Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.
Humans ; Salvia miltiorrhiza/metabolism* ; Biosynthetic Pathways ; Quinones/metabolism* ; Plant Roots/metabolism* ; Gene Expression Regulation, Plant

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.
Oxidoreductases ; Biosynthetic Pathways ; Molecular Docking Simulation ; Reproducibility of Results ; Salvia miltiorrhiza/chemistry* ; Amino Acids/metabolism* ; Plant Roots/genetics*

14-3-3 proteins are important proteins in plants, as they regulate plant growth and development and the response to biotic or abiotic stresses. In this study, a 14-3-3 gene(GenBank accession: OM683281) was screened from the cDNA library of the medicinal species Salvia miltiorrhiza by yeast two-hybrid and cloned. The open reading frame(ORF) was 780 bp, encoding 259 amino a cids. Bioinformatics analysis predicted that the protein was a non-transmembrane protein with the molecular formula of C_(1287)H_(2046)N_(346)O_(422)S_9, relative molecular weight of 29.4 kDa, and no signal peptide. Homologous sequence alignment and phylogenetic tree analysis proved that the protein belonged to 14-3-3 family and had close genetic relationship with the 14-3-3 proteins from Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. The 14-3-3 gene was ligated to the prokaryotic expression vector pGEX-4 T-1 and then transformed into Escherichia coli BL21 for the expression of recombinant protein. Real-time fluorescent quantitative PCR showed that the expression of this gene was different among roots, stems, leaves, and flowers of S. miltiorrhiza. To be specific, the highest expression was found in leaves, followed by stems, and the lowest expression was detected in flowers. S. miltiorrhiza plants were treated with 15% PEG(simulation of drought), and hormones salicylic acid, methyl jasmonate, and ethephon, respectively, and the expression of 14-3-3 gene peaked at the early stage of induction. Therefore, the gene can quickly respond to abiotic stresses such as drought and plant hormone treatments such as salicylic acid, jasmonic acid, and ethylene. This study lays the foundation for revealing the molecular mechanism of 14-3-3 protein regulating tanshinone biosynthesis and responding to biotic and abiotic stresses.
14-3-3 Proteins/metabolism* ; Amino Acid Sequence ; Cloning, Molecular ; Ethylenes/metabolism* ; Gene Expression Regulation, Plant ; Hormones/metabolism* ; Phylogeny ; Plant Growth Regulators/pharmacology* ; Plant Proteins/metabolism* ; Recombinant Proteins/genetics* ; Salicylic Acid/metabolism* ; Salvia miltiorrhiza/metabolism*

The solid wastes of Chinese materia dedica industrialization represented by Salvia miltiorrhiza residues have a strong small-molecule bio-recalcitrance in the process of high-value utilization of biotransformation. Highly tolerant strains were bred to break bio-recalcitrance of Salvia miltiorrhiza residues and produce high-value added cellulose, which has a significant significance for recycling and industrial utilization of solid waste. In this study, a strain of fungus, Penicillium expansum SZ13, was found with small-molecule antibacterial substance tanshinone contained in Salvia miltiorrhiza residues by a biological method. The optimal enzyme production process and peak period of SZ13 were determined. It was found that SZ13 could maintain peak enzyme production for 5 days by degrading residues under the conditions of temperature 35 ℃, rotation speed 180 r·min~(-1), 5% of residues addition, and 5% seed solution addition. Meanwhile, the ability of SZ13 to degrade the enzyme production of multiple types of residues was explored. The results showed a high enzyme activity and stable enzyme production of SZ13 in the process of degrading residues. SZ13 could efficiently utilize various types of Chinese medicine residues, such as Salvia miltiorrhiza residues, to realize the high-value utilization of cellulose in multiple types of residues.
Cellulase/biosynthesis* ; China ; Drug Industry ; Drugs, Chinese Herbal ; Fermentation ; Materia Medica ; Penicillium/metabolism* ; Salvia miltiorrhiza ; Solid Waste

Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos are commonly traditional Chinese medicines with invigorating blood circulation and eliminating blood stasis,but they are different in effects due to differences in five tastes and four properties. In this study,Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos are selected as research vectors to obtain the active ingredients and targets through TCMD,TCMSP database and Ch EMBL database. The protein interaction information of the target is used to obtain from STRING online database,then imported into Cytoscape software to plot the protein interaction network and perform GO enrichment analysis.The results show that the heart-liver protein interaction network,involving blood circulation and hepatic lipid metabolism,thereby exerting the effect of activating blood circulation. The cold-bitter protein interaction network involves the biological process of vasoconstriction,thereby exerting cooling blood and the efficacy of eliminating phlegm. The warm-pungent protein interaction network involves blood coagulation,lipid metabolism and other biological processes to play the role of phlegm pain. Through analysis,it is found that the relationship between pharmacological efficacy and medicinal properties has a certain degree of specificity,which facilitates the subsequent scientific and systematic study of medicinal properties on the basis of this study.
Blood Coagulation ; Carthamus ; chemistry ; Databases, Pharmaceutical ; Drugs, Chinese Herbal ; pharmacology ; Flowers ; chemistry ; Humans ; Lipid Metabolism ; Medicine, Chinese Traditional ; Plant Roots ; chemistry ; Rhizome ; chemistry ; Salvia miltiorrhiza ; chemistry

This study is to investigate the compatibility mechanism of Danshen-Chuanxiong drug pair on the pharmacokinetics of four phenolic acids. A UPLC-MS/MS method for quantitative determination of salvianolic acid B( Sal B),rosmarinic acid( RA),lithospermic acid( LA) and ferulic acid( FA) in plasma and heart tissue of rats was established. After single salvianolic acids and Chuanxiong-extract or combined intravenous infusion was given to rats,plasma samples and heart tissues in different time were collected. The chromatographic separation was performed on a BEH C18 column using 0. 15% formic acid-acetonitrile as mobile phase for gradient elution. A triple-quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating on multiple-reaction monitoring( MRM) scanning in negative ionization mode. Full validation of UPLC method including calibration curves,accuracy,precision,repeatability and matrix effect was investigated to comply with quantitative analysis requirements for biological samples. There were significant differences in the major pharmacokinetic parameters of Sal B,FA and RA for intravenous infusion of salvianolic acids and Chuanxiong-extract or combined in rat plasma. The AUC of Sal B and FA were increased above 40% and100%,respectively. Their Vd and CL were dropped evidently. t1/2 and Vd of RA increased above 130%. The concentration of four phenolic acids were all increased obviously in heart tissue comparing with single infusion. These results demonstrated that the compatibility mechanism of Danshen-Chuanxiong drug pair showed synergistic effect.
Animals ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Drugs, Chinese Herbal/metabolism* ; Heart/physiology* ; Hydroxybenzoates ; Rats ; Rats, Sprague-Dawley ; Reproducibility of Results ; Salvia miltiorrhiza ; Tandem Mass Spectrometry

Postmenopausal women, who have reduced circulating estrogen levels, are more prone to develop obesity and related metabolic diseases than premenopausal women. The absence of safe and effective treatments for postmenopausal obesity has changed the focus to natural products as alternative remedies. Total salvianolic acids (TSA) are the major water-soluble ingredients of Danshen. Salvianolic acid (SA) is the major constituent of the TSA. Salvianolic acids, including TSA and SA, are widely used in traditional Chinese medicine. In the present study, ovariectomized rats and LO2 cells were used to study the effects of salvianolic acids on body weight gain and hepatic steatosis. Salvianolic acids reduced ovariectomy (OVX)-induced body weight gain, attenuated the expressions of hepatic lipogenic genes, such as sterol regulatory element binding protein (SREBP)1, fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD)1, and decreased the liver triglyceride (TG) and total cholesterol (TC). For the molecular mechanisms, OVX and high glucose-induced phosphorylation of signal transducer and activator of transcription (STAT)-3 was inhibited by salvianolic acids treatment. In LO2 cells, inhibition of STAT-3 by siRNA attenuated the increased expression of SREBP1 and TG induced by high glucose. Salvianolic acids reduced the upregulation of SREBP1 and TG induced by high glucose in LO2 cells. In conclusion, these findings illustrated that salvianolic acids markedly alleviated the lipid metabolism disorders and protected against the postmenopausal obesity. The underlying mechanism was probably associated with the regulation of STAT-3 signaling.
Alkenes ; administration & dosage ; Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Lipid Metabolism ; drug effects ; Liver ; drug effects ; metabolism ; Obesity ; drug therapy ; genetics ; metabolism ; Ovariectomy ; Polyphenols ; administration & dosage ; Postmenopause ; drug effects ; genetics ; metabolism ; Rats ; STAT3 Transcription Factor ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; Signal Transduction ; drug effects ; Sterol Regulatory Element Binding Protein 1 ; genetics ; metabolism ; Triglycerides ; metabolism

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; Blood-Brain Barrier ; drug effects ; enzymology ; immunology ; Brain ; Brain Ischemia ; drug therapy ; enzymology ; genetics ; Caffeic Acids ; administration & dosage ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Lactates ; administration & dosage ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; enzymology ; genetics ; immunology ; prevention & control ; Salvia miltiorrhiza ; chemistry ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; immunology

Danshen, the dried root of Salvia miltiorrhiza Bunge (Lamiaceae), is one of the traditional Chinese medicines (TCMs) most commonly used for the treatment of cardiovascular and cerebrovascular diseases. However, little is known about the chemical and metabolic profiles of danshen in vitro or in vivo. In particular, more information is needed in relation to the 50% ethanol extracts usually used in danshen formulations such as Fufang Xueshuantong Capsules and Fufang Danshen tablets. High-performance liquid chromatography coupled with a linear ion trap-Orbitrap mass spectrometer (HPLC-LTQ-Orbitrap) provides a sensitive and accurate method for analyzing the composition of samples. This method was used to determine the in vitro and in vivo chemical and metabolic profiles of danshen. Sixty-nine components of danshen extract and 118 components of danshen in rat plasma, urine, feces, and bile were unambiguously or tentatively identified. These results not only revealed the material composition of danshen, but also provided a comprehensive research approach for the identification of multi-constituents in TCMs.
Animals ; Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/metabolism* ; Male ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; Spectrometry, Mass, Electrospray Ionization

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA, MeJA and Ag, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.
Amino Acid Sequence ; Benzofurans ; Biosynthetic Pathways ; genetics ; Cinnamates ; Depsides ; Gene Expression Regulation, Plant ; genetics ; Oxidoreductases ; genetics ; Phenylpropionates ; metabolism ; Phenylpyruvic Acids ; metabolism ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; chemistry ; enzymology ; genetics ; metabolism ; Plants, Genetically Modified ; Recombinant Proteins ; analysis ; biosynthesis ; Salvia miltiorrhiza ; chemistry ; enzymology ; genetics ; metabolism ; Sequence Alignment