Danhong Injection Research Articles

Specific Combination of Salvianolic Acids As Core Active Ingredients of Danhong Injection for Treatment of Arterial Thrombosis and Its Derived Dry Gangrene.

Although single-targeting anti-platelet agents are used extensively in clinics, their limitations in resistance and bleeding have started a trend of combination therapy. Danhong injection (DHI) is a widely prescribed injection medicine for cardiovascular and cerebrovascular diseases in China. However, its precise clinical efficacy and functional components remain unexplored. In this study, we investigated the anti-thrombotic role and its chemical basis of DHI. In a photochemically-induced thrombosis model, DHI effectively dissolved thrombus and ameliorated its derived dry gangrene. DHI inhibited multiple GPCR agonists-induced platelet adhesion, aggregation and downstream Ca2+ and cAMP signaling pathways. A functional screen of DHI library identified its major active components as a cluster of seven salvianolic acids. A combination of salvianolic acid A and C synergistically inhibited platelet aggregation in vitro while salvianolic acid B antagonized this effect. Our study revealed the anti-thrombotic activity of DHI. The multi-targeting mechanism of DHI proves the effectiveness of a natural anti-thrombotic combination therapy. The identification of salvianolic acids as a core anti-thrombotic activity of DHI and the discovery that their different combinations could either synergistically or antagonistically provide a better guidance for safer clinical application and paves the way for further development of DHI.

Protective and therapeutic effects of Danhong injection on acute pancreatitis‑associated lung injury.

Lung functional impairment caused by acute pancreatitis (AP) is the primary contributor to AP‑associated mortality. Previous studies have reported that AP‑associated lung injury is associated with systemic inflammatory response syndrome and oxidative stress. In the present study, the protective effects of Danhong injection (DHI), a widely used Chinese Traditional Medicine preparation, on AP‑associated lung injury in rats was examined. The myeloperoxidase activity, malondiadelhyde level and superoxide dismutase activity determination demonstrated the anti‑inflammatory and anti‑oxidative properties of DHI. The results of western blotting and reverse‑transcription‑semi‑quantitative polymerase chain reaction indicated that DHI could protect rats against AP‑associated lung injury, and the protective effect was associated with the suppression of nuclear factor‑κB activation and cell adhesion molecule expression, and the reduction of neutrophil infiltration and oxidative stress levels. As demonstrated by HE staining, DHI inhibited the pancreas and lung tissue injury. Therefore, DHI could be a potential candidate for the treatment of patients with AP‑associated lung injury.

Toward a scientific understanding of the effectiveness, material basis and prescription compatibility of a Chinese herbal formula Dan-hong injection

Since traditional Chinese medicine (TCM) is a complex mixture of multiple components, the application of methodologies for evaluating single-components Western medicine in TCM studies may have certain limitations. Appropriate strategies that recognize the integrality of TCM and connect to TCM theories remain to be developed. Here we use multiple unique approaches to study the scientific connotation of a TCM formula Dan-hong injection (DHI) without undermining its prescription integrity. The blood circulation improving and healing promoting effects of DHI were assessed by a qi stagnation blood stasis rat model and a mouse model of laser irradiation induced cerebral microvascular thrombosis. By UFLC-PDA-Triple Q-TOF-MS/MS and relevance analysis between chemical characters and biological effects, 82 chemical constituents and nine core components, whose blood circulation promoting effects were found comparable to that of whole DHI, were successfully identified. What’s more, the rationality of DHI prescription compatibility could be reflected not only in the maximum efficacy of the original ratio, but also in the interactions of compounds from different ingredient herbs, such as complementary activities and facilitating tissues distribution. This study provides scientific evidences in explanation of the clinical benefits of DHI, and also gives a good demonstration for the comprehensive evaluation of other TCM.

The clinical study of Danhong injection combined with edaravone in the treatment of acute ischemic stroke

Objective To evaluate the clinical efficacy of Danhong injection combined with edaravone in the treatment of acute ischemic stroke. Methods 90 patients with acute ischemic stroke were randomly divided into control group and observation group, 45 cases in each group.The control group was given edaravone treatment on the basis of conventional treatment, the observation group was treated with Danhong injection combined with edaravone treatment, the two groups were treated for 14 days.The changes of main physiological indices, clinical efficacy and adverse reactions etc.were observed. Results After treatment, in the two groups, the differences were statistically significant in the platelet aggregation and plasma viscosity compared with before treatment (t=4.412, 4.215, 3.984, 4.608, all P 0.05). Conclusion On the basis of conventional therapy, Danhong injection combined with edaravone in the treatment of acute ischemic stroke can significantly improve the clinical symptoms of the patients, improve the clinical efficacy and has good safety and great clinical significance. Key words: Stroke; Danhong injection; Edaravone

Simultaneous determination of eight Danshen polyphenols in rat plasma and its application to a comparative pharmacokinetic study of DanHong injection and Danshen injection.

Polyphenols derived from Danshen are responsible for the therapeutic effects of DanHong injection, a two-herb combination of Danshen and Honghua. Whether the pharmacokinetics of Danshen polyphenols is changed by coexisting Honghua constituents remains unknown. A sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed in this study for simultaneous determination of eight Danshen polyphenols (i.e., protocatechuic aldehyde, protocatechuic acid, tanshinol, salvianolic acid D, rosmarinic acid, salvianolic acid A, lithospermic acid, and salvianolic acid B) in rat plasma and applied to a comparative pharmacokinetic study of DanHong injection and Danshen injection. Liquid chromatography conditions, mass spectrometry parameters, and sample preparation were optimized step by step. The calibration curves showed good linearity (r > 0.99) for all the polyphenols. The mean extraction efficiencies ranged from 62.2 to 88.7% with negligible matrix effects. The intrabatch and interbatch precision at all the quality control levels were less than 15% of the nominal concentrations with accuracy of 88.8-114%, except that precision and accuracy at lower limit of quantitation were 3.2-17.3 and 95.7-119%, respectively. Comparative pharmacokinetic study suggested that the coexisting Honghua constituents might have negligible influences on the pharmacokinetics of Danshen polyphenols from DanHong injection. The bioanalytical method could also be applied to pharmacokinetic studies of other Danshen herbal products.

Relative quantification of neuronal polar lipids by UPLC-MS reveals the brain protection mechanism of Danhong injection

Promising results from clinical trials have fueled a growing acceptance of Danhong injection (DHI) as a Chinese Materia Medica standardized product for the treatment of ischemic stroke.

Coordinated Activation of VEGF/VEGFR-2 and PPARδ Pathways by a Multi-Component Chinese Medicine DHI Accelerated Recovery from Peripheral Arterial Disease in Type 2 Diabetic Mice.

Diabetic mellitus (DM) patients are at an increased risk of developing peripheral arterial disease (PAD). Danhong injection (DHI) is a Chinese patent medicine widely used for several cardiovascular indications but the mechanism of action is not well-understood. We investigated the therapeutic potential of DHI on experimental PAD in mice with chemically induced as well as genetic (KKAy) type 2 DM and the overlapping signaling pathways regulating both therapeutic angiogenesis and glucose homeostasis. Compared with normal genetic background wild type (WT) mice, both DM mice showed impaired perfusion recovery in hind-limb ischemia (HLI) model. DHI treatment significantly accelerated perfusion recovery, lowered blood glucose and improved glucose tolerance in both DM models. Bioluminescent imaging demonstrated a continuous ischemia-induced vascular endothelial growth factor receptor 2 (VEGFR-2) gene expressions with a peak time coincident with the maximal DHI stimulation. Flow cytometry analysis showed a DHI-mediated increase in endothelial progenitor cell (EPC) mobilization from bone marrow to circulating peripheral blood. DHI administration upregulated the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor-2 (VEGFR-2) in ischemic muscle. A cross talk between ischemia-induced angiogenesis and glucose tolerance pathways was analyzed by Ingenuity Pathway Analysis (IPA) which suggested an interaction of VEGF-A/VEGFR-2 and peroxisome proliferator-activated receptor δ (PPARδ)/peroxisome proliferator-activated receptor γ (PPARγ) genes. We confirmed that upregulation of VEGF-A/VEGFR-2 by DHI promoted PPARδ gene expression in both type 2 diabetic mice. Our findings demonstrated that a multi-component Chinese medicine DHI effectively increased blood flow recovery after tissue ischemia in diabetic mice by promoting angiogenesis and improving glucose tolerance through a concomitant activation of VEGF-A/VEGFR-2 and PPARδ signaling pathways.

Drug-protein binding of Danhong injection and the potential influence of drug combination with aspirin: Insight by ultrafiltration LC–MS and molecular modeling

Danhong injection (DHI) is a widely used Chinese medicine injection (CMI) for the clinical treatment of cardiovascular and cerebrovascular diseases. In this study, a simple and efficient in vitro method based on ultrafiltration LC–MS and molecular modeling has been developed to study the human serum albumin (HSA) binding of the compounds in DHI. Seven major components including protocatechuic aldehyde, p-coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid and salvianolic acid B were identified as HSA ligands and their binding degrees in the proposed non-saturated model were 26.17, 37.69, 99.77, 91.78, 96.91, 99.42 and 98.10%, respectively. Considering the drug-HSA binding property of the compounds in DHI may change during drug combination therapy, competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-HSA binding. Experimental results revealed that the salvianolic acids in DHI had stronger binding ability to HSA than sodium salicylate. To further verify the results above, molecular modeling and probe displacement assay were conducted to investigate the optimum binding site and binding affinity of the ligands on HSA. Our findings suggested that the established method could be a powerful tool to study the drug-HSA binding property of CMIs.

Efficacy and safety of Danhong injection for idiopathic pulmonary fibrosis：Meta-analysis

To systematically review the efficacy and safety of Danhong injection for patients with idiopathic pulmonary fibrosis(IPF), two researchers electronically searched PubMed, EMbase, Web of Science, Cochrane Library, CNKI, CBM, WanFang Data and VIP databases from the date of establishment to May 2016 for all randomized controlled trials(RCTs) and quasi-RCTs on the use of Danhong injection in patients with IPF. Manual search in relevant journals and search of relevant literature on other websites were also performed. The data extraction and quality assessment of included RCTs and quasi-RCT were conducted by two reviewers independently. Then, Meta-analysis was conducted by using RevMan 5.3 software. A total of 12 RCTs involving 844 patients were included, 423 cases in experiment group and 421 cases in control group. The results of meta-analysis indicated that the Danhong injection group was superior than the control group in clinical effectiveness(RR=1.36, 95%CI 1.25 to 1.49, P<0.000 01), increased DLCO value(MD=4.25, 95%CI 3.32 to 5.18, P<0.000 01), and increased PaO2 value(MD=14.51, 95%CI 12.35 to 16.68, P<0.000 01). The analysis results showed that Danhong injection could significantly reduce the level of TGF-β1 in serum. There were no serious or frequently happened adverse effects in the Danhong injection group, indicating high safety and good tolerance of Danhong injection in treatment of IPF. The current evidences suggested that Danhong injection in short term use(<12 weeks) could increase clinical effectiveness, improve DLCO and PaO2, and decrease the level of TGF-β1 in serum of IPF patients, with less adverse effects. However, these results should be carefully interpreted due to the low methodology quality and small sample size of trials, and this conclusion had to be further verified by high quality, large scale and double blinded RCTs.

Amelioration of cardiac dysfunction and ventricular remodeling after myocardial infarction by danhong injection are critically contributed by anti-TGF-β-mediated fibrosis and angiogenesis mechanisms

Ethnopharmacological relevanceDanhong injection (DHI) is a standardized product extracted from Radix et Rhizoma Salviae Miltiorrhizae and Flos Carthami , which has been long applied mainly used to treat ischemic encephalopathy and cardiac diseases including myocardial infarction and angina in clinical practice. Aim of the studyAim of this study was to investigate the salutary effects of DHI by slowing ventricular remodeling and improving cardiac function after myocardial infarction (MI) in rats. Materials and methodsIn this study, Male Sprague-Dawley rats were subjected to ligation on left anterior descending coronary artery to establish MI models and valsartan was selected as positive control. Cardiac function examination was conducted at the 1st, 3rd, 7th, 14th and the 28th days after LAD. Haematoxylin and Eosin (HE) staining and Masson staining were conducted to observe cardiac pathology and morphological changes levels of VEGF, TGF-β, MMP-2, and MMP-9 in the myocardial tissue were determined in gene and protein expressions. ResultsAfter 3 days post-treatment and thereafter, EF and FS in DHI group were greater than that of model group (p<0.05). Compared with the MI group, ratio of infarct was markedly decreased in treated-DHI group(p<0.05). TGF-β1 protein and fibrosis-related proteins MMP-2 and MMP-9 were up-regulated after MI, and they were significantly suppressed by the administration of DHI(p<0.05 and p<0.01, respectively). Moreover, DHI improved the mRNA expression of VEGF and increased the blood vessel density of myocardial infarct border zone. DHI decreased the expression of cell apoptosis protein of caspase-3 and increased the anti-apoptotic protein, bcl-2. ConclusionsWe provided direct evidences that DHI improves cardiac remodeling and preserves ventricular function post-MI in rats. DHI conferred cardio-protection in rats with MI via anti-myocardial apoptosis, angiogenesis, reduction of myocardial fibrosis and many other aspects of joint actions.

Effect of danhong injection on the mobilisation of endothelial progenitor cells to vascular repair after percutaneous coronary intervention: a randomised controlled trial

BackgroundEndothelial progenitor cells (EPCs) derived from bone marrow are associated with the repair of vascular injury by accelerating re-endothelisation, and represent a potential therapeutic target for ischaemic disease. Danhong injection (DHI), a Chinese Materia Medica standardised product extracted from Radix Salvia miltiorrhiza and Flos Carthamus tinctorius L, is effective in the treatment of ischaemic heart disease. We assessed the effect of DHI on the mobilisation of EPCs and vascular repair mediated by EPCs after percutaneous coronary intervention (PCI). MethodsIn a prospective, randomised clinical trial (ChiCTR-ONRC-14004100), patients with coronary heart disease and chest pain (n=54) who were referred to coronary angiography and underwent PCI were enrolled; 12 dropped out because of incomplete blood sample collection. The remaining 42 patients (age 35–75 years) were randomly assigned by random number table method to either a control group (14 men, seven women) that received standard medical treatment or a DHI group (14 men, seven women) that received standard medical treatment combined with DHI (40 mL per day) for 7 days at the affiliated cardiovascular hospital of Qingdao University. The primary outcomes were endothelial lesion and endothelial regeneration. Endothelial lesion was assessed by enumeration of CD45– CD146+ KDR+ circulating endothelial cells (CECs) and inflammatory factors (interleukin 6, C-reactive protein [CRP], tumour necrosis factor α [TNFα]). Endothelial regeneration was evaluated by measurement of CD45dim CD34+ KDR+ EPCs, as determined by flow cytometry. Measurements were taken before PCI, 1 day after PCI, and 7 days after treatment by two investigators masked to treatment assignment. All healthy participants and those with coronary heart disease gave informed consent to participate in the study. All protocols were approved by each of the local ethics committees. FindingsBaseline cytokines, CECs, and EPCs did not differ between DHI and control groups. PCI increased interleukin 6 (p=0·004 in control group; p=0·045 in DHI group), TNF α (p=0·001 in control group; p=0·16 in DHI group), and CECs concentrations (p=0·008 in control group; p=0·005 in DHI group), whereas EPCs decreased (p=0·002 in control group; p=0·001 in DHI group). Compared with the control group, the mean expression levels of cytokines were significantly reduced (interleukin 6 14·98 pg/mL [SD 13·51] vs 7·25 pg/mL [8·03], p=0·001; TNFα 594·17 pg/mL [368·34] vs 388·48 pg/mL [210·24], p<0·0001; CRP 38·07 ng/mL [18·93] vs 22·42 ng/mL [5·94], p=0·04), and CECs were significantly reduced (0·1228% of PBMC [0·0669] vs 0·0564% of PBMC [0·0445], p<0·0001). EPC population significantly increased (0·0962% of PBMC [0·044] vs 0·2569 of PBMC [0·1215], p<0·0001) in DHI group after treatment. InterpretationThese initial results suggest that DHI is effective in improving endothelial repair and protecting the endothelial lesion by mobilising EPCs, by inhibiting the inflammatory response, and by inhibiting of shedding of endothelial cells after PCI in patients with coronary heart disease. FundingChina National Basic Research Program (973 programme, number 2012CB518404).

Systematic investigation of transcription factors critical in the protection against cerebral ischemia by Danhong injection.

Systematic investigations of complex pathological cascades during ischemic brain injury help to elucidate novel therapeutic targets against cerebral ischemia. Although some transcription factors (TFs) involved in cerebral ischemia, systematic surveys of their changes during ischemic brain injury have not been reported. Moreover, some multi-target agents effectively protected against ischemic stroke, but their mechanisms, especially the targets of TFs, are still unclear. Therefore, a comprehensive approach by integrating network pharmacology strategy and a new concatenated tandem array of consensus transcription factor response elements method to systematically investigate the target TFs critical in the protection against cerebral ischemia by a medication was first reported, and then applied to a multi-target drug, Danhong injection (DHI). High-throughput nature and depth of coverage, as well as high quantitative accuracy of the developed approach, make it more suitable for analyzing such multi-target agents. Results indicated that pre-B-cell leukemia transcription factor 1 and cyclic AMP-dependent transcription factor 1, along with six other TFs, are putative target TFs for DHI-mediated protection against cerebral ischemia. This study provides, for the first time, a systematic investigation of the target TFs critical to DHI-mediated protection against cerebral ischemia, as well as reveals more potential therapeutic targets for ischemic stroke.

A high content screening method for detection of anaphylactoid reaction of injection formulations

Anaphylactoid reaction (AR) is the most common adverse reaction of injection formulations, however, there are obvious drawbacks in available methods for AR detection. A novel in vitro detection method for AR was established based on fluorescent labeling and high content screen (HCS) system in present study. With the use of RBL-2H3 cells degranulation model, positive cell count was determined with specific cellular membrane fluorescent dye FM4-64 labeling vesicle recycle, and total cells count was determined with specific nucleus fluorescent dye Hochest 3334, and then the ratio of cells degranulation after drug stimulation was calculated. In order to verify the reliability of this HCS method, positive drug Compound 48/80 was first used to confirm the consistence of HCS method with the traditional β-hexosaminidase release test and the Evans blue staining ears test in mice. The results showed high consistence between HCS method and traditional testing methods, and the HCS method showed higher sensitivity than the other two tests. Then 30 samples of Danhong injection (DHI) with clinical allergy symptoms further were used to confirm the reliability of this HCS method. The HCS results showed high consistence with the clinical report, and the HCS method had the advantage in reducing the interference by drug color. Therefore, this HCS method is reliable, sensitive, simple and high-throughput method in detection of AR, applicable for the AR evaluation of injection formulations, and can provide guidance for safety of clinical application in clinical practice.

Establishment of a ternary network system for evaluating the antioxidant fraction of Danhong injection.

Oxidative stress plays a crucial role in numerous cardiovascular diseases. As an effective therapy, Danhong injection (DHI) is considered to act through an antioxidant mechanism for the treatment of cardiovascular disease. In our study, we focused on the potential contribution of the antioxidant capacity of DHI fractions (Frs) and established an innovative screening method based on a 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay. A ternary network evaluation system, which was constructed based on the radical scavenging activity, the area under the activity-concentration curve and the solid content of the fractions, was implemented to select the fractions that posed the greatest antioxidant effect. As a result, Frs 5-7 and Frs 17-19 were shown to exhibit superior antioxidant activity according to the regression area of the ternary network, which was >0.5. Furthermore, the active fractions were characterized by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry combined with nuclear magnetic resonance. This study provided an effective method for the comprehensive evaluation of the antioxidant effect of DHI fractions. Copyright © 2016 John Wiley & Sons, Ltd.

Danhong injection attenuates cardiac injury induced by ischemic and reperfused neuronal cells through regulating arginine vasopressin expression and secretion

Ischemic stroke is associated with cardiac myocyte vulnerability through some unknown mechanisms. Arginine vasopressin (AVP) may exert considerable function in the relationship of brain damage and heart failure. Danhong injection (DHI) can protect both stroke and heart failure patients with good efficacy in clinics. The aim of this study is to investigate the mechanism of DHI in heart and brain co-protection effects to determine whether AVP plays key role in this course. In the present study, we found that both the supernatant from oxygen-glucose deprivation (OGD) and reperfused primary rat neuronal cells (PRNCs) and AVP treatment caused significant reduction in cell viability and mitochondrial activity in primary rat cardiac myocytes (RCMs). Besides, DHI had the same protective effects with conivaptan, a dual vasopressin V1A and V2 receptor antagonist, in reducing the RCM damage induced by overdose AVP. DHI significantly decreased the injury of both PRNCs and RCMs. Meanwhile, the AVP level was elevated dramatically in OGD and reperfusion PRNCs, and DHI was able to decrease the AVP expression in the injured PRNCs. Therefore, our present results suggested that OGD and reperfusion PRNCs might induce myocyte injury by elevating the AVP expression in PRNCs. The ability of DHI to reinstate AVP level may be one of the mechanisms of its brain and heart co-protection effects.

Effects of Danhong Injection (丹红注射液) and its main components on anticoagulation and fibrinolysis in cultured vein endothelial cells

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). VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N and 5% CO 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-PGF1α) 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 (PAI) were measured by the method of chromogenic substrate. The contents of endothelin (ET) and nitric oxide (NO) were detected by non-equilibrium RIA and enzymelinked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. Compared with the normal control group, the floating cells number, PAI activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1α 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-PGF1α content and SOD activity, regulated PAI and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate (P<0.05 or P<0.01). 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.

The mixture of Salvia miltiorrhiza–Carthamus tinctorius (Danhong injection) alleviates low-dose aspirin induced gastric mucosal damage in rats

BackgroundDanhong injection (DHI) is quite often used in combination with low-dose aspirin (ASA, 75–325mg daily) in clinic, particularly for the treatment of cardiovascular diseases. Exploring their interaction profile is of great clinical importance. PurposeThe current study aims to explore the interaction between DHI and low-dose ASA in rats. MethodsSixty four rats were randomly divided into eight groups. Stomach and other four vital organs were collected for histological evaluation. Organs which exhibited histological changes were selected for a further study to evaluate the damage score and mode of action. We tested the protective effect of DHI on gastric mucosal damage in different regimes of administration. COX activity, gastric mucus secretion, pepsin activity, antioxidant activity and ROS level were assayed to reflect the protective effect of DHI on gastric mucosal damage induced by ASA. ResultsStomach was the target organ of interaction when DHI and ASA were used in combination. DHI alleviated gastric mucosal damage by 55.8% when DHI was injected before ASA (Group E) and by 53.5% when DHI was injected 2h after ASA administration (Group F). Additionally, if DHI treatment was appended to the long-term administration of ASA, DHI still decreased the gastric mucosal damage score in 52.0% from 2.50 to 1.20. DHI improved gastric mucus secretion, as well as decreased pepsin activity to maintain the integrity of gastric mucosal barrier (P<0.05). Furthermore, DHI recovered antioxidant activity which was impaired by ASA. In details, DHI decreased gastric mucosal ROS level, increased CAT, GSH-Px and SOD activity, and reduced MDA concentration (P<0.05). When ASA (71.9µM) was used in combination with DHI (23–fold dilution, presented in terms of concentrations of DSS, PA, SaD RA, SaB and SaA were 6.45–6.92, 1.10–1.14, 1.09–1.10, 0.86–0.90, 16.76–19.38 and 1.83–1.94µg/ml, respectively) in vitro, the inhibition rate of ASA increased from 38.6% (ASA alone) to 62.8% (ASA-DHI) on COX-1 and from 28.9% (ASA alone) to 38.8% (ASA-DHI) on COX-2 (P<0.05). DHI strengthened the inhibition activity of ASA on both COX-1 and COX-2, which showed that DHI alleviated ASA induced gastric mucosal damage but not antagonized anti-COX effect of ASA. ConclusionsGastric protective benefits were clearly produced when DHI and ASA were used in combination, which provided rational guidance for clinical combined application of DHI and ASA.

Danhong injection attenuates isoproterenol-induced cardiac hypertrophy by regulating p38 and NF-κb pathway

Ethnopharmacological relevanceDanhong injection (DHI), derived from Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Salvia militiorrhiza Bunge), is an extensively-used Chinese material standardized clinical product for treatment of cardiovascular diseases. Aim of the studyCardiac hypertrophy (CH) is an adaptive response of cardiomyocytes. Long-lasting cardiac hypertrophy results in the loss of compensation by cardiomyocytes which could ultimately develop into heart failure. In the present study, we aimed to investigate the effect and exact mechanisms of DHI on isoproterenol (ISO)-induced CH. Materials and methodsH9c2 cells and male Wistar rats were stimulated by ISO in the present study to establish CH models in vitro and in vivo. CCk-8 assay, Western blot, real time-polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA) and Echocardiography were used in the present study. ResultsDHI significantly attenuated ISO-induced CH of H9c2 cells (p<0.01). DHI decreased ISO-induced atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elevation both at the mRNA and protein levels (p<0.05 and p<0.01, respectively). Western blot showed that DHI down-regulated the phosphorylation of p38. Furthermore, we found that DHI inhibited the nuclear translocation and activation of NF-κb. Echocardiography from ISO-induced CH rats showed that DHI significantly decreased left ventricle (LV) mass, the thickness of the LV end-systolic posterior wall (LVPWs), and the LV end-diastolic posterior wall (LVPWd) elevated by ISO (p<0.01 and p<0.05, respectively). ConclusionThese data demonstrate that DHI might exert anti-cardiac hypertrophic effects by regulating p38 and NF-κb pathway.

Application of a high-resolution benchtop quadrupole-Orbitrap mass spectrometry for the pharmacokinetics and tissue distribution of Danhong injection in guinea pig

Danhong injection (DHI) is a compound preparation of traditional Chinese medicine Salviae miltiorrhiza (a.k.a. Danshen) and Flos Carthami tinctorii. Phenolic acids, such as salvianic acid A sodium (SAS), protocatechualdehyde (PAL), salvianolic acid B (SAB) and flavonoids, such as rutin, are the main bioactive ingredients of DHI. This paper presents an application of ultra high-performance liquid chromatography and quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HR MS) to quantify salvianic acid A sodium, protocatechualdehyde, salvianolic acid B, and rutin in guinea pig plasma and tissues for pharmacokinetic and tissue distribution studies. Biological samples were processed with methanol extraction, and calibration curves were determined with indometacin as internal standard (I.S.). The analytes were separated by a Hypersil GOLD aQ C18 column and detected using negative ion full MS/dd-MS2 (data-dependent MS2) mode. The full MS scan acquired data for identification and quantification, and dd-MS2 scan obtained product ion spectra for confirmation. The response showed good linear relationship over the concentration range between 10.22 and 5110ng/mL for salvianic acid A sodium, 10.06–5030ng/mL for salvianolic acid B, 11.12–5560ng/mL for protocatechualdehyde, and 10.58–5290ng/mL for rutin, all the coefficients of correlation (r2) >0.9990. The lower limit of quantification (LLOQ) was 10ng/mL for the analytes. Overall, the novel UHPLC-Q-Orbitrap has demonstrated great performance for rapidity, accuracy, high sensitivity, and high selectivity. It was successfully applied to the pharmacokinetic and tissue distribution studies of Danhong injection. These acquired data would provide helpful information for the clinical applications, mechanism studies, and technology promotion of DHI in the future.

Rationality analysis of clinical application of Danhong injection in affiliated hospital of Nanjing university of Chinese medicine from 2013 to 2014

It is necessary to investigate the influence of the rationality of clinical drug use on the benefit and risk factors of traditional Chinese medicine injections. The retrospective survey was based on the medical records and information of 4 950 patients who used Danhong injection in the HIS database of the first affiliated hospital of Nanjing University of Chinese Medicine from 2013 to 2014. The basic statistical methods and associated rules analysis were utilized to analyze the HIS information of these patients, including the basic information, the diagnosis, the department, the dosage, the usage of medication, the drug combination and the adverse reactions. And the rationality analysis of the clinical application of Danhong injection was carried out to investigate relevant factors of the adverse reactions. The results showed that most cases came from the department of cardiology (51.95%) and encephalopathy center (20.67%). In the statistical period, the patients aged above 40 years old accounted for 96.65%. And the two western medicine diagnosis items with the highest confidence level were coronary heart disease and angina pectoris (97.15%), while the three items were coronary heart disease, angina pectoris and hypertension (97.02%). The irrational indications were mainly hypertension (12.93%) and diabetes (4.55%). All of them were diagnosed as blood stasis syndrome by the traditional Chinese medicine. About 98.93% of the single dosage was within the range stipulated on package insert, the duration mainly ranged between 1 and 21 days, and 97.64% of the menstrua contained 0.9% NS and 5% GS. According to the medication records,99.26% were the use of combined drugs, with 8.41 drugs on average. Antiplatelet drugs (72.04%) were the most frequently combined with western medicine, followed by the cholesterol-regulating drugs (64.86%) and the cerebrovascular drugs (60.26%). When used in the combination with antibiotics for the infection, cephalosporin antibiotics were the most frequently applied (8.81%). When used with traditional Chinese medicines, traditional Chinese medicines for activating blood circulation and removing blood stasis or monomer traditional Chinese medicine injections (28.93%) were the dominance, in which Gastrodin injection was the most frequently applied (16.23%). And 12 cases of adverse reactions were reported, with the ADR rate of 0.24%. The indications, solvent compatibility and irrational drug combination may be the potential risk factors for ADRs induced by Danhong injection. Further experiments are required to evaluate the benefits and risks in these three aspects.