Identification and Expression Pattern of Salvia miltiorrhiza Terpenoid Synthase Gene Family / 中国实验方剂学杂志

ObjectiveBioinformatics methods were used to systematically identify the Salvia miltiorrhiza terpenoid synthase （SmTPS） gene family members and predict their functions from the perspective of the genome. MethodThe genome and transcriptome data of S. miltiorrhiza， Arabidopsis thaliana， and tomato were obtained from the national genomics data center （NGDC）， national center for biotechnology information （NCBI）， the Arabidopsis information resource （TAIR）， and tomato functional genomics database （TFGD）， and the whole genome identification and bioinformatics analysis of the SmTPS gene family member were carried out with the help of Perl language programming， Tbtools， and other bioinformatics tools. ResultA total of 52 TPS gene family members were identified， and they were distributed on eight chromosomes of S. miltiorrhiza. Their coding amino acid number was 207-822 aa. The isoelectric points were 4.76-9.16. The molecular mass was 24.11-94.81 kDa， and all members are hydrophilic proteins. Gene structure analysis showed that there were significant differences in the number of introns among different subfamilies. The number of introns in 72.6% of TPS-a， b， and g subfamilies was 6， and that in 88.9% of TPS-c and e/f subfamilies was more than 10. Protein motifs were conserved among TPS subfamilies. The analysis of promoter cis-acting elements showed that all promoters of the SmTPSs contained a large number of light-responsive elements， and most of them had hormone-responsive elements. Gene expression analysis showed that SmTPS gene family members exhibited tissue-specific expression， and 24 of them responded to exogenous methyl jasmonate. ConclusionBased on the published S. miltiorrhiza genome， 52 SmTPS gene family members were identified， and their functions were predicted based on the phylogenetic analysis and expression patterns. This paper provides reference information for the further biosynthesis pathway and regulatory mechanism analysis of terpenoids in S. miltiorrhiza.

Advances in pharmacological effects of tanshi-none ⅡA on pain / 中国药理学与毒理学杂志

Pain is one of the most serious problems plaguing human health today.Drug therapy is one of the main ways to treat pain in clinic.The analgesic drugs commonly used in clinical treatment of pain are often accompanied by many side effects,the analgesic effect is still not ideal.Salvia miltiorrhiza is a traditional medici-nal material with the same origin as food and medicine.It has the functions of promoting blood circulation and removing blood stasis,relieving pain through menstrual circulation,and contains many effective ingredients such as tanshinone and salvianolic acid.Tanshinone is a kind of rosin diterpenoid compound,which mainly consists of o-quinone type and p-quinone type parent nucleus,and tanshinone Ⅱ A is the representative compound.The pharmacological mechanism of tanshinone ⅡA in labor pain mainly includes:① Regulate inflammatory factors.Inflammatory cytokines played an important role in the occurrence and progression of pain.It was found that the analgesic effect of tanshinone ⅡA was related to the anti-inflammatory effect.Tanshinone ⅡA showed anti-injuri-ous activity in various pain models,such as bone cancer pain and sciatic nerve ligation,and related studies found that tanshinone ⅡA could inhibit the expression of inflammatory factors TNF-α,IL-1β and IL-6 in the spinal cord of model rats.In the spinal nerve ligation model,tanshinone ⅡA also promoted the release of anti-inflam-matory cytokine IL-10 in the spinal cord of rats.② Regu-late signal pathways related to regulating spinal cord oxi-dation and apoptosis.Apoptosis and oxidation played an important role in the process of pain.When nerve injury was caused by stimulation,oxidative stress and apopto-sis of nerve cells were involved in the mechanism of hyperalgesia.Tanshinone ⅡA sodium sulfonate could relieve pain by regulating apoptosis-related pathways.In neuralgia model,tanshinone ⅡA could reduce the apop-tosis of spinal cord neurons by inhibiting oxidative stress response in rat spinal cord tissue.In addition,tanshinone ⅡA also decreased the expression of pro-apoptotic protein in spinal dorsal horn of CCI rats.They included caspase-3,Bcl-2,Bax protein,and enhancer binding protein homologous protein,Increased the expres-sion of anti-apoptosis protein Bcl-2.③ Inhibit the activa-tion of spinal cord glial cells.tanshinone ⅡA could exert its labor pain effect by inhibiting the activation of astro-cytes,including inhibiting the expression of chemo-therapy-induced neuralgia,inflammatory pain and inflam-matory cytokines IL-6,IL-1β and TNF-α,and inhibiting the activation of inflammatory signaling pathways related to astrocyte activation.Such as NF-κB signaling path-way,c-Jun N-terminal kinase signaling pathway,etc.In addition,tanshinone ⅡA also inhibited the activation of microglia by inhibiting the expression of CX3CR1 receptor on the surface of microglia and inhibiting the phosphoryla-tion of ERK,JNK and p38 signaling pathways.④ Decr-ease the expression of glutamate receptors in spinal cord.NMDA is an ionic glutamate receptor in the central nervous system,and its subunit NR2B is closely related to pain.The overexpression of NR2B in spinal cord could lead to the decrease of pain threshold,which was an important mechanism of pain generation.The mechani-cal threshold and thermal threshold of CCI rats were increased by tanshinone ⅡA,and the expression of spi-nal dorsal horn 2B subunit was significantly decreased after tanshinone ⅡA treatment in CCI rats.Therefore,it was concluded that the analgesic effect of tanshinone ⅡA on CCI model may be related to the decreased expres-sion of NR2B in spinal dorsal horn.In conclusion,tanshi-none ⅡA can effectively play the role of labor pain,and has great potential for development in the field of medi-cine and health products.

SmHPPR1 from Salvia miltiorrhiza regulated the biosynthesis of salvianolic acids / 药学学报

italic>Salvia miltiorrhiza Bunge is a traditional Chinese medicinal herb widely used to treat cardiovascular and cerebrovascular diseases at clinic. Its main water-soluble components are rosmarinic acid (RA) and salvianolic acid B (SAB), which are produced by phenylpropanoid pathway. 4-Hydroxyphenylpyruvate reductase (HPPR) is a key enzyme in phenylpropanoid metabolism pathway. SmHPPR1 was cloned from S. miltiorrhiza and was constructed into plant expression vector pJR-SmHPPR1. On this basis, SmHPPR1 transgenic Arabidopsis plants were induced and the content of 4-hydroxyphenyllactic acid (pHPL) was determined. SmHPPR1-overexpressing (SmHPPR1-OE) hairy roots of S. miltiorrhiza were obtained and the concentration of active components and transcriptome analysis were performed. The results showed that the concentration of pHPL in SmHPPR1 transgenic Arabidopsis T1 was 0.594 mg·g-1 dry weight. The concentration of RA, SAB and total salvianolic acid in SmHPPR1-OE-3 hairy roots were 1.09, 1.29, 1.15 times of that in control-3, respectively, and the content of Danshensu was 36.26% of that in control-3. Transcriptomic analysis revealed that overexpression of SmHPPR1 caused the upregulation of other phenylpropanoid pathway genes like SmTAT2. Protein-protein interaction indicated CYT (TR74706_c0_g1), NADP+ (TR26565_c0_g1) and NADP+ (TR68771_c0_g1) is the central node of the network and participated in metabolic process and cellular process. The tracking work in this study proved that SmHPPR1 could catalyze the reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid in SmHPPR1 transgenic Arabidopsis, and SmHPPR1-overexpressing in hairy roots of S. miltiorrhiza could increase the concentration of salvianolic acids through synergistically regulating other pathway genes.

Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza / 药学学报

Superoxide dismutase (SOD) is a key enzyme that scavenge superoxide anion free radical (O2·-) in vivo, and plays an important role in plant growth and development and stress. In this study, according to the genome and transcriptome data of Salvia miltiorrhizae, 9 SOD genes were identified and the expression patterns of SOD family genes were further analyzed, including 5 Cu/Zn-SOD, 2 Fe-SOD and 2 Mn-SOD. On the basis of proteomic analysis, combined with transcriptome data, one full-length cDNA of Mn-SOD gene, namely SmMSD2 was cloned from Salvia miltiorrhizae. The results of amino acid sequence alignment and phylogenetic analysis showed that SmMSD2 protein belongs to the manganese superoxide dismutase (Mn-SOD) subfamily, and SmMSD2 protein shares high sequence identity with the Mn-SOD proteins of various plants that all contain a C-terminal conserved metal-binding domain "DVWEHAYY". The prokaryotic expression vector pMAL-c2X-SmMSD2 was constructed and transformed into E. coli BL21 expressing strain, and the target recombinant protein was successfully induced and its enzymatic properties were analyzed. Spatiotemporal expression analysis showed that SmMSD2 gene was expressed in all tissues, indicating that SmMSD2 gene was constitutively expressed at a stable level. Real-time quantitative PCR indicated that drought (15% PEG6000), abscisic acid (ABA) and indole-3-acetic acid (IAA) could induce the expression of SmMSD2 gene, suggesting that SmMSD2 may be involved in the response of Salvia miltiorrhizae to abiotic stress such as drought, as well as the signaling pathways of phytohormone ABA and IAA. These results lay the foundation for further elucidating the involvement of superoxide dismutase in the stress response and accumulation of active components of Salvia miltiorrhiza.

Guanxinning Tablet and their active substances alleviate ox-LDL-induced endothelial cell injury / 药学学报

This study investigated the intervention effect of Guanxinning Tablet on human umbilical vein endothelial cells (HUVECs) injury induced by oxidized low density lipoprotein (ox-LDL), providing experimental basis for Guanxinning Tablet in the treatment of atherosclerosis-related diseases. Under the damage of HUVECs by ox-LDL, the cell viability was detected by CCK-8 (cell counting kit-8) assay; lactate dehydrogenase (LDH) in the cell culture supernatant was detected by the corresponding kit; the cell morphology of different groups was observed by common phase contrast microscope; reactive oxygen species (ROS) and NO levels in the cells were detected by DCFH-DA and DAF-FM DA probes, respectively; monocyte adhesion assay was used to detect the recruitment of THP-1 in HUVECs, and TMRM dye was used to detect the level of mitochondrial membrane potential; interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) secretion in the cells was detected by ELISA assay. The results showed that Guanxinning Tablet had a concentration-dependent proliferative effect on HUVECs. Under the stimulation of 100 μg·mL-1 ox-LDL, the morphology of endothelial cells was significantly changed. At this time, NO level was significantly decreased, ROS level was significantly increased and accompanied by a decrease in mitochondrial membrane potential. The recruitment of THP-1 cells by endothelial cells and IL-6, ICAM-1 and MCP-1 were also significantly increased, resulting in oxidative stress and inflammatory injury. Guanxinning Tablet and its composed extracts could significantly improve cell morphology, increase NO level, decrease ROS production, and also reduce the secretion of inflammation-related proteins IL-6 and MCP-1. Salvia miltiorrhiza and Ligusticum striatum DC. have significant synergistic effects on NO. Among them, salvianolic acid B and salvianic acid A exerted the main effects, and the combined efficacy of salvianic acid A and ferulic acid was superior to that of single administration. The above results showed that Guanxinning Tablet and their active substances had the effects of improving endothelial basal function, resisting oxidative stress, and alleviating inflammatory injury, and Salvia miltiorrhiza and Ligusticum striatum DC. synergized, which may be related to their regulation of oxidative stress and inflammation and have application prospects in the treatment of atherosclerosis-related diseases.

Nanocrystals self-stabilized Pickering emulsion loaded with active components of Tongmai prescription: preparation, characterization and evaluation by Caco-2 cell model / 药学学报

It is of great significance to apply the nanocrystals self-stabilized Pickering emulsion (NSSPE) to traditional Chinese medicine (TCM) compounds, and to study the effect of NSSPE on the oral absorption of various components with different solubility and permeability. In the study, NSSPE of Tongmai prescription was prepared by the high pressure homogenization method with nanocrystals of main active components (puerarin, ferulic acid, salvianolic acid B and tanshinone IIA) of Tongmai prescription as solid particle stabilizers and a mixture of Ligusticum chuanxiong essential oil and Labrafil M 1944 CS as oil phase. The NSSPE had better physical stability than nanocrystals suspension and blank emulsion. The adsorption of nanocrystals on the surface of oil droplets was confirmed by scanning electron microscopy and fluorescence microscopy. The surface adsorption rates of puerarin, ferulic acid, salvianolic acid B and tanshinone ⅡA in NSSPE were 15.40% ± 3.19%, 15.39% ± 5.07%, 10.97% ± 3.70% and 31.51% ± 1.60%, respectively. When solid active components were prepared into nanocrystals suspension, the cellular uptake and transport across Caco-2 cells were increased significantly for puerarin and tanshinone IIA. The uptake rates of ferulic acid, ligustilide and tanshinone IIA in NSSPE were further increased compared with the physical mixture of nanocrystals suspension and oil, and the transports of ligustilide and tanshinone IIA were also significantly improved. The main absorption mechanisms of NSSPE were passive diffusion and caveolin-mediated endocytosis, which were determined mainly by the microstructure of NSSPE. In conclusion, NSSPE could be applied to complicated TCM. The "micro" and "nano" synergistic microstructure with drug nanocrystals adsorbed on the surface of micron-sized oil droplets could not only improve the physical stability of NSSPE, but also promote the absorption of various components in NSSPE, which made NSSPE a promising oral drug delivery system for TCM.

Effects of temperature and humidity on infection of Fusarium oxysporum in seedlings of Salvia miltiorrhiza / 中国中药杂志

Wilt disease is a major disease of cultivated Salvia miltiorrhiza, which is caused by Fusarium oxysporum. Since the infection process of F. oxysporum in plants is affected by environment factors, this study was conducted to reveal the relationship between disease severity and concentration of the pathogen in plants in the infection process of F. oxysporum in seedlings of S. miltiorrhiza by pot experiments and to reveal the effects of temperature and humidity on the infection process. The results showed that, after inoculation of S. miltiorrhiza seedlings with F. oxysporum, the pathogen in different parts was detected at different time, and it was first detected in substrates. With the continuous propagation of the pathogen(4-5 d), it gradually infected the roots and stems of the seedlings, and the plants had yellowing leaves and withering. The number of the pathogen reached the maximum in each part after 7-8 d, and then gradually decreased in the later stage of the disease. The concentration of the pathogen in substrates, roots and stems of S. miltiorrhiza showed a trend of decreasing after increasing with the aggravation of the disease and reached the maximum in the samples of moderate morbidity, while the concentration in the samples of severe morbidity decreased. In addition, the infection of F. oxysporum in seedlings of S. miltiorrhiza was affected by temperature and humidity. The suitable temperature was 25-30 ℃ and the suitable humidity was 80%-90%. This study could provide guidance for the experiments on pathogenicity of F. oxysporum, screening of biocontrol bacteria and controlling of wilt.

Effect of Rhizophagus intraradices on growth of Salvia miltiorrhiza / 中国中药杂志

The study aimed to explore the effects of inoculation of Rhizophagus intraradices on the biomass, effective component content, and endogenous hormone content of Salvia miltiorrhiza through pot experiments. The number of leaves, plant height, dry weight of aboveground and underground parts, branch number, root number, root length, root diameter, and other biomass were mea-sured by weighing and counting methods. The content of salvianolic acid B, caffeic acid, rosmarinic acid, tanshinone Ⅰ, tanshinone Ⅱ_A, cryptotanshinone, and other effective components was determined by ultra-high performance liquid chromatography. The content of ABA and GA_3 was determined by triple quadrupole mass spectrometry. The correlations between biomass and effective components and between effective components and plant hormones ABA and GA_3 were analyzed. The results showed that R. intraradices significan-tly increased the aboveground dry weight, leaf number, and root number of S. miltiorrhiza by 0.24-0.65 times, respectively. The content of salvianolic acid B and rosmarinic acid in the aboveground part and the content of salvianolic acid B, caffeic acid, rosmarinic acid, tanshinone Ⅰ, and tanshinone Ⅱ_A in the underground part were significantly increased by 0.44-1.78 times, respectively. R. intraradices infection significantly increased the GA_3/ABA values of aboveground and underground parts by 3.82 and 76.47 times, respectively. The correlation analysis showed that caffeic acid, the effective component of the aboveground part, was significantly positively correlated with plant height, tanshinone Ⅱ_A, the effective component of the underground part, was significantly positively correlated with biomass root number, cryptotanshinone, and dry weight, while rosmarinic acid was significantly negatively correlated with dry weight. There were significant positive correlations between cryptotanshinone and ABA, tanshinone Ⅱ_A and ABA and GA_3, and caffeic acid and GA_3. In conclusion, R. intraradices can promote the accumulation of biomass and secondary metabolites of S. miltiorrhiza and regulate the balance between plant hormones ABA and GA_3, thereby promoting the growth of S. miltiorrhiza.

Investigation on anti-hepatitis B virus active components and mechanism of Salvia miltiorrhiza based on network pharmacology / 药学学报

We predicted the anti-hepatitis B virus (HBV) active components and mechanism of Salvia miltiorrhiza based on network pharmacology. The active components of S. miltiorrhiza were obtained through TCMSP, PubChem database and literature research. The potential targets of the active components and HBV infection were predicted by SwissTargetPrediction and GeneCards databases, respectively. The protein-protein interaction (PPI) network was constructed by String database. Cytoscape software was adopted to construct a visual network of active component-disease target and perform topological analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using DAVID platform. The molecular docking of key components and core targets was carried out by AutoDock Vina software. We screened out a total of 38 active components and 178 disease-component overlapping targets. Enrichment analyses obtained 405 related GO items and 68 signaling pathways, such as T/B cell receptor signaling pathways, PI3K/AKT signaling pathway, and mTOR signaling pathway. According to the results of molecular docking, most characteristic components of S. miltiorrhiza (miltionone Ⅱ, miltirone, protocatechuic acid, lithospermic acid, protocatechualdehyde) showed good affinity with the key targets (PIK3CA, APP, STAT3,AKT1 and mTOR). Furthermore, the anti-HBV activity of lithospermic acid, the representative active component of S. miltiorrhiza, and its regulation on PI3K/AKT and mTOR signaling pathways were investigated in an HBV replicating mouse model. Animal welfare and experimental procedures follow the regulations of the Animal Ethics and Welfare Committee of Hubei University. The results showed that lithospermic acid significantly inhibited HBV DNA replication, reduced serum HBsAg and HBeAg levels, and decreased the phosphorylation protein expression levels of AKT and mTOR in liver, indicating that lithospermic acid might exert the anti-HBV activity by regulating PI3K/AKT and mTOR signaling pathways.

Visualizing the spatial distribution and alteration of metabolites in continuously cropped Salvia miltiorrhiza Bge using MALDI-MSI / 药物分析学报

Salvia miltiorrhiza Bge(SMB)has long been used in traditional Chinese medicine to treat cardiovascular and cerebrovascular diseases.Growing clinical usage has led to a huge demand for artificial planting of SMB.Thus,continuous cropping of SMB is an important challenge that needs to be addressed.Contin-uous cropping can alter the metabolic profile of plants,resulting in poor growth and low yield.In this study,we tried to image the spatial location and variation of endogenous metabolites in continuously cropped SMB using matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI).Spatially resolved expressions of tanshinones,salvianolic acids,polyamines,phenolic acids,amino acids,and oligosaccharides in normal and continuously cropped SMB roots were compared.The ex-pressions of dihydrotanshinone Ⅰ,tanshinone Ⅱ A,dehydromiltirone,miltirone,dehydrotanshinone ⅡA,spermine,salvianolic acid B/E,tetrasaccharide,and pentasaccharide in continuously cropped SMB roots were much lower than those in normal roots.There was little difference in the expressions of caffeic acid and salvianolic acid A in normal and continuously cropped SMB roots.Ferulic acid was more widely distributed in xylem of normal SMB but strongly expressed in xylem,phloem,and cambium of continuously cropped SMB.The spatially resolved metabolite information enhances our understanding of the metabolic signature of continuously cropped SMB and also provides insights into the metabolic ef-fects of continuous cropping in other plants.

Cloning, induction pattern and prokaryotic expression of a small heat shock protein SmHSP21.8 gene from Salvia miltiorrhiza / 药学学报

In order to reveal the molecular mechanism of the small heat shock proteins (sHSPs) involved in stress resistance and active ingredients accumulation in Salvia miltiorrhiza, a small heat shock protein gene was cloned from Salvia miltiorrhiza by reverse transcription PCR according to the transcriptome data of orange root Salvia miltiorrhiza. The gene is named SmHSP21.8 based on the molecular weight of the protein, and it contains an open reading frame of 585 bp, which encodes 194 amino acids. The results of phylogenetic analysis and amino acid sequence alignment showed that SmHSP21.8 protein belongs to the endoplasmic reticulum (ER) subfamily, and contains a conserved endoplasmic reticulum-specific DPFR-I/V-LE-H/Q-x-P motif at N-terminus. The prokaryotic expression vector pMAL-c2X-SmHSP21.8 was constructed and transformed into E. coli BL21 competent cells. The recombinant protein was successfully expressed after inducted. Temporal and spatial expression analysis showed that SmHSP21.8 gene was the highest expressed in flowers and had significant tissue specificity. The relative expression of the gene was significantly increased in seedlings after induction by 38 ℃, PEG6000, abscisic acid(ABA), and indole-3-acetic acid (IAA), indicating that SmHSP21.8 gene may be involved in abiotic stress such as high temperature and drought, as well as the response to exogenous hormones ABA and IAA. These results lay the foundation for further research on the molecular mechanism of small heat shock proteins involved in adversity stress.

Study on the changes of HPLC fingerprints and main chemical compounds before and after the compatibility of Salvia miltiorrhiza and Salvia miltiorrhiza / 国际中医中药杂志

Objective:To establish methods for HPLC fingerprints and simultaneous determination of multi-index components before and after compatibility of Salvia miltiorrhiza and Angelica sinensis, so as to analyze the dissolution rate of the main compounds. Methods:The extracts of Salvia miltiorrhiza, Angelica sinensis and their compatibility were prepared. The separation was performed on an Eclipse XDB-C18 column (4.6 mm×250 mm,5 μm), mobile phase with 0.1% phosphoric acid aqueous solution-acetonitrile for gradient elution, flow rate at 1.0 ml/min, column temperature was maintained at 35 ℃, and the detection wavelength was set at 280 nm. The HPLC fingerprint were established before and after the compatibility of Salvia miltiorrhiza and Angelica sinensis, and the shared patterns of the fingerprint were obtained to gain chromatographic peaks. The content of 9 components Danshensu, caffeic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A, tanshinone Ⅱ A, ferulic acid, chlorogenic acid and Yangchuanxiong lactone were determinated, and the changes of dissolution rate of each compound before and after the compatibility were analyzed. Results:The determination method for the multi- components with HPLC is precise and the components (waiting to be determinate) in the solution were stable within 48 hours, and the RSD values of each chromatographic peak were <5.0%. The nine components showed good linear relationships within their own ranges, and the recovery rate was in compliance with regulations. The fingerprint similarities of each sample were ?0.9. After the compatibility of Salvia miltiorrhiza and Angelica sinensis, a total of seventeen common peaks were calibrated, ten of which were from Salvia miltiorrhiza, seven from Angelica sinensis. No new components was found under this chromatographic condition. After the combination of these two material medicica decoction, the average dissolution rates of rosmarinic acid, salvianolic acids and Danshensu in Salvia miltiorrhiza were significantly lower than those of the single decoction group ( P<0.05 or P<0.01); the average dissolution rates of caffeic acid in Salvia miltiorrhiza was significantly higher than that of the single decoction group ( P<0.01); the average dissolution rates of chlorogenic acid and ferulic acid in Angelica sinensis were significantly higher than that of the single decoction group ( P<0.05 or P<0.01); the average dissolution rate of Yangchuanxiong lactone after the compatibility was not statistically different than that of single decoction group. Conclusion:The characteristic peaks of HPLC fingerprint of the compatibility of Salvia miltiorrhiza and Angelica sinensis did not increase under this chromatographic condition, which had a significant effect on the dissolution of index components.

Variation of photosynthesis, secondary metabolites and antioxidant activities in third generation of spaceflight-induced Salvia miltiorrhiza / 中草药·英文版

Objective: Spaceflight has long been perceived as an effective way to improve the quantity and quality of plants with wide applications. In order to obtain stable and inheritable descendants of spaceflight-induced Salvia miltiorrhiza lines, we investigated and analyzed four lines m16, m50, m51, m57 (three individuals of each line) and the ground control (three individuals) of the third generation of spaceflight-induced S. miltiorrhiza from primary/secondary metabolism and antioxidative abilities. Methods: A portable photosynthesis system (Li-6400) with red/blue LED light source was used to perform the photosynthetic characteristics to evaluate their primary productivity. The secondary metabolites (phenolic acids, tanshinones, total phenolics and flavonoids) and antioxidant activity of roots were analyzed to assess their quality. Results: Compared with control, line m16 presented weak photosynthetic ability, but high apparent quantum yield (AQY), higher contents of secondary metabolites, and stronger antioxidative abilities. Line m57 had a strong gas exchange ability, relatively higher secondary metabolites contents, and ascending antioxidative abilities. Lines m50 and m51 were in the middle level of lines m16 and m57. The principal component analysis for all the original data revealed three components including a root-related index, a leaf-related index, and a CO

Bioaccessibility analysis of active components in Salvia miltiorrhiza / 药学学报

In this study, a method for simultaneous quantitative analysis of 6 salvianolic acids and 4 tanshinones in extracts of Salviae Miltiorrhizae Radix et Rhizoma was established by ultra-high performance liquid chromatography (UHPLC). The semi-biomimetic method was applied to simulate digestion process in vitro, to explore the digestion and transport characters of oral administration through the gastrointestinal tract, and to explain the content ratio changes and bioaccessibility of active ingredients in Salviae Miltiorrhizae Radix et Rhizoma. The results showed that the 10 index components have a good linear relationship in the corresponding concentration range, and the average recovery rate was 91.35% to 105.65%. After simulated digestion in vitro, types of chemical composition in simulated gastric fluid and simulated intestinal fluid digested extracts of Salviae Miltiorrhizae Radix et Rhizoma did not change significantly. While the content ratio of salvianolic acid B and rosmarinic acid decreased, and the content ratio of protocatechuic aldehyde and danshensu increased. In the simulated gastric fluid digestion extract of Salviae Miltiorrhizae Radix et Rhizoma, the order of bioaccessibility was: danshensu (50.19%) > salvianolic acid B (33.44%) > lithospermic acid (27.34%) > salvianolic acid A (21.71%) > rosmarinic acid (12.31%). In the simulated intestinal fluid digestion extract of Salviae Miltiorrhizae Radix et Rhizoma, the order of bioaccessibility was: 15,16-dihydrotanshinone Ⅰ (5.45%) > tanshinone Ⅰ (3.67%) > cryptotanshinone (3.29%) > tanshinone ⅡA (3.01%) > salvianolic acid A (2.39%) > lithospermic acid (1.57%) > salvianolic acid B (1.02%) > danshensu (0.41%) > rosmarinic acid (0.34%). In conclusion, the UHPLC method established in this study can be applied for accurately and sensitively detecting the contents of 6 salvianolic acids and 4 tanshinones in Salviae Miltiorrhizae Radix et Rhizoma. The results of semi-biomimetic extraction showed that not all components were extracted with simulated gastric fluid and simulated intestinal fluid, especially rosmarinic acid and salvianolic acid B. Therefore, in the quality study of Salviae Miltiorrhizae Radix et Rhizoma and its extract, bioavailability should be considered at the same time when select quality markers and determine their content limits.

Mechanism of 'Invigorating Qi and Promoting Blood Circulation' Drug Pair Ginseng-Danshen on Treatment of Ischemic Heart Disease Based on Network Pharmacology / 中国结合医学杂志

OBJECTIVE@#Using network pharmacology to explore the mechanism of the 'invigorating qi and promoting blood circulation' drug pair Ginseng-Danshen (Salvia miltiorrhiza) on treatment of ischemic heart disease (IHD).@*METHODS@#The chemical constituents of ginseng and Danshen drug pair were identified by searching the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the potential targets of the pair were identified. The pharmacodynamics of the pair was analyzed using network pharmacology. The targets of IHD were identified by database screening. Using protein-protein interaction network, the interaction targets of Ginseng-Danshen on IHD were constructed. A "constituent-target-disease" interaction network was constructed using Cytoscape software, Gene Ontology (GO) term enrichment analysis and biological pathway enrichment analysis were carried out, and the mechanism of improving myocardial ischemia by the Ginseng-Danshen drug pair was investigated.@*RESULTS@#Seventeen active constituents and 53 targets were identified from ginseng, 53 active constituents and 61 targets were identified from Danshen, and 32 protein targets were shared by ginseng and Danshen. Twenty GO terms were analyzed, including cytokine receptor binding, cytokine activity, heme binding, and antioxidant activity. Sixty Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways were analyzed, including phosphatidylinositol 3-kinase-serine-threonine kinase (PI3K-AKT) signaling pathway, p53 signaling pathway, interleukin 17 signaling pathway, tumor necrosis factor signaling pathway, and the advanced glycation end product (AGE)-the receptor for AGE (RAGE) signaling pathway in diabetic complications.@*CONCLUSION@#The specific mechanism of Ginseng-Danshen drug pair in treating IHD may be associated with improving the changes of metabolites inbody, inhibiting the production of peroxides, removing the endogenous oxygen free radicals, regulating the expression of inflammatory factors, reducing myocardial cell apoptosis and promoting vascular regeneration.

Effect of combined application of inorganic and organic fertilizers on growth and quality of Salvia miltiorrhiza / 中国中药杂志

The study is aimed through field experiments to study the effect of combined application of organic and chemical fertilizers on the growth and quality of Salvia miltiorrhiza, provide ideas for reducing fertilization while increasing the efficiency as well as improving the quality of produces. The experiment included 6 treatments viz., no fertilization(CK), full application of chemical fertilizer(F), 25% orga-nic fertilizer with 75% chemical fertilizer(M25), 50% organic fertilizer with 50% chemical fertilizer(M50), 75% organic fertilizer with 25% chemical fertilizer(M75), and fully apply organic fertilizer(M100). The results showed that:(1)from the perspective of yield and economic benefits, M75 was the best and M100 second;(2)for effective components, the combined application of organic and chemical fertilizers increased the content of main water-soluble components and the total content of effective components, among which M25 and M50 were better.

Effect of different fungicides on efficiency of mycorrhizal symbiosis on Salvia miltiorrhiza / 中国中药杂志

Arbuscular mycorrhizal fungi provided is beneficial to Salvia miltiorrhiza for increasing yield, promoting the accumulation of active ingredients, and alleviating S. miltiorrhiza disease etc. However, the application of fungicides will affect the benefit of arbuscular mycorrhizal fungi and there is little research about it. This article study the effect of four different fungicides: carbendazim, polyoxin, methyl mopazine, and mancozeb on mycorrhiza benefit to S. miltiorrhiza by the infection intensity of arbuscular mycorrhizal fungi, the growth of S. miltiorrhiza, and the content of active ingredients. RESULTS:: showed that different fungicides had different effects. The application of mancozeb had the strongest inhibitory effect on the mycorrhizal benefit to S. miltiorrhiza. Mancozeb significantly reduced the mycorrhizal colonization and the beneficial effect of arbuscular mycorrhizal fungi on the growth and the accumulation of active components of S. miltiorrhiza. The application of polyoxin had no significant effect on mycorrhizal colonization. Instead, it had a synergistic effect with the mycorrhizal benefit to promoting the growth and accumulation of rosmarinic acid of S. miltiorrhiza. The inhibitory strengths of four fungicides are: mancozeb>thiophanate methyl, carbendazim>polyoxin. Therefore, we recommend applying biological fungicides polyoxin and avoid applying chemical fungicides mancozeb for disease control during mycorrhizal cultivation of S. miltiorrhiza.

Content determination of phenolic acids in fresh Salvia miltiorrhiza and effect of polyphenol oxidase on content / 中国中药杂志

There is no consensus on the content, accumulation, transformation and content determination methods of phenolic acids in fresh Salvia miltiorrhiza. In order to find out the true content of phenolic acids in fresh S. miltiorrhiza, a variety of treatment me-thods were used in this study to prepare sample solution. The content changes of phenolic acids in S. miltiorrhiza samples with different dehydration rates were investigated during drying and shade drying processes. Polyphenol oxidase(PPO) of S. miltiorrhiza was extracted and purified by ammonium sulfate precipitation and dialysis to investigate the enzymatic properties. The content of rosmarinic acid, lithosperic acid and S. nolic acid B in S. miltiorrhiza was determined by UPLC. The results showed that the content of phenolic acids in fresh S. miltiorrhiza was highest when it was homogenized with 1 mol·L~(-1) HCl solution or 1 mol·L~(-1) HCl methanol solution. There was no significant difference in the content of phenolic acids in S. miltiorrhiza with different dehydration rates, indicating that there was no correlation between phenolic acid content and dehydration rate. The optimum pH of S. miltiorrhiza PPO was 7.6 and the optimum temperature was 40 ℃. With catechol as substrate, S. miltiorrhiza PPO had the enzymatic browning reaction which was in compliance with Michaelis equation, with Michaelis constant K_m of 0.12 mol·L~(-1) and V_(max) of 588.23 U·min~(-1). The inhibitory effect of citric acid, disodium ethylenediamine tetraacetate, ascorbic acid and sodium sulfite on S. miltiorrhiza PPO increased with the increase of inhibitor concentration, and sodium sulfite showed the strongest inhibitory effect. The present study proved that there were a large number of phenolic acids in fresh S. miltiorrhiza, which were the secondary metabolite of primitive accumulation during the growth of S. miltiorrhiza, rather than the induced product of postharvest drying and dehydration stress. This study has reference value and significance for the cultivation, harvest and processing of S. miltiorrhiza.

Determination of inhibitory activity of Salvia miltiorrhiza extracts on xanthine oxidase with a paper-based analytical device / 药物分析学报

A novel paper-based analytical device(PAD)was prepared and applied to determine the xanthine oxi-dase(XOD)inhibitory activity of Salvia miltiorrhiza extracts(SME).First,polycaprolactone was 3D printed on filter paper and heated to form hydrophobic barriers.Then the modified paper was cut according to the specific design.Necessary reagents including XOD for the colorimetric assay were immobilized on two separate pieces of paper.By simply adding phosphate buffer,the reaction was performed on the double-layer PAD.Quantitative results were obtained by analyzing the color intensity with the specialized device system(consisting of a smartphone,a detection box and sandwich plates).The 3D-printed detection box was small,with a size of 9.0 cm x 7.0 cm x 11.5 cm.Color component G performed well in terms of linearity and detection limits and thus was identified as the index.The reaction con-ditions were optimized using a definitive screening design.Moreover,a 10％glycerol solution was found to be a suitable stabilizer.When the stabilizer was added,the activity of XOD could be maintained for at least 15 days under 4℃or-20℃storage conditions.The inhibitory activity of SME was investigated and compared to that of allopurinol.The results obtained with the PAD showed agreement with those ob-tained with the microplate method.In conclusion,the proposed PAD method is simple,accurate and has a potential for point-of-care testing.It also holds promise for use in rapid quality testing of medicinal herbs,intermediate products,and preparations of traditional Chinese medicines.

Research progress of the effect of Salvia miltiorrhiza on VEGF/VEGFR in angiogenesis / 国际中医中药杂志

The signal transduction pathway of VEGF combing with vascular endothelial growth factor receptor (VEGFR) is not only key pathway to regulate angiogenesis, but also the focus of basic research and important target of clinical treatment. Salvia miltiorrhiza extract and its main compound salvianolic acid B also have two-way regulative effect on VEGF/VEGFR signal pathway in different diseases. Tanshinone Ⅰ , tanshinone Ⅱ A and cryptotanshinone could inhibit the angiogenesis throughthis pathway, and sodium tanshinone ⅡA sulfonate could promote the angiogenesis through this pathway.