr. The transcription level of the dxr gene in the hairy roots was up-regulated after exposure to hyperosmotic stress and a yeast elicitor in parallel with increased tanshinone accumulation in the hairy roots. This is the first report, to our knowledge, of elicitor-induced dxr transcription and its correlation with the accumulation of diterpenoid tanshinones in S. miltiorrhiza roots.

Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of Salvia miltiorrhiza hairy roots

Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.

Enhancement of tanshinone production in Salvia miltiorrhiza Bunge (red or Chinese sage) hairy‐root culture by hyperosmotic stress and yeast elicitor

The present study evaluates the use of hyperosmotic stress and a biotic elicitor YE (yeast elicitor; polysaccharide fraction of yeast extract) to stimulate diterpenoid tanshinone production in hairy-root culture of Salvia miltiorrhiza Bunge (Lamiaceae). Sorbitol was applied as an osmoticum at various concentrations (30-100 g/l) to the hairy-root culture in MS (Murashige and Skoog) medium [Murashige and Skoog (1962) Physiol. Plant. 15, 473-497] containing 30 g/l sucrose, and it increased the TT (total tanshinone) content of roots most dramatically at 50-70 g/l (medium osmolality 410-500 mmol/kg; 1 mol/kg approximately 1 osmol/kg), to 4.5-fold of that in the control. The hairy roots showed strong tolerance to hyperosmotic stress, retaining a stable or higher dry weight of roots at osmolality up to 500 mmol/kg. Most remarkably, the combined use of sorbitol (50 g/l) and YE (100 mg/l) increased the TT content 10-fold (1481.6 versus 146.4 microg/g dry root) and the volumetric tanshinone yield 9-fold (16.3 versus 1.77 mg/l) compared with the control. The results suggest that the combined use of hyperosmotic stress and a biotic elicitor can effectively enhance secondary metabolite production in hairy-root cultures.

Induction and potentiation of diterpenoid tanshinone accumulation in Salvia miltiorrhiza hairy roots by β-aminobutyric acid

The non-protein amino acid beta-aminobutyric acid (BABA) is a proven inducer of plant defense against pathogens. This work examines its effect on the production of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures, both separately and in combination with a yeast elicitor (YE, the carbohydrate fraction of yeast extract). In the absence of YE, BABA at 0.1, 1 and 2 mM caused a dose-dependent enhancement of tanshinone accumulation, with up to a 4.5-fold increase (from 0.24 to 1.09 mg/g DW) in total content of three major tanshinones (cryptotanshinone, tanshinone I and tanshinone IIA) in the hairy roots. The combination of BABA with YE treatment further enhanced tanshinone production, but only when the BABA treatment was applied to the culture a few days before the YE treatment. Compared with methyl jasmonate, BABA was more effective in enhancing tanshinone production. A 3-day pretreatment with 1 mM BABA followed by YE-treatment, increased the total tanshinone content of roots by 9.4 times to 2.26 mg/g cells, and the volumetric tanshinone yield of culture by 6.3 times (from 3.2 to 20.1 mg/l). The results suggest that BABA can strongly potentiate elicitor-induced secondary metabolism in plant tissue cultures.