Flos Carthami Research Articles

Danhong formula alleviates endothelial dysfunction and reduces blood pressure in hypertension by regulating MicroRNA 24 - Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase axis

Ethnopharmacological relevanceEssential hypertension (EH) is one of the important risk factors of cardio-cerebrovascular diseases, and it can significantly increase the incidence and mortality of acute myocardial infarction, cerebral infarction and hemorrhage. Danhong Formula (DHF) was consisting of Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese) (Plant names have been checked with http://www.the plant list.org on June 28th, 2023) was approved by State Food and Drug Administration of China, that has been used for thousands of years in the treatment of cardiovascular diseases in China with proven safety and efficacy. Though our previous studies have found that DHF improved endothelial dysfunction (ED) and decreased high blood pressure (BP), the underlying mechanisms of its antihypertensive effect still remain unclear. Aim of the studyThis study investigated whether DHF regulated MicroRNA 24- Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase (miR-24 - PI3K/AKT/eNOS) axis to produce antihypertensive effect and improve endothelial dysfunction. Materials and methodsFirstly, the chemical components of DHF were analyzed by UHPLC-MS. After that, BP was continuously monitored within the 1st, 3rd, and 4th week in SHR to evaluate the antihypertensive effect of DHF intraperitoneal injection. In addition, not only the contents of serum nitric oxide (NO), prostacyclin (PGI2), and angiotensin II (Ang II) were detected, but also the isolated aorta ring experiment was conducted to evaluate the vasomotoricity to evaluate of DHF on improving endothelial dysfunction. Key proteins or mRNA expression associated with miR-24 - PI3K/AKT/eNOS axis in aorta were detected by capillary Western blot, immunohistochemistry or RT-PCR to explore the underlying mechanisms. Index of NO, Ang II PGI2 and key proteins or mRNA expression were also conducted in miR-24-3p over-expression HUVECs model. ResultsCompared with SHR control group, DHF (4 mL/kg/day, 2 mL/kg/day, 1 mL/kg/day) treatment significantly reduced high BP in SHR and selectively increased acetylcholine (Ach) induced vasodilation, but not sodium nitroprusside (SNP) in a manner of concentration dependency in isolated aorta ring. DHF (4 mL/kg/day, 1 mL/kg/day) treatment was accompanying an increment of NO and PGI2, and lowering AngII in SHR. Moreover, DHF treatment significantly up-regulated expression of p-PI3K, p-AKT, mTOR, eNOS and p-eNOS, but down-regulated miR-24-3p expression in aorta. Compared with miR-24-3p over-expression HUVECs model group, DHF treatment inhibited miR- 24-3p expression and up-regulated p-PI3K, p-AKT, mTOR and eNOS mRNA expression. Similarly, DHF treatment increased PI3K, AKT, mTOR and eNOS protein expression in HUVECs by Western blot. ConclusionsThese findings suggest that DHF alleviates endothelial dysfunction and reduces high BP in SHR mediated by down-regulating miR-24 via ultimately facilitating up-regulation of PI3K/AKT/eNOS axis. This current study firstly demonstrates a potential direction for antihypertensive mechanism of DHF from microRNA aspect and will promote its clinical applications.

The mechanism of Semen Persicae-Flos Carthami in treating hypertrophic scar: A study based on network pharmacological analysis and invitro experiments.

Traditional medicine believes that hypertrophic scar (HS) falls into the category of "blood stasis". Chinese herbs for promoting blood circulation and removing blood stasis, activating meridians, and relieving pain are usually selected to treat HS by traditional Chinese medicine (TCM). Both Semen Persicae (SP) and Flos Carthami (FC) are confirmed to be effective for HS. Clinically, SP and FC are often used in combination with each other. However, the pharmacodynamic mechanism and molecular target of SP-FC in the treatment of HS are still unclear. Therefore, this study is intended to explore the mechanism and target of SP-FC in the treatment of HS through network pharmacology combined with invitro cell and molecular biology experiments. Target genes of SP-FC were obtained from the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), and targets of HS-related diseases were searched from databases such as Disgenet and GeneCards. Based on the targets searched and obtained, a Venn diagram was plotted to acquire common targets of SP-FC-HS. Next, STRING 11.0 was employed for protein-protein interaction (PPI) network analysis of common targets; and cytoscape 3.9.0 for connection relationship analysis of PPI and plotting of a "drug-component-target" network diagram. Besides, a modified explant culture method was applied to separate primary hypertrophic scar fibroblasts (HSFs); MTT assay to detect cell viability of HSFs after treatment by SP-FC for 24 h; Annexin V-FITC/PI double staining combined with flow cytometry to test apoptosis; western blot to check the protein expression level of p53; and real-time fluorescence quantitative PCR to determine mRNA level of p53. In the analysis of network pharmacology, 269 pharmacological targets of SP, 449 pharmacological targets of FC, and 2569 targets of HS-related diseases were screened from the databases. After plotting the Venn diagram, 116 common targets of SP-FC-HS were acquired. In vitro experiments showed that the expression of p53 in HSFs was decreased. SP-FC significantly reduces the viability of HSFs, increases p53 levels in HSFs, and promotes apoptosis. SP-FC can reduce scar formation by promoting p53 expression.

Salvianolic acid A from Danhong Injection induces vasorelaxation by Regulating L-type calcium channel in isolated mouse arteries

Ethnopharmacological relevanceDanhong injection (DHI), which is a Chinese clinical prescription consists of Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese)(Plant names have been checked with http://www.theplantlist.org on March 1st, 2022), has been mainly used in the clinical therapy of cardiovascular diseases, including hypertension in China for many years. Aim of the studyCardiovascular diseases (CVDs) are the major causes of death all around the world. Due to the various stimulation, a series of vasoconstrictor substances are secreted to regulate the vasoconstriction function and then change blood pressure. The representative substances leading to abnormal vasoconstriction include renin-angiotensin system, endothelin, vasopressin and adrenaline, which act on the corresponding receptors on vascular smooth muscle to constrict blood vessels. Finally, blood pressure increases, followed by a series of cardiovascular diseases, including hypertension. However, little is known about Danhong injection’s specific vasodilating mechanisms and active substances. The aims of the study were to determine the vasodilating substances of Danhong injection and explain its molecular mechanism of vasodilation. Materials and methodsThe effects of DHI and its active components on vascular tension were measured by myograph system in the aortic or mesenteric rings of mice. Based on this, the pharmacodynamic substances were analyzed and effective molecules were found. Combined with multiple types of vascular myograph experiments and network pharmacological analysis, the molecular pathway was preliminarily determined. With molecular biology experiments, it was verified that the relevant mechanisms were closely related to calcium-mediated vasoconstriction in smooth muscle cells. ResultsDHI could relax endothelium-removed aortic rings pre-constricted with PE and 3 possible active vasodilator substances, including salvianolic acid A, salvianolic acid B and danshensu, were screened out by network pharmacology and vascular myograph experiments, among which the effects of salvianolic acid A were dominant. Meanwhile, salvianolic acid A could dilate mesenteric artery in a pressure-dependent manner. Interestingly, salvianolic acid A could still relax the vascular rings under the stimulation of KCl and Bayk8644, two agonists of L-type calcium channel. By contrast, inhibitors of Kir, Kv, Katp and BKCa channels did not block the effect of salvianolic acid A on vasodilation. Salvianolic acid A alleviated Ca2+ transient, referring to changes of intracellular calcium, induced by PE, Bayk8644 and high K+ in the VSMCs. Salvianolic acid A could partially restore the vasodilation function of vascular smooth muscle damaged by AngII and ET-1 induced hypertension situation. ConclusionsOur results indicate that salvianolic acid A is the major vasodilator substance in DHI and the vasorelaxation pharmacology mechanism involved in inhibiting the L-type calcium channel signaling in smooth muscle cell. Hence, there are potential therapeutic effects of taking salvianolic acid A preparation which may be beneficial to protect cardiovascular system and reduce blood pressure.

Compatibility of ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) and their protective effects on cerebral ischemia-reperfusion injury in rats.

Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) (Danhong) are two drugs commonly prescribed together, which are often used in the treatment of cerebrovascular diseases in China. Due to the complexity of the ingredients of Danhong, the present study focused on performing the orthogonal compatibility method on the primary effective molecules of this drug: Tanshinol, salvianolic acid A, salvianolic acid B and hydroxysafflor yellow A. These four molecules were studied to determine their protective effects and to screen for the most compatible ingredients to improve cerebral ischemia-reperfusion injury (IR) in rats. Focal middle cerebral artery occlusion was performed to establish the cerebral IR model in rats. Male Sprague-Dawley rats were randomly divided into sham operation group, IR group and nine orthogonal administration groups with different ratios of Danhong effective ingredients and Danhong injection group. Neurological deficit score and cerebral infarction volume were measured postoperatively. Morphological pathological alterations were observed via H&E staining. Bcl-2 and Bax were quantified using ELISA. Immunohistochemistry was conducted to analyze the expression of caspase-3 in the hippocampus. The expression levels of cytochrome c, apoptotic peptidase activating factor 1 (apaf-1), caspase-9, caspase-3 and p53 mRNA in the hippocampus were assessed via reverse transcription-quantitative PCR. The results demonstrated that different compatibility groups significantly reduced the neurological function score and decreased the volume of cerebral infarct compared with the IR group. These groups were also indicated to improve the pathological damage to the brain tissue. In addition, certain compatibility groups significantly decreased the number of caspase-3 positive cells in the hippocampus and the expression levels of cytochrome c, apaf-1, caspase-9, caspase-3 and p53 mRNA in the brain tissue. Orthogonal group 4 (30 mg/kg tanshinol; 2.5 mg/kg salvianolic acid A; 16 mg/kg salvianolic acid B; 8 mg/kg hydroxysafflor yellow A) was indicated to be the most effective. The four effective ingredients of Danhong exhibited a protective effect on rats with cerebral IR injury, potentially through the inhibition of apoptosis via the downregulation of key targets upstream of the caspase-3 pathway. In addition, the present study provided novel insights for the continued study of the drug compatibility rules of TCM.

Metabolomics characterization of different geographical origins of Flos Carthami using UPLC-QTOF/MS and their correlation with climate factors

A UPLC-QTOF/MS method was developed to characterize chemical differences of Flos Carthami from different geographical origins. PCA, PLS-DA and heatmap analysis were used to differentiate and identify differential metabolites of Flos Carthami samples from different geographical origins. Grey correlation analysis (GCA) was conducted between differential metabolites and climate factors.

Danhong injection in cardiovascular and cerebrovascular diseases: Pharmacological actions, molecular mechanisms, and therapeutic potential.

Cardiovascular and cerebrovascular diseases are the main cause of mortality worldwide, currently with less than optimum therapeutic options. Danhong injection (DHI) is a medicinal preparation based on two eminent Chinese herbal medicines, Salviae Miltiorrhizae (Dan Shen; family: Lamiaceae) and Flos Carthami (Hong Hua; family: Compositae/Asteraceae). DHI has been mainly used in the clinical therapy of cardiovascular (such as acute coronary syndrome and angina pectoris) and cerebrovascular diseases (such as stroke) in China for many years. The pharmacological properties of DHI include anti-inflammatory, anti-oxidant, anti-coagulatory, hypolipidemic, anti-apoptotic, vasodilatory, and angiogenesis-promoting actions. DHI offers a safe and effective therapeutic agent against cardiovascular and cerebrovascular diseases by modulating multiple disease-relevant signaling pathways and molecular targets. Herein, we provide a comprehensive review of the phytochemistry, therapeutic effects, molecular mechanisms, and adverse reactions of DHI in cardiovascular and cerebrovascular diseases. We also highlight the latest pharmacological advances and therapeutic potential of this promising herb-derived cardiovascular drug preparation.

Protective effects of effective ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) compatibility after rat hippocampal neurons induced by hypoxia injury

OBJECTIVETo investigate the effective ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) (Danhong) on protective properties towards neonatal rat hippocampal neurons under hypoxia condition. METHODSPrimary culture of neonatal rat hippocampal neurons was used to model hypoxia damage on the hippocampus. Methyl thiazolyl tetrazolium (MTT) assay and preliminary experiments were conducted to identify the four effective ingredients of Danhong, along with the injection of positive drug onto hippocampal neurons at a non-toxic dosage level. The cultured cells were randomly divided into 12 groups: the normal group, model group, positive drug control group and nine compatibility groups of the four effective ingredients. Different test methods were applied to determine lactate dehydrogenasein (LDH), total superoxide dismutase (T-SOD), malondialdehyde (MDA), 6-keto prostaglandin Fla (6-keto-PGF1a), thromboxane B2 (TXB2), mitochondrial membrane potential (MMP), free calcium ions concentration ([Ca2+]i) and early onset cell apoptosis. RESULTSDifferent compatibility groups could inhibit the content of LDH and intracellular calcium overload, increase activity in T-SOD, decrease level of MDA and TXB2, improve level of 6-keto-PGF1a and MMP, and prevent the early onset cell apoptosis. CONCLUSIONThe compatibility of four effective ingredients of Danhong had protective effect toward hippocampi hypoxia. The mechanism might be related to inhibit oxidative stress damage and cell apoptosis, resist thrombosis, and reduce the intracellular calcium ion of overload.

Network pharmacology exploration reveals endothelial inflammation as a common mechanism for stroke and coronary artery disease treatment of Danhong injection

Although Danhong injection (DHI) is the most widely prescribed Chinese medicine for both stroke and coronary artery disease (CAD), its underlying common molecular mechanisms remain unclear. An integrated network pharmacology and experimental verification approach was used to decipher common pharmacological mechanisms of DHI on stroke and CAD treatment. A compound-target-disease & function-pathway network was constructed and analyzed, indicating that 37 ingredients derived from DH (Salvia miltiorrhiza Bge., Flos Carthami tinctorii and DHI) modulated 68 common targets shared by stroke and CAD. In-depth network analysis results of the top diseases, functions, pathways and upstream regulators implied that a common underlying mechanism linking DHI’s role in stroke and CAD treatment was inflammatory response in the process of atherosclerosis. Experimentally, DHI exerted comprehensive anti-inflammatory effects on LPS, ox-LDL or cholesterol crystal-induced NF-κB, c-jun and p38 activation, as well as IL-1β, TNF-α, and IL-10 secretion in vascular endothelial cells. Ten of 14 predicted ingredients were verified to have significant anti-inflammatory activities on LPS-induced endothelial inflammation. DHI exerts pharmacological efficacies on both stroke and CAD through multi-ingredient, multi-target, multi-function and multi-pathway mode. Anti-endothelial inflammation therapy serves as a common underlying mechanism. This study provides a new understanding of DHI in clinical application on cardiovascular and cerebrovascular diseases.

Effects of Danhong Injection on platelet aggregation in hyperlipidemia rats

Ethnopharmacological relevanceDanhong Injection (DHI), a Chinese medical product extracted from Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese), has been reported to have effects on inflammatory, anti-fibrinolytic properties, antithrombotic and decrease blood-lipid. It is extensively used for the clinical treatment of cardiovascular disease. This study aimed to investigate the effects of DHI on blood-lipid levels and platelet aggregation rate in hyperlipidemia rats. Materials and methodsRats were randomly divided into 6 groups: normal control (NC), model control (MC), DHI-treated control at doses of 1.0mL/kg, 2.0mL/kg, 4.0mL/kg, respectively, and Simvastatin positive control at dose of 2.0mg/kg. All DHI treated groups were intraperitoneally injected for 7 days. The effects of DHI on serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were evaluated. And platelet activating factor (PAF), platelet membrane glycoprotein IIb/IIIa (GP IIb/IIIa) and 6-keto-prostaglandin F1а (6-K-PGF1а) were determined by enzyme-linked immunosorbent assay (ELISA). Moreover, the expression of prostaglandin I-2 (PGI2), prostaglandin E-2 (PGE2) and thromboxane A2 (TXA2) in liver was determined by real-time PCR. ResultsCompared with the MC group, the rats treated with DHI had significantly reduced TC, TG, LDL-C, FIB, GP IIb/IIIa and platelet aggregation. Meanwhile, the thrombin time (TT), activated partial thrombin time (APTT), prothrombin time (PT), 6-K-PGF1а was significantly increased. Expression of PGI2 and PGE2 mRNA was significantly increased, whereas the TXA2 was significantly reduced. ConclusionsThis study demonstrated that the blood lipid and platelet aggregation has a regulatory effect after DHI treatment. The insights gained from this study will improve understanding of the mechanisms involved in the effect of DHI on hyperlipidemia and the pharmacological rationale for the use of DHI in diseases caused by formation of thrombosis and lipid metabolic disorders.

Development of an on-line mixed-mode gel liquid chromatography × reversed phase liquid chromatography method for separation of water extract from Flos Carthami

A novel on-line comprehensive two-dimensional liquid chromatography (2D-LC) method by coupling mixed-mode gel liquid chromatography (MMG-LC) with reversed phase liquid chromatography (RPLC) was developed. A mixture of 17 reference compounds was used to study the separation mechanism. A crude water extract of Flos Carthami was applied to evaluate the performance of the novel 2D-LC system. In the first dimension, the extract was eluted with a gradient of water/methanol over a cross-linked dextran gel Sephadex LH-20 column. Meanwhile, the advantages of size exclusion, reversed phase partition and adsorption separation mechanism were exploited before further on-line reversed phase purification on the second dimension. This novel on-line mixed-mode Sephadex LH-20×RPLC method provided higher peak resolution, sample processing ability (2.5mg) and better orthogonality (72.9%) versus RPLC×RPLC and hydrophilic interaction liquid chromatography (HILIC)×RPLC. To the best of our knowledge, this is the first report of a mixed-mode Sephadex LH-20×RPLC separation method with successful applications in on-line mode, which might be beneficial for harvesting targets from complicated medicinal plants.

Efficacy of Topical Compound Danxiong Granules for Treatment of Dermatologic Toxicities Induced by Targeted Anticancer Therapy: A Randomized, Double-Blind, Placebo-Controlled Trial.

Dermatologic toxicities resulting in dose reduction or discontinuation of treatment pose challenges for targeted anticancer therapies. We conducted this randomized, double-blind, placebo-controlled trial to investigate the efficacy of topical application of Compound Danxiong Granules (CDG) for treatment of dermatologic toxicities associated with targeted anticancer therapies. One hundred and ten patients with dermatologic toxicities induced by targeted anticancer therapies were randomly assigned to CDG or placebo group. Each crude herb (Rhizoma Chuanxiong, Paeonia suffruticosa Andr., Cortex Phellodendri, Geranium sibiricum L., and Flos Carthami) was prepared as an instant herbal powder. Application of the CDG via topical washes lasted 20 minutes, twice daily, for 10 days. The primary outcome was the total effective rate, defined as reduction in at least one grade of skin toxicity. The total effective rate was 77.61% (52/67) in the CDG group and 27.27% (9/33) in the placebo group (P < 0.0001). Compared to the placebo treatment, CDG treatment achieved a higher total effective rate for hand-foot skin reaction (95.45% versus 27.27%), acneiform eruption (69.23% versus 30.78%), and paronychia (68.42% versus 22.22%). Topical application of CDG can effectively attenuate dermatologic toxicities induced by targeted anticancer therapies. The effect of CDG was more pronounced in hand-foot skin reaction.

Rapid Fingerprint Analysis of Flos Carthami by Ultra-Performance Liquid Chromatography and Similarity Evaluation.

Flos Carthami (FC), the dried flower of Carthamus tinctorius L., has widely been used for the treatment of cardiovascular diseases in China. A rapid fingerprint analysis method of FC was established by ultra-performance liquid chromatography (UPLC) for the quality control. The analysis time was shortened by 45 min, from 75 min on conventional high-performance liquid chromatography (HPLC) to 30 min on UPLC. Ten batches of FC samples from various sources were analyzed by the UPLC method. Coexisting peaks in FC chromatograms were chosen to calculate their relative retention time (RRT) and relative peak area (RPA). Thirteen characteristic peaks were illustrated on UPLC fingerprints. In the 24-h stability test, the relative standard deviation (RSD) for the RRT and RPA was <0.08% and 4.41%, respectively. The RSD for precision of the RRT and RPA was <0.05% and 4.26%, respectively. And the RSD for repeatability was <0.05% for the RRT and 3.91% for the RPA. The similarities of the 10 batches of FC were ranged from 0.938 to 0.995.Compared with the HPLC method, UPLC provided shorter analysis time, higher resolution and better separation performance, which is feasible for the quality control of FC.

Biosynthetic approach to combine early steps of cardenolide biosynthesis in Saccharomyces cerevisiae

A novel on-line comprehensive two-dimensional liquid chromatography (2D-LC) method by coupling mixed-mode gel liquid chromatography (MMG-LC) with reversed phase liquid chromatography (RPLC) was developed. A mixture of 17 reference compounds was used to study the separation mechanism. A crude water extract of Flos Carthami was applied to evaluate the performance of the novel 2D-LC system. In the first dimension, the extract was eluted with a gradient of water/methanol over a cross-linked dextran gel Sephadex LH-20 column. Meanwhile, the advantages of size exclusion, reversed phase partition and adsorption separation mechanism were exploited before further on-line reversed phase purification on the second dimension. This novel on-line mixed-mode Sephadex LH-20 × RPLC method provided higher peak resolution, sample processing ability (2.5 mg) and better orthogonality (72.9%) versus RPLC × RPLC and hydrophilic interaction liquid chromatography (HILIC) × RPLC. To the best of our knowledge, this is the first report of a mixed-mode Sephadex LH-20 × RPLC separation method with successful applications in on-line mode, which might be beneficial for harvesting targets from complicated medicinal plants.

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.

Chinese Herbal Medicine Safflower (Flos carthami) Does Not Increase Bleeding Complications: A Population-based Cohort Study

Safflower (Flos carthami; FC) has been used widely as a food additive, as a coloring and flavoring agent. It has also been used as a Chinese herbal medicine for improving blood flow and resolving thrombosis. This study investigated whether the risk of bleeding complications is increased among FC users. We conducted a retrospective study involving a test group that used FC and a control (non-FC users) group. The participants were aged 18 years and above, and were recruited from the 2000 – 2006. The participants were from beneficiaries of LHID2000. The FC cohort included participants who had been prescribed FC accumulated for more than 30 days, whereas the non-FC cohort included people who were not using FC prescriptions. The compared cohort individuals were randomly selected at a ratio of 1:4 and frequency matched by age, gender, and index year from FC user cohort group. The primary outcome was a new diagnosis of bleeding disorders including gastrointestinal (GI) bleeding (ICD-9-CM: 578.0, 578.1, 578.9), intracranial hemorrhage (ICD-9-CM: 432.0, 432.9), and blood transfusions (ICD-9-CM op-code: 99.0). The results showed that the proportions of participants with bleeding disorders were 2.6% and 2.3% in the FC and non-FC cohorts, respectively (P = 0.6675). In univariate and multivariate Cox’s proportional hazard regression models, the adjusted hazard ratio for bleeding disorders was 0.86 for the FC cohort relative to the non-FC cohort (P = 0.6094). In conclusion, the risk of bleeding complications was not increased among Chinese herbal medicine FC users.

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.

Hydroxysafflor Yellow A Attenuates Neuron Damage by Suppressing the Lipopolysaccharide-Induced TLR4 Pathway in Activated Microglial Cells.

Microglia activation initiates a neurological deficit cascade that contributes to substantial neuronal damage and impairment following ischemia stroke. Toll-like receptor 4 (TLR4) has been demonstrated to play a critical role in this cascade. In the current study, we tested the hypothesis that hydroxysafflor yellow A (HSYA), an active ingredient extracted from Flos Carthami tinctorii, alleviated inflammatory damage, and mediated neurotrophic effects in neurons by inducing the TLR4 pathway in microglia. A non-contact Transwell co-culture system comprised microglia and neurons was treated with HSYA followed by a 1mg/mL lipopolysaccharide (LPS) stimulation. The microglia were activated prior to neuronal apoptosis, which were induced by increasing TLR4 expression in the activated microglia. However, HSYA suppressed TLR4 expression in the activated microglia, resulting in less neuronal damage at the early stage of LPS stimulation. Western blot analysis and immunofluorescence indicated that dose-dependently HSYA down-regulated TLR4-induced downstream effectors myeloid differentiation factor 88 (MyD88), nuclear factor kappa b (NF-κB), and the mitogen-activated protein kinases (MAPK)-regulated proteins c-Jun NH2-terminal protein kinase (JNK), protein kinase (ERK) 1/2 (ERK1/2), p38 MAPK (p38), as well as the LPS-induced inflammatory cytokine release. However, HSYA up-regulated brain-derived neurotrophic factor (BDNF) expression. Our data suggest that HSYA could exert neurotrophic and anti-inflammatory functions in response to LPS stimulation by inhibiting TLR4 pathway-mediated signaling.

Hydroxysafflor yellow A exerts neuroprotective effects in cerebral ischemia reperfusion-injured mice by suppressing the innate immune TLR4-inducing pathway

The innate immune response, which is tightly regulated by Toll-like receptor 4 (TLR4) pathway, has been shown to play a critical role in brain damage following cerebral ischemia-reperfusion injury. Hydroxysafflor yellow A (HSYA) is the active component extracted from the Flos Carthami and has been reported to decrease neurological deficit scores following ischemia-reperfusion injury. However, the precise mechanism by which it exerts these neuroprotective effects remains poorly understood. In this study, we demonstrated that the administration of HSYA could significantly down-regulate TLR4 expression in middle cerebral artery occlusion (MCAO) mice. Following the down-regulation of TLR4 by HSYA treatment, cerebral infarction and inflammatory neuronal damage was alleviated. The number of apoptotic neurons in the HSYA-treated group was significantly decreased along with the decrease in TLR4 expression in MCAO mice. Activation of the NF-κB and MAPK signaling pathways was observed at 1h following ischemia and at 24h post-reperfusion. HSYA could significantly inhibit NF-κB p-p65, ERE1/2, JNK and p38 phosphorylation, which coincided with the suppressed secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nitric oxide (NO). Moreover, brain-derived neurotrophic factor (BDNF) was up-regulated following 1h of ischemia and continued to increase initially during reperfusion but was down-regulated at later stages. Following treatment with HSYA, BDNF was up-regulated relative to control MCAO mice at 1h post-ischemia and at 12 and 24h post-reperfusion. Our data suggest that HSYA exerts neurotrophic and anti-inflammatory functions against ischemic stroke by inhibiting TLR4 pathway-mediated signaling responses.

Danhong injection protects cardiomyocytes against hypoxia/reoxygenation- and H2O2-induced injury by inhibiting mitochondrial permeability transition pore opening

Ethnopharmacological relevanceDanhong injection (DHI), a Chinese medical product extracted from Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese), has been widely used for the treatment of ischemic heart disease, and clinical and experimental studies have demonstrated the protective effects against myocardial ischemia/reperfusion injury. Nevertheless, the underlying cellular mechanisms responsible for this protective effect are poorly understood. Aim of the studyThe present study aimed to examine the mechanism of DHI in regulating hypoxia/reoxygenation- and H2O2-induced cardiomyocytes injury. Materials and methodsNeonatal rat cardiomyocytes were subjected to hypoxia (9h)-reoxygenation (2h) or H2O2 (100μM) in the presence or absence of DHI (2.5, 5, 10μg/mL). Intracellular reactive oxygen species (ROS), cytosolic and mitochondrial Ca2+ concentrations, mitochondrial membrane potential (ΔΨm) and mitochondrial permeability transition pore (mPTP) opening were monitored using CMH2DCFDA, Fluo-4 and rhod-2, JC-1 and calcein, respectively. Cell survival was evaluated using the 2-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and apoptosis was detected by Annexin V/propidium iodide (PI) staining. ResultsDHI improved cell survival following H/R and H2O2 injury and reduced H/R-induced cytochrome c release and apoptosis when compared with non-DHI treated cells. In addition, DHI attenuated H/R-induced ROS generation, H2O2-induced cytosolic and mitochondrial Ca2+ overload, and cellular ROS generation when compared with H/R- and H2O2-only groups. Moreover, DHI significantly inhibited both mPTP opening and ΔΨm depolarization. ConclusionThese data demonstrate that the protective mechanism of DHI against H/R- and H2O2-induced injury is mediated by the inhibition of mPTP opening via mitigating Ca2+ overload and ROS generation.

Traditional chinese medicine Xuebijing treatment is associated with decreased mortality risk of patients with moderate paraquat poisoning.

Paraquat poisoning causes multiple organ injury and high mortality due to severe toxicity and lack of effective treatment. Xuebijing (XBJ) injection, a traditional Chinese medicine preparation of five Chinese herbs (Radix Salviae Miltiorrhiae, Rhizoma Chuanxiong, Flos Carthami, Angelica Sinensis and Radix Paeoniae Rubra), has an anti-inflammatory effect and is widely used in the treatment of sepsis. This retrospective study was designed to evaluate the effects of XBJ combined with conventional therapy on mortality risk of patients with acute paraquat poisoning. Out of 68 patients, 27 were treated with conventional therapy (control group) and 41 were treated with intravenous administration of XBJ (100 ml, twice a day, up to 7 days) plus conventional therapy (XBJ group). Vital organ function, survival time within 28 days and adverse events during the treatment were reviewed. Results indicated that XBJ treatment significantly increased median survival time among patients ingesting 10-30 ml of paraquat (P=0.02) compared with the control group. After adjustment for covariates, XBJ treatment was associated significantly with a lower mortality risk (adjusted HR 0.242, 95% CI 0.113 to 0.516, P=0.001) compared with the control group. Additionally, compared with Day 1, on Day 3 the value of PaO2/FiO2 was significantly decreased, and the values of serum alanine aminotransferase, creatinine and troponin T were significantly increased in the control group (all P<0.05), but these values were significant improved in the XBJ group (all P<0.05). Only one patient had skin rash with itch within 30 minutes after injection and no severe adverse events were found in the XBJ group. In conclusion, XBJ treatment is associated with decreased mortality risk of patients with moderate paraquat poisoning, which may be attributed to improved function of vital organs with no severe adverse events.