A High-Performance Liquid Chromatography Method for the Simultaneous Determination of Five Index Components in Danhong Injection

The purpose of this study was to establish a high-performance liquid chromatography (HPLC) method for the simultaneous determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid in Danhong injection. The chromatographic method employed was as follows: the column was a Welch Ultimate XB-C18 column (250 mm × 4.6 mm, 10 μm), the mobile phase was a gradient elution of 0.4% formic acid aqueous solution (A) and acetonitrile (B), the detection wavelengths were 280 nm for sodium danshensu, protocatechuic aldehyde, and salvianolic acid B and 326 nm for 4-coumaric acid and rosmarinic acid, the sample volume was 10 μL, the flow rate was 1.0 mL/min, and the column temperature was 35°C. This method can realize the separation and determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid within 50 minutes. The linear relationships of the five peak areas and their concentrations are good (R2> 0.9997). The precision RSD values are all less than 1.0%. The reproducibility RSD values are all less than 1.3%. The stability RSD values are all less than 2.2%. The recovery values ranged from 92.4% to 99.4%. This method is simple, accurate, and reproducible. It can be used for the determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid in Danhong injection.

Pretreatment with Danhong injection protects the brain against ischemia-reperfusion injury

Danhong injection （DHI）, a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is widely used in China for treating acute isch-emic stroke. In the present study, we explored the neuroprotective efficacy of DHI in a rat model of temporary middle cerebral artery ocdusion, and evaluated the potential mechanisms under-lying its effects. Pretreatment with DHI （0.9 and 1.8 mL/kg） resulted in a significantly smaller infarct volume and better neurological scores than pretreatment with saline. Furthermore, DHI significantly reduced the permeability of the blood-brain barrier, increased occludin protein expression and decreased neutrophil infiltration, as well as profoundly suppressing the upreg-ulation of matrix metallopeptidase-9 expression seen in rats that had received vehicle. Matrix metallopeptidase-2 expression was not affected by ischemia or DHI. Moreover, DHI （1.8 mL/kg） administered 3 hours after the onset of ischemia also improved neurological scores and reduced infarct size. Our results indicate that the neuroprotective efficacy of DHI in a rat model of cerebral ischemia-reperfusion injury is mediated by a protective effect on the blood-brain barrier and the reversal of neutrophil infiltration.

Danhong injection A modulator for Golgi structural stability after cerebral ischemiareperfusion injury

The cerebral ischemia-reperfusion model was established using the suture occlusion method, and rats were intraperitoneally given 8 mL/kg Danhong injection once a day prior to model establishment Rat brain tissues were harvested at 6, 24, 48, 72 hours after reperfusion. Immunohistochemical staining showed that transforming growth factor-J31 expression increased, while Golgi matrix protein GM130 expression decreased after cerebral ischemia-reperfusion. Danhong injection was shown to significantly up-regulate the expression of transforming growth factor-131 and GM130, and expres- sion levels peaked at 7 days after reperfusion. At 7 days after cerebral ischemia-reperfusion, Golgi morphology was damaged in untreated rats, while Golgi morphology breakage was not observed after intervention with Danhong injection. These experimental findings indicate that Danhong injec- tion can up-regulate the expression of transforming growth factor-131 and GM130, and maintain Golgi stability, thus playing a neuroprotective role in rats after cerebral ischemia-reperfusion.

Efficacy and safety of Danhong injection in the treatment of arteriosclerosis obliterans:a meta-analysis of randomized controlled trials

Aims:To systematically evaluate the clinical efficacy and safety of danhong injection in the treatment of arteriosclerotic occlusion.Methods:Computer retrieval China knowledge network(CNKI),and Wiper Chinese journal database(VIP),and Wanfang data knowledge service platform,the Chinese biomedical literature database(CBM),Pubmed,science citation index(SCI)database,the Cochrane Library,including eight Chinese English database,to conform to the criteria for the two researchers use the recommended bias risk assessment method to evaluate the Cochrane bias risk,extracted data,and apply the Review Manager software Meta analysis and system evaluation.Results:Finally,13 studies were included,with a total number of 884 cases.The results showed that the clinical efficacy of danhong injection in the treatment of arteriosclerotic occlusion was higher than that of conventional treatment,and the difference was statistically significant[OR=3.08,95%CI(2.07,4.60),P<0.00001].The improvement of arteriosclerosis was more obvious[OR=0.15,95%CI(0.12,0.19),P<0.00001].The improvement of hemodynamics was more obvious[OR=-0.19,95%CI(-0.33,-0.04),P=0.01].No adverse reactions occurred.Conclusions:Danhong injection has higher clinical efficacy in the treatment of atherosclerotic occlusion,more obvious improvement in arteriosclerosis degree and hemodynamics,and higher safety.However,due to the low quality of literature research,more high-quality,large samples and randomized controlled double-blind clinical trials are needed to provide high-level evidence to further verify the efficacy and safety of danhong injection in the treatment of atherosclerotic occlusion.

Progresses in the research of Danhong Injection against the cardial ischemia-referfusion injury

Researches in the field of the myocardial ischemia-reperfusion injury are attracting the attentions of clinicians for the treatments that protect cardiac muscle cells from being injured can not only help the patients get recovery but also keep them in health. By clearing the free radicals and reducing calcium overload of myocardial cell, treatments with Danhong Injection will help myocardial cells survive from inflammatory reactions which are triggered by ischemia reperfusion so as that endothelial function will be improved and myocardial cell apoptosis will be inhibited. In all, Danhong Injection is an ideal medicine for protecting myocardial cell against ischemia reperfusion injury.

Danhong Injection Up-Regulates miR-125b in Endothelial Exosomes and Attenuates Apoptosis in Post-Infarction Myocardium

Objective:To investigate the involvement of endothelial cells(ECs)-derived exosomes in the antiapoptotic effect of Danhong Injection(DHI)and the mechanism of DHI-induced exosomal protection against postinfarction myocardial apoptosis.Methods:A mouse permanent myocardial infarction(MI)model was established,followed by a 14-day daily treatment with DHI,DHI plus GW4869(an exosomal inhibitor),or saline.Phosphatebuffered saline(PBS)-induced ECs-derived exosomes were isolated,analyzed by miRNA microarray and validated by droplet digital polymerase chain reaction(ddP CR).The exosomes induced by DHI(DHI-exo),PBS(PBS-exo),or DHI+GW4869(GW-exo)were isolated and injected into the peri-infarct zone following MI.The protective effects of DHI and DHI-exo on MI hearts were measured by echocardiography,Masson's trichrome staining,and TUNEL apoptosis assay.The Western blotting and quantitative reverse transcription PCR(qR T-PCR)were used to evaluate the expression levels of miR-125b/p53-mediated pathway components,including miR-125b,p53,Bak,Bax,and caspase-3 activities.Results:DHI significantly improved cardiac function and reduced infarct size in MI mice(P<0.01),which was abolished by the GW4869 intervention.DHI promoted the exosomal secretion in ECs(P<0.01).According to the results of exosomal miRNA microarray assay,30 differentially expressed miRNAs in the DHI-exo were identified(28 up-regulated miRNAs and 2 down-regulated miRNAs).Among them,DHI significantly elevated miR-125b level in DHI-exo and DHI-treated ECs,a recognized apoptotic inhibitor impeding p53 signaling(P<0.05).Remarkably,treatment with DHI and DHI-exo attenuated apoptosis,elevated miR-125b expression level,inhibited capsase-3 activity,and down-regulated the expression levels of proapoptotic effectors(p53,Bak,and Bax)in post-MI hearts,whereas these effects were blocked by GW4869(P<0.05 or P<0.01).Conclusion:DHI and DHI-induced exosomes inhibited apoptosis,promoted the miR-125b expression level,and regulated the p53 apoptotic pathway in post-infarction myocardium.

Rapid separation and identification of multiple constituents in Danhong Injection by ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry

To characterize and identify multiple constituents in Danhong injection(DHI), a fast ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry(UHPLC-ESI-QTOF/MS) method was established and validated in the present study. A total of 63 compounds, including 33 phenolic acids, 2 C-glycosyl quinochalcones, 6 flavonoid O-glycosides, 4 iridoid glycosides, 6 organic acids, 5 amino acids, and 3 nucleosides, were identified or tentatively characterized. In conclusion, the UHPLC-ESI-QTOF/MS method is useful and efficient for in-depth structural elucidation of chemical compounds in complex matrices of herbal medicines such as DHI.

Effects of Danhong Injection(丹红注射液) and Its Main Components on Anticoagulation and Fibrinolysis in Cultured Vein Endothelial Cells

Objective： To observe the effects of Danhong Injection （丹红注射液） and its main components, including daiclzein and hydroxysafflor yellow A （HSYA）, on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells （VECs）. Methods： VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N2 and 5% CO2 mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α （6-keto-PGF1 α） level was measured by the method of radioimmunoassay （RIA）. Superoxide dismutase （SOD） activity was tested by water soluble tetrazolium salt. The content of malondialdehyde （MDA） was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator （t-PA） and plasminogen activator inhibitor （PAl） were measured by the method of chromogenic substrate. The contents of endothelin （ET） and nitric oxide （NO） were detected by non-equilibrium RIA and enzyme- linked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. Results： Compared with the normal control group, the floating cells number, PAl activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1 α and NO contents, and t-PA and SOD activities were decreased significantly （P〈0.01）. Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1 α content and SOD activity, regulated PAl and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate （P〈0.05 or P〈0.01）. Conclusions： Danhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.

Synergistic Protection of Danhong Injection (丹红注射液) and Ischemic Postconditioning on Myocardial Reperfusion Injury in Minipigs

Objective： To explore the synergistic protection of Danhong Injection （丹红注射液,DHI） and ischemic postconditioning on myocardial reperfusion injury in minipigs.Methods： Acute myocardial infarction model was made by balloon occlusion in left anterior descending coronary artery （LAD） of minipigs,and then postconditioning was simulated through inflation/deflation of the angioplasty balloon.Minipigs were divided into four groups： the sham operation group （SH group）,the ischemia/reperfusion group （I/R group）,the ischemic postconditioning group （POC group） and DHI combined with ischemic postconditioning group （PAD group,DHI 20 mL through ear vein）,six in each group.After 24-h continuous observation,myocardial infarction size was assessed by triphenyltetrazolium staining （TTC）.Morphological changes of ischemic myocardium were observed by light microscopy,and cardiomyocyte ultrastructure was studied with electron microscopy.The superoxide dismutase （SOD） and malondialdehyde （MDA） activity in heart homogenates were measured by a biochemical method.Results： The myocardial infarction size was smaller in the POC group than in the I/R group （0.26±0.02 vs.0.37±0.09,P〈0.05）,and the PAD group （0.14±0.08） displayed a significantly reduced infarction size relative to the I/R group （P〈0.01） and POC group （P〈0.05）.The damage of myocardial tissue was severe in the I/R group shown by light and electron microscopy： myocardial fibers disorder,sarcoplasmic dissolution,myofilament fracture,mitochondria swelling and even vacuolization formation and a large number of inflammatory cell infiltrations.Compared with the I/R group,reduction of reperfusion injury in the PAD group included more orderly arranged myocardial fibers,less infiltration of inflammatory cells and maintenance of mitochondrial integrity.Compared with the I/R group,the damage of myocardial tissue in the POC group was improved,but not as significant as that in the PAD group.SOD levels in the POC group and the PAD group were significantly higher than those in the I/R group （96.96±13.43,112.25±22.75 vs.76.32±10.63,P〈0.05）,and MDA was significantly lower in the POC group and the PAD group compared to the I/R group （1.27±0.19,1.09±0.21 vs.1.47±0.16,P〈0.05）.Conclusion： DHI and ischemic postconditioning show a synergistic cardioprotection on myocardial reperfusion injury in minipigs.

Effect of Danhong Injection(丹红注射液)on Improving Coronary Microcirculation Injury after Percutaneous Coronary Intervention

Objective:To explore the effectiveness of Danhong Injection(丹红注射液)on improving microcirculatory injury after percutaneous coronary intervention(PCI)in patients with coronary heart disease(CHD).Methods:A randomized controlled trial was conducted and 90 patients were enrolled.A random sequence was generated using statistical analysis software.Patients with microcirculatory injuries after PCI were randomly divided into 3 groups for treatment(30 subjects in each group):Danhong Injection group:after PCI,Danghong Injections were given with intravenous administration with 40 mL twice a day for a week;statins intensive group:after PCI,atorvastatin calcium tablets were given oral medication with 80 mg once,and then atorvastatin 40 mg daily for 1 week;the control group:after PCI,atorvastatin calcium tablets were given oral medication with 10–20 mg daily for 1 week.The index of microcirculation resistance(IMR)was used to assess microcirculatory injury during PCI.The IMR of the target vessel was reexamined after 1 week of drug treatment.Results:After one week's drug treatment,IMR was significantly decreased in both statins intensive group and Danhong Injection group compared with the control group(P<0.01),but no difference was found between statins intensive group and Danhong injection group(14.03±2.54 vs.16.03±5.72 U,P=0.080).Conclusions:The efficacy of Danhong Injection is non-inferior to statin.Early use of Danhong Injection after PCI can effectively improve coronary microcirculation injury after PCI.

Effect of Danhong injection on expressions of macrophage scavenger receptor 1 and sub-family A member 1 in human U937 cells

OBJECTIVE： To study the effects of Danhong injec- tion （DHI） on expression of the macrophage scaven- ger receptor 1 （MSR1） and ATP-binding cassette, sub-family A member 1 （ABCA1） genes, which en- code scavenger receptor-A I （SR-AI） and ATP-bind- ing cassette transporter 1 （ABCA1）, respectively, as a potential anti-atherosclerotic mechanism. METHODS： Human U937 cells were stimulated by in- cubation with 100 nM phorbo112-myristate 13-ace- tate （PMA） for 48 h.These stimulated, monocyte-like cells were then incubated for 24 h with 50 mg/L oxi- dized low-density lipoprotein （ox-LDL, to induce foam cell formation）, together with a liver X recep- tor （LXR） agonist or with different DHI concentra- tions. MSR1 and ABCA1 mRNA levels were mea- sured by fluorescence-based quantitative PCR. RESULTS： Compared with control cells （which re- ceived only ox-LDL）, cells treated with both ox-LDL and 10 IJmol/L LXR agonist showed lower MSR1 ex-pression （but this effect was not statistically signifi- cant, P〉0.05） and higher ABCA1 expression （P〈 0.01）. Cells that received ox-LDL and 3 mL/L DHI possessed higher MSR1 mRNA levels than the con- trols, whereas cells treated with ox-LDL and higher DHI concentrations （10, 30 or 60 mL/L） showed low- er MSR1 expression levels （but the differences ob- served between DHI concentration groups were not statistically significant, P〉0.05）. ABCA1 expression in cells treated with ox-LDL and 3, 10 or 30 mL/L DHI was higher than in the control cells, and increased with increasing DHI concentration （P〈0.05）. ABCA1 expression in cells treated with ox-LDL and the highest DHI concentration tested （60 mL/L） was not significantly different from that in the controls. ABCA1 mRNA levels in cells treated with ox-LDL and DHI were similar to, or lower than, those in cells treated with ox-LDL and the LXR agonist. CONCLUSION： DHI does not affect MSR1 mRNA lev- els in ox-LDL-treated U937 cells. However, at certain concentrations （10 and 30 mL/L）, DHI significantly increases ABCA1 mRNA levels. Therefore, the an- ti-atherosclerotic action of DHI might be mediated by an increased expression of ABCA1.

Danhong Injection Improves Elective Percutaneous Coronary Intervention in UA Patients with Blood Stasis Syndrome Revealed by Perioperative Metabolomics

Objective:To observe the effect of Danhong injection(DHI)on perioperative metabolomics of unstable angina pectoris(UA)with blood stasis syndrome.Materials and Methods:A prospective,randomized,controlled,and single-blind clinical trial was conducted.Sixty-one UA patients with traditional Chinese medicine blood stasis syndrome undergoing elective percutaneous coronary intervention(PCI)were randomly divided into the Danhong and control groups,and 10 healthy volunteers were included as baseline.The Danhong group received western medicine+DHI treatment,while the control group received western medicine+saline.Nontargeted metabolomics was used to analyze the serum metabolites of healthy volunteers in the Danhong and control groups before and 5 days after PCI.Results:Before treatment,there was no significant difference in serum metabolites between the Danhong and control groups,but there was a significant difference between the two groups and the healthy group.Differential metabolites were clustered mainly in glycerophospholipid,sphingolipid,purine,and amino acid groups,which were generated in their metabolic pathways.After 5 days of PCI,the profiles of serum metabolites were significantly closer between the Danhong-or control-treated groups and that of the healthy group.Furthermore,DHI treatment converted the serum metabolite profile more to that of the healthy group than the control treatment.Conclusion:The beneficial effect of DHI on patients with unstable angina is reflected at the level of serum metabolic biomarkers.

Exploring the mechanism of action of DHI on myeloproliferative neoplasms based on network pharmacology

Objective:The aim of this study is to explore the active ingredients and mechanism of action of danhong injection(DHI)in treating myeloproliferative neoplasms using network pharmacology.Methods:The TCMSP platform and relevant literature were used to search for the active ingredients and targets of Radix Salviae and Carthami Flos in DHI.Disease targets related to myeloproliferative neoplasms were obtained from the GEO database,GeneCards,and DisGeNET database.The queried component targets were normalized using the UniProt database.Potential targets were identified by constructing protein-protein interactions networks using STRING 11.5 and visualized and analyzed using Cytoscape 3.9.1.GO and KEGG analysis were performed using the Metascape platform,and visualization was done using the built-in plug-in CluoGO or SangerBox platforms with Cytoscape 3.9.1.Results:The active ingredients of DHI for treating myeloproliferative neoplasms mainly consist of flavonoids and o-benzoquinones,including quercetin,luteolin,kaempferol,stigmasterol,tanshinone iia,cryptotanshinone,beta-carotene,2-isopropyl-8-methylphenanthrene-3,4-dione,and neocryptotanshinone ii.The potential targets are JUN,TP53,STAT3,AKT1,MAPK1,RELA,TNF,MAPK14,IL6,and FOS.The relevant signaling pathways involved are mainly TNFαsignaling pathway,PI3K-Akt signaling pathway,apoptosis,IL-17 signaling pathway,cellular senescence,MAPK signaling pathway,p53 signaling pathway,JAK-STAT signaling pathway,and NF-kappa B signaling.Conclusions:DHI acts mainly through flavonoids and o-benzoquinones to treat myeloproliferative neoplasms in a multi-targeted and multi-pathway manner.

Development and validation of global prediction models for monitoring the manufacturing process of herbal medicine by ultraviolet spectroscopy

Objective: Process monitoring for traditional Chinese medicine(TCM) preparations is necessary to ensure the quality of the product. A typical pharmaceutical process of TCM preparations consists of multiple manufacturing units, such as ethanol precipitation, concentration, and water precipitation, among others. Compared with the traditional practice of one prediction model for one unit, the global model covers the variation from samples with different backgrounds or processes and can be used to monitor intermediates from substeps.Methods: We used ultraviolet(UV) spectroscopy to establish global models for a typical TCM preparation-Danhong injection.The concentrations of danshensu, protocatechualdehyde, rosmarinic acid, salvianolic acid A, salvianolic acid B, and hydroxyl safflor yellow A and the total phenolic and total sugar contents were quantified for every intermediate from operation units of Danhong injection. New samples prepared by mixing different intermediates were introduced for the calibration set to cover more variations. An accuracy profile was employed to validate the developed method from the aspects of specificity, trueness, precision,accuracy, linearity, and robustness.Results: The developed models showed a high determination coefficient(R2) value up to 0.97 and a low root-mean-square error of the prediction set. Five components of the models passed all validation tests, whereas the total sugar was not suitable for modeling with UV and did not apply to the whole process.Conclusions: This study indicates that the global models of UV spectroscopy for the quantitative determination of phenolic acids are feasible and reliable with a simple, rapid, and non-destructive method.Graphical abstract: http://links.lww.com/AHM/A22.