Shuang-Huang-Lian Research Articles

Lipopolysaccharide aggravating anaphylactoid reactions caused by traditional Chinese Medicine injections via p38/ERK/NF-κB signaling pathways

Ethnopharmacological relevanceCurrently, adverse reactions limit the development of traditional Chinese medicine injections (TCMI), and severe anaphylactoid shock is one of the serious adverse reactions, which presents a significant challenge. The presence of abnormal inflammatory mediators before the administration of TCMI will most likely result in severe anaphylactoid reactions. Not only that, the lack of clinically relevant safety evaluations impedes the widespread use of TCMI, and there is an urgent need for studies to reveal the mechanisms of anaphylactoid shock caused by TCMI. Aim of the studyTo investigate the effects and underlying mechanisms of lipopolysaccharide (LPS)-induced inflammation, which aggravates anaphylactoid reactions caused by TCMI, utilizing a guinea pig model. MethodsThe dose and duration of LPS administration and different doses of compound 48/80 (C48/80) were examined by using guinea pigs as a model. Shuanghuanglian (SHLI) and Qingkailing (QKLI) injections, these two representative TCMI, were used for validation. The plasma biochemical indices, including histamine, 5-hydroxytryptamine, tumor necrosis factor-α, interleukin 6, immunoglobulin E, C5a, tryptase, and platelet activating factor, as well as the pathological characteristics of the lung, were analyzed. Futhermore, plasma metabolomics was employed to reveal changes in metabolic pathways in vivo when inflammation co-exists with TCMI. In addition, Western blot analysis was conducted to assess the expression of critical signaling pathways. ResultsStimulation with 2 mg/kg of LPS for 12 h induced inflammatory responses in the guinea pig model. C48/80 (3.0 mg/kg) in combination with LPS resulted in an increase in anaphylactoid-related indicators in the plasma. High doses of SHLI and QKLI aggravated plasma indices and lung histological injury caused by LPS-induced inflammation. A total of 36 and 63 differential metabolites were significantly altered after LPS stimulation in the SHLI-and QKLI-treated guinea pig groups, respectively. The associated metabolic pathways include central carbon metabolism in cancer, the tricarboxylic acid cycle, glyoxylate and dicarboxylate metabolism. The p38/ERK/NF-κB signal pathway may be significantly affected by TCMI in vivo after LPS stimulation. ConclusionLPS-induced inflammation aggravated anaphylactoid reactions caused by SHLI and QKLI, with a correlation to dosage. After the presence of LPS, the administration of TCMI interferes with the immune response by over-activating the p38/ERK/NF-κB signaling pathway. This activation leads to an excessive release of inflammatory factors and anaphylactoid mediators. These results present a new direction for mitigating adverse clinical reactions associated with TCMI.

Exploring the pharmacological mechanisms of Shuanghuanglian against T-cell acute lymphoblastic leukaemia through network pharmacology combined with molecular docking and experimental validation

Context Due to the poor prognosis of T-cell acute lymphoblastic leukaemia (T-ALL), there is an urgent need to identify safer and more cost-effective drugs. Objective This study evaluated the antitumour activity of Shuanghuanglian (SHL) on T-ALL cells and elucidated the mechanism. Materials and methods Jurkat and Molt4 cells were treated with SHL (0.1, 0.2 and 0.4 mg/mL) for 24 and 48 h. The controls were treated with RPMI 1640 containing 10% foetal bovine serum. Cell viability was evaluated through Cell Counting Kit-8 assay. Patterns of death and signalling pathway alterations caused by SHL were identified by network pharmacology combined with GO enrichment analysis and then were verified by Hoechst 33342 staining, Annexin V-FITC/PI staining and Western blotting. Interactions of the active ingredients with targets were analysed by molecular docking. Results The IC50 values of SHL in Jurkat and Molt4 cells were 0.30 ± 0.10 and 0.48 ± 0.07 mg/mL, respectively, at 24 h and 0.27 ± 0.05 and 0.30 ± 0.03 mg/mL at 48 h. In T-ALL, 117 target genes of SHL were mainly enriched in the apoptosis and NOTCH signalling pathways. SHL induced apoptosis was confirmed by Hoechst 33342 staining and flow cytometry. The protein levels of cleaved caspase-7 and cleaved PARP were significantly increased but those of cleaved NOTCH1 and MYC were reduced. The active ingredients of SHL can interact with γ-secretase. Discussion and conclusions: SHL induces apoptosis in T-ALL cells via the NOTCH1-MYC pathway and may be a potential drug for the treatment of T-ALL.

Study on the mechanism of anti-acute lung injury of Shuanghuanglian oral liquid based on identification of transitional components in blood and network pharmacology

Acute lung injury (ALI) is a common clinical disease that seriously affects people's health and endangers their lives. Shuanghuanglian (SHL) oral liquid is a well-known traditional Chinese medicine (TCM) preparation that is often used clinically to treat respiratory infections. SHL oral liquid has good efficacy, but its mechanism is still unclear. A strategy combining the identification of transitional components in blood and network pharmacology was proposed and applied to explore the potential anti-ALI mechanism of SHL oral liquid. A UHPLC-Q-Exactive Orbitrap-MS method was first developed to characterize the metabolic profiling of rat serum after gavage administration of SHL oral liquid. Then, based on the identified compounds, network pharmacology was used to establish a component-target-pathway network to explore the molecular mechanism of SHL oral liquid in the treatment of ALI. As a result, 92 transitional components in blood after oral administration of SHL oral liquid were identified, including 28 prototype components and 64 metabolites, and the metabolic pathways were also estimated and analyzed. Based on network pharmacology, the key anti-ALI targets of SHL oral liquid were screened as ADORA1, PTGS2, EGFR, ALOX5 and TNF, and the key pathway was PI3K-Akt signal pathway. This study provided a basis and strategy for the follow-up study of the anti-ALI molecular mechanism of SHL oral liquid and revealing the mechanism of TCM.

Inhibition of Human UDP-Glucuronosyltransferases1A1-Mediated Bilirubin Glucuronidation by the Popular Flavonoids Baicalein, Baicalin, and Hyperoside Is Responsible for Herb (Shuang-Huang-Lian)-Induced Jaundice.

Bilirubin-related adverse drug reactions (ADRs) or malady (e.g., jaundice) induced by some herbs rich in certain flavonoids have been widely reported. However, the causes and mechanisms of the ADRs are not well understood. The aim of this paper was to explore the mechanism of Shuang-huang-lian (SHL) injections and its major constituents-induced jaundice via inhibiting human UDP-glucuronosyltransferases1A1 (hUGT1A1)-mediated bilirubin glucuronidation. The inhibitory effects of SHL and its major constituents in the herbal medicine, including baicalein (BAI), baicalin (BA), and hyperoside (HYP), on bilirubin glucuroBBREVInidation were investigated. This study indicated that the average formation rates of bilirubin glucuronides [i.e., mono-glucuronide 1 (BMG1), BMG2, and bilirubin diglucuronide] displayed significant differences (P < 0.05). Specifically, the formation of BMGs was favored regardless of whether an inhibitor was absent or present. SHL, BAI, BA, and HYP dose-dependently inhibit bilirubin glucuronidation, showing the IC50 values against total bilirubin glucuronidation were in the range of (7.69 ± 0.94)-(37.09 ± 2.03) μg/ml, (4.51 ± 0.27)-(20.84 ± 1.99) μM, (22.36 ± 5.74)-(41.35 ± 2.40) μM, and (15.16 ± 1.12)-(42.80 ± 2.63) μM for SHL, BAI, BA, and HYP, respectively. Both inhibition kinetics assays and molecular docking simulations suggested that SHL, BAI, BA, and HYP significantly inhibited hUGT1A1-mediated bilirubin glucuronidation via a mixed-type inhibition. Collectively, some naturally occurring flavonoids (BAI, BA, and HYP) in SHL have been identified as the inhibitors against hUGT1A1-mediated bilirubin glucuronidation, which well explains the bilirubin-related ADRs or malady triggered by SHL in clinical settings. SIGNIFICANCE STATEMENT: Herbal products and their components (e.g., flavonoids), which been widely used across the entire world, may cause liver injury. As a commonly used herbal products rich in flavonoids, SHL injections easily lead to symptoms of liver injury (e.g., jaundice) owing to significant inhibition of hUGT1A1-mediated bilirubin glucuronidation by its flavonoid components (i.e., baicalein, baicalin, and hyperoside). Herb-induced bilirubin-related ADRs and the associated clinical significance should be seriously considered.

A Comprehensive Review of Pharmacokinetics and Pharmacodynamics in Animals: Exploration of Interaction with Antibiotics of Shuang-Huang- Lian Preparations.

As a traditional Chinese medicine (TCM), Shuang-Huang-Lian (SHL) has been widely used for treating infectious diseases of the respiratory tract such as encephalitis, pneumonia, and asthma. During the past few decades, considerable research has focused on pharmacological action, pharmacokinetic interaction with antibiotics, and clinical applications of SHL. A huge and more recent body of pharmacokinetic studies support the combination of SHL and antibiotics have different effects such as antagonism and synergism. SHL has been one of the best-selling TCM products. However, there is no systematic review of SHL preparations, ranging from protection against respiratory tract infections to interaction with antibiotics. Since their important significance in clinical therapy, the pharmacodynamics, pharmacokinetics, and interactions with antibiotics of SHL were reviewed and discussed. In addition, this review attempts to explore the possible potential mechanism of SHL preparations in the prevention and treatment of COVID-19. We are concerned about the effects of SHL against viruses and bacteria, as well as its interactions with antibiotics in an attempt to provide a new strategy for expanding the clinical research and medication of SHL preparations.

Enhancing activity of antibiotics against Staphylococcus aureus with Shuang-Huang-Lian

Staphylococcus aureus is a major pathogen in both hospitals and the community and can cause systemic infections such as pneumonia. Multi-drug resistant strains, such as Methicillin-resistant S. aureus (MRSA) are particularly worrisome. Bacterial antibiotic resistance can be attributed to various mechanisms including antibiotic inactivation. In order to reduce the development of bacterial resistance, we hypothesized that two selected traditional Chinese medicines, Shuang-Huang-Lian (SHL) and Lan-Qin, would be effective against S. aureus. In this study, we carried out the zone of inhibition determination of five antibiotics including amoxicillin, gentamicin, penicillin, clarithromycin, cefazolin and the traditional Chinese medicines SHL and Lan-Qin. The preliminary screening results showed that all the drugs except Lan-Qin exhibited antibacterial effects. Then, we conducted quantitative determination experiments to further explore the bacteriostasis of different drugs. The results of minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) determination indicated that penicillin had the best inhibitory effect on bacterial growth, and clarithromycin killed all the bacteria at the lowest concentration. Also, we tested the combined effect of SHL with different antibiotics against S. aureus and calculated the fractional inhibitory concentration (FIC) of the antibiotics. The results showed that SHL had a synergistic effect with gentamicin as well as additive effects with penicillin and cefazolin against S. aureus by decreasing MICs of antibiotics compared with using antibiotics alone. Our study provides a reference for the clinical treatment of S. aureus infection in combination with traditional Chinese medicines.

Shuanghuanglian oral preparations combined with azithromycin for treatment of Mycoplasma pneumoniae pneumonia in Asian children: A systematic review and meta-analysis of randomized controlled trials.

BackgroundMycoplasma pneumoniae is one of the main causes of community-acquired pneumonia. Due to the imperfect immune system of children, this also causes Mycoplasma pneumoniae pneumonia (MPP) to be more common in children. Globally, the incidence of MPP in children is gradually increasing. This study was the first to systematically review the clinical efficacy and safety of Shuanghuanglian (SHL) oral preparations combined with azithromycin in the treatment of MPP in children.MethodsThis study fully retrieved 3 Chinese databases and 5 English databases to search the randomized controlled trials (RCTs) of SHL oral preparations combined with azithromycin in the treatment of children with MPP. The search time is from the inception to September 2020. Data extraction and risk bias evaluation were performed independently by two researchers. We conducted a Meta-analysis of all the outcome indicators. Besides, Meta-regression, subgroup analysis, and heterogeneity analysis were used for the primary outcomes to find the possible potential confounding factors.ResultsFinally, we included 27 RCTs involving 2884 patients. SHL oral preparations combined with azithromycin were better than azithromycin alone in response rate (RR = 1.14, 95% CI[1.11, 1.18]; low certainty evidence), disappearance time of fever(MD = -1.72, 95% CI[-2.47, -0.97]; low certainty evidence), disappearance time of cough (MD = -2.95, 95% CI[-3.55, -2.34]; low certainty evidence), and disappearance time of pulmonary rales (MD = -2.13, 95% CI[-2.88, -1.38]; low certainty evidence). The Meta-regression results showed that the course of disease, age, and method of administration may be the source of heterogeneity. Subgroup analysis and sensitivity analysis have found that the results were stable. For other related clinical symptoms, T lymphocytes, and Serum inflammatory factors, SHL oral preparations combined with azithromycin was better than azithromycin alone, and the difference was statistically significant. For adverse events with low certainty evidence, safety needs further verification.ConclusionBased on the results of meta-analysis with low certainty evidence, we believed that SHL oral preparations combined with azithromycin likely be effectively improved clinical symptoms compared with azithromycin alone. Low certainty evidence showed that SHL may safety with no serious adverse events. Due to these limitations, the safety needs further verification. More high-quality, multicenter, and large-sample RCTs should be tested and verified in the future.

Systematic investigation of the pharmacological mechanism of Tanreqing injection in treating respiratory diseases by UHPLC/Q-TOF-MS/MS based on multiple in-house chemical libraries coupled with network pharmacology

Tanreqing injection (TRQI), a drug approved by the National Drug Regulatory Authority of China (China SFDA, number: Z20030045), is widely used clinically to treat respiratory diseases. However, as a complex system, the pharmacological mechanism of TRQI for the treatment of respiratory diseases is still unclear. TRQI contains three Chinese medicines that make up the classic Chinese compound formulas Shuang-Huang-Lian (SHL). Moreover, it is known that SHL components are beneficial for characterizing the chemical compounds of TRQI. Therefore, in this study, we applied UHPLC/Q-TOF-MS/MS analysis based on multiple chemical compound libraries to identify the chemical profiles of TRQI and used network pharmacology to predict the potential targets of TRQI active compounds. First, three chemical libraries related to TRQI were created, including the TRQI in-house library, SHL in-house library, and targeted Metlin library. An integrated TRQI library was established by combining three chemical libraries for the identification and characterization of the chemical profiles of TRQI. Second, the potential targets of TRQI active compounds were predicted with the Swiss Target Prediction and TCMSP databases, and targets of respiratory disease were collected from the GeneCards database. Then, the network between the active compounds and common targets was established by Cytoscape 3.7.1. The common targets were imported into the STRING database to construct protein-protein interaction (PPI) networks and select core targets of TRQI against respiratory diseases. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analyses of the core targets were performed by the Omicsbean analytic system and DAVID database, respectively. As a result, a total of 126 compounds were identified, and network pharmacological analysis showed that luteolin, wogonin, baicalein, chenodeoxycholic acid, l-serine, aspartic acid, oroxylin A, syringin, phenylalanine, and glutamic acid could be the active compounds of TRQI; GABBR1, MAPK3, GRM5, FOS, DRD2, GRM1, VEGFA, GRM3 and 92 other potential core targets for the treatment of respiratory diseases by modulating pathways in cancer, the calcium signaling pathway, cAMP signaling pathway, estrogen signaling pathway and TNF-α signaling pathway.

Effects of Shuanghuanglian oral liquids on patients with COVID-19: a randomized, open-label, parallel-controlled, multicenter clinical trial.

We conducted a randomized, open-label, parallel-controlled, multicenter trial on the use of Shuanghuanglian (SHL), a traditional Chinese patent medicine, in treating cases of COVID-19. A total of 176 patients received SHL by three doses (56 in low dose, 61 in middle dose, and 59 in high dose) in addition to standard care. The control group was composed of 59 patients who received standard therapy alone. Treatment with SHL was not associated with a difference from standard care in the time to disease recovery. Patients with 14-day SHL treatment had significantly higher rate in negative conversion of SARS-CoV-2 in nucleic acid swab tests than the patients from the control group (93.4% vs. 73.9%, P = 0.006). Analysis of chest computed tomography images showed that treatment with high-dose SHL significantly promoted absorption of inflammatory focus of pneumonia, which was evaluated by density reduction of inflammatory focus from baseline, at day 7 (mean difference (95% CI), −46.39 (−86.83 to −5.94) HU; P = 0.025) and day 14 (mean difference (95% CI), −74.21 (−133.35 to −15.08) HU; P = 0.014). No serious adverse events occurred in the SHL groups. This study illustrated that SHL in combination with standard care was safe and partially effective for the treatment of COVID-19.Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s11684-021-0853-6 and is accessible for authorized users.

Comparison of plasma and pulmonary availability of chlorogenic acid, forsythiaside A and baicalin after intratracheal and intravenous administration of Shuang-Huang-Lian injection

Ethnopharmacological relevanceThe off-label nebulization of Shuang-Huang-Lian (SHL) injection is often utilized to treat respiratory tract infections in China. However, the pulmonary biopharmaceutics of SHL was generally unknown, limiting the rational selection of therapeutic dose and dose frequency. Aim of the studyTo characterize the size distribution of nebulized aerosols and to compare the pharmacokinetics and the lung distribution of three chemical makers of SHL, chlorogenic acid (CHA), forsythiaside A (FTA) and baicalin (BC), after intratracheal and intravenous administration of SHL to rats. Materials and methodsThe droplet size distribution profiles over nebulization process were dynamically monitored using a laser diffraction method whereas the levels of CHA, FTA and BC in plasma, lung tissues and bronchoalveolar lavage fluids (BALF) were determined by a validated LC-MS/MS assay. The pulmonary anti-inflammatory efficacy was evaluated using a lipopolysaccharide (LPS) induced lung inflammation model as indicated by the level of tumor necrosis factor-α (TNF-α) in BALF. ResultsThe nebulization of SHL showed good inhalability and allowed the aerosols to reach the upper or lower respiratory tract dependent on the performance of selected nebulizers. Following intratracheal administration of SHL at different doses, CHA, FTA and BC were absorbed into the bloodstream with the mean absorption time being 67.5, 63.5 and 114 min, respectively, rendering mean absolute bioavailabilities between 42.4% and 61.4% roughly independent of delivered dose. Relative to the intravenous injection, the intrapulmonary delivery increased the lung-to-plasma concentration ratios of CHA, FTA and BC by more than 100 folds and markedly improved the lung availability by 563–676 folds, leading to enhanced and prolonged lung retention. The production of TNF-α in BALF was decreased by ~50% at an intratracheal dose of 125 μL/kg SHL to LPS-treated mice. ConclusionThe nebulization delivery of SHL is a promising alternative to the intravenous injection for the treatment of respiratory tract infections.

Combination of multi-model statistical analysis and quantitative fingerprinting in quality evaluation of Shuang-huang-lian oral liquid.

A model consisting of quantitative fingerprinting integrated with fundamental statistical analysis was established to carry out quality control analysis of Shuang-huang-lian (SHL) oral liquid. The quantitative fingerprinting approach was developed by systematic investigation of the chromatographic condition and optimization of a gradient using a complex sample analysis software system (CSASS). Five pivotal components from three traditional Chinese medicines of SHL oral liquid were determined at dual wavelengths, including phillyrin, baicalin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid. Among them, neochlorogenic acid and cryptochlorogenic acid were quantified by quantitative analysis of multi-components with a single marker (QAMS)method. Further, the developed quantitative fingerprinting approach was validated. Good linearity with correlation coefficients (R2) higher than 0.9999 were achieved for phillyrin, baicalin and chlorogenic acid. Recoveries of the three analytes were between 96% and 108%. Relative standard deviation (RSD) values were below 3% regarding the stability and intra-day and inter-day precision. Besides, the feasibility of the QAMS method was verified by an external standard method (ESM) using 18 batches of SHL oral liquid. Fifty-nine batches of SHL oral liquid from nine manufacturers were then analyzed. Effective distinction was realized based on a linear principal component analysis (linear-PCA) model by the combination of the quantitative data and chromatographic fingerprinting. The linear-PCA model based on quantitative chromatographic fingerprinting exhibited great advantage over conventional similarity analysis to distinguish between different samples. The strategy provided a particularly simple and effective approach for quality evaluation of SHL oral liquid from various manufacturers. Graphical abstract.

Mechanism of Shuang-Huang-Lian Oral Liquid for Treatment of Mycoplasmal Pneumonia in Children on Network Pharmacology.

Mycoplasmal pneumonia (MP) can lead to inflammation, multiple system immune damage, and mixed infection in children. The pathogenesis is still unclear. Shuang-Huang-Lian (SHL) oral liquid can treat acute upper respiratory tract infection, acute bronchitis and light pneumonia. However, our current understanding of the molecular mechanisms supporting its clinical application still lags behind due to the lack of researches. It is difficult to understand the overall sensitization mechanism of SHL oral liquid. The purpose is to explain the mechanism of action of drugs in this study, which is useful to ensure the safety of medication for children. The therapeutic mechanism of SHL oral liquid was investigated by a system pharmacology approach integrating drug-likeness evaluation, oral bioavailability prediction, ADMET, protein-protein interaction worknet, Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes database pathway performance, C-T-P network construction and molecular docking. A total of 18 active ingredients contained in SHL oral liquid and 53 major proteins were screened out as effective players in the treatment of M. pneumoniae disease through some related pathways and molecular docking. The majority of targets, hubs and pathways were highly related to anti-mycoplasma therapy, immunity and inflammation process. This study shows that the anti-bacterial effect of SHL oral liquid has multicomponent, multi-target and multi-pathway phenomena. The proposed approach may provide a feasible tool to clarify the mechanism of traditional Chinese medicines and further develop their therapeutic potentials.

A rational usage of combined using of herbal medicines and synthetic medicines: A time interval window research

The combined use of herbal and traditional medicine with synthetic drugs is a common clinical issue in China, as well as in other countries around the world. The improper combination of Traditional Chinese Medicine (TCM) with synthetic drugs may increase the risk of severe adverse drug reactions (ADRs). In China, the combination of Shuanghuanglian (SHL) and Ribavirin injections (RB) is one such example that has led to a higher risk of pseudo-allergic ADRs, observed during post-marketing safety monitoring. Such combination-related ADRs may be associated with concurrent drug exposure concentrations in vivo . Thus, it is important to administer both formulations at acceptable intervals to prevent ADRs. In this study, we propose an appropriate time interval of administration to reduce ADR risk in combination drug use. SHL and RB combination was then tested in rats. This combination was tested at different administration intervals. Ear weight was chosen as the quantitative index to indicate tissue edema and intensity of pseudo-allergic reactions. Compared to the control group, rats that were simultaneously administered with SHL and RB intravenously, showed different behaviors and symptoms such as ear edema and increase in weight, swelling of limbs and huddling, which were similar to those seen in pseudo-allergic reactions. By contrast, rats administered solely with either RB or SHL did not exhibit obvious pseudo-allergic reactions. With respect to the combination groups at different administration intervals, there were two different types of responses. One of them was similar to the concurrent administration of drug pairs, which occurred in groups at intervals of 0, 1.5, 2, 3.5 and 4 h. The other type was similar to the sole use of RB or SHL, which occurred in groups with intervals of 6, 8, 12 and 24 h. We also determined serum concentrations of complement C5 (C5) and thrombomodulin (THBD), proteins that mediate the complement system and coagulation cascade-depended pseudo-allergic reactions. Results showed that there was significant increase in these two indices in combination groups, with intervals below or equal to 4 h and non-significant increase in combination groups with intervals above or equal to 6 h. These results indicate that a “safe window” of administration interval exists when using the drug pair: SHL and RB, thus reducing the risk of pseudo-allergic reactions. Moreover, we tested serum metabolomic profiles of each group. The principal component analysis revealed that there was an obvious distinction between the rat groups, of both clusters: One cluster included the normal control group where either SHL or RB group was administered and the combination groups that were administered both SHL and RB at intervals of over or equal to 6 h. The other cluster involved combination groups that were administered both drugs at intervals below or equal to 4 h. Thus, the metabolomic results also elucidated a “safe window” administration interval, when using such drug pairs. We then screened out metabolites that might be used to distinguish these two clusters of rat groups. The results of ROC curves showed that compounds of m / z 154.0592, m / z 433.2357, and m / z 331.0528 could distinguish rats where SHL was used after and before 6 h. These metabolites were identified as L-2-Amino-3-(1-pyrazolyl) propanoic acid, [2-hydroxy-1-(6-hydroxy-1-oxo-1H-isochromen-3-yl) butoxy] sulfonic acid and LysoPA (0:0/18:2 (9Z, 12Z)) according to their MS/MS spectrum, respectively. This study verified the hypothesis of the “safe window” of administration interval in combination drug use, including TCM and synthetic drugs. Administered at appropriate intervals, it can reduce ADR risk and prevent safety-related issues from occurring. This study thus provides a new perspective for improving safety, when using Chinese herbal medicines in combination with synthetic medicine.

Safety Concerns of Traditional Chinese Medicine Injections Used in Chinese Children.

Objective To explore risks underlying traditional Chinese medicine (TCM) injection-related adverse drug reactions (ADRs) in Chinese children, and to discuss the implications of postmarketing reevaluation studies. Methods We identified potential cases of exposure to TCM injections for children (<18 years of age) and adults (18 years and upwards) from database of ADRs. First, the associations between TCM injection-related ADRs and three administration routes (i.e., intravenous or intramuscular administration, oral administration, and external use) and the imbalance of TCM injection-related ADRs between the paediatric and adult populations were tested using the Chi-square (χ2) test. Second, the proportional reporting ratio (PPR) was applied to identify statistically significant associations between drugs and anaphylactic shock in the paediatric population. Results The χ2 test revealed that the highest frequency of paediatric ADRs was due to 5 types of herbal injections (i.e., Shuanghuanglian (SHL), Yuxingcao (YXC), Qingkailing (QKL), Xiyanping (XYP), and Reduning (RDN) herbal injections) (P<0.000), and the reports of ADRs attributed to the XYP and RDN herbal injections in children accounted for a greater proportion than the reports for adults (P<0.000). The PPR identified 5 types of herbal injections-anaphylactic shock pairs (i.e., the SHL, XYP, QKL, YXC, and Fufang Danshen herbal injections) that met the minimum criteria (i.e., a PPR of at least 2 and χ2 of at least 4 and three or more cases), which suggested that TCM injections were significantly associated with anaphylactic shock. Conclusions TCM injections pose graver risks to the paediatric population than the adult population. To achieve optimal benefits and minimal risk to children treated with TCM injections, we suggest reevaluating the effectiveness and safety, monitoring the risks, and promoting rational use of TCM injections in Chinese children.

Pharmacokinetic interaction between shuanghuanglian and azithromycin injection: a nonlinear mixed-effects model analysis in rats

1. This study aimed to evaluate the pharmacokinetic interaction of shuanghuanglian (SHL) and azithromycin in rats, and to provide experimental support for rational drug use in clinics.2. High-performance liquid chromatography with ultraviolet detection (HPLC-UV) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) approaches were respectively developed to detect the forsythiaside (active component of SHL) and azithromycin concentrations. Both non-compartmental and compartmental analyzes were employed to calculate pharmacokinetic parameters. A nonlinear mixed-effects modeling method was applied to fit the drug concentration-time data. The influence of drug coadministration on pharmacokinetic parameters was tested using forward inclusion and backward elimination procedures.3. After drug co-administration, areas under the drug concentration–time curve (AUC) and half-lives (T1/2) of both azithromycin and forsythiaside increased significantly, meanwhile, the drug clearance (CL) decreased compared to single drug administration. Both forsythiaside and azithromycin exposures increased after coadministration. Two-compartment models were suitable to describe the in vivo behavior of both azithromycin and forsythiaside. The coadministration of SHL could significantly decrease the central volume of azithromycin (VCA) and forsythiaside clearance (CLF) decreased after co-intravenous administration of azithromycin.4. Co-intravenous administration of forsythiaside and azithromycin could significantly increase drug exposures for both drugs. Lower dose can provide sufficient drug exposure to obtain antibacterial activity. The coadministration may be a potential method to increase therapy efficiency while decrease adverse drug reactions.

Shuang-Huang-Lian Attenuates Airway Hyperresponsiveness and Inflammation in a Shrimp Protein-Induced Murine Asthma Model.

Shuang-Huang-Lian (SHL), an herbal formula of traditional Chinese medicine, is clinically used for bronchial asthma treatment. Our previous study found that SHL prevented basophil activation to suppress Th2 immunity and stabilized mast cells through activating its mitochondrial calcium uniporter. Sporadic clinical reports that SHL was used for the treatment of bronchial asthma can be found. Thus, in this study, we systematically investigated the effects of SHL on asthmatic responses using a shrimp protein (SP)- induced mouse model. SHL significantly inhibited airway inspiratory and expiratory resistance, and histological studies suggested it reduced thickness of airway smooth muscle and infiltration of inflammation cells. It also could alleviate eosinophilic airway inflammation (EAI), including reducing the number of eosinophils and decreasing eotaxin and eosinophil peroxidase levels in the bronchoalveolar lavage fluid (BALF). Further studies indicated that SHL suppressed SP-elevated mouse mast cell protease-1 and IgE levels, prevented Th2 differentiation in mediastinal lymph nodes, and lowered Th2 cytokine (e.g., IL-4, IL-5, and IL-13) production in BALF. In conclusion, SHL attenuates airway hyperresponsiveness and EAI mainly via the inhibition of mast cell activation and Th2 immunity, which may help to elucidate the underlying mechanism of SHL on asthma treatment and support its clinical use.

Chlorogenic acid in the oxidative stress injury triggered by Shuang-Huang-Lian injection.

Injections of Chinese herbs are a novel approach to prepare traditional Chinese medicines. However, as injections of Chinese herbs have been extensively used, adverse drug reactions (ADRs) have been on the increase. Additionally, the mechanism for injections of Chinese herbs remains unclear. This study explored the potential role played by chlorogenic acid (CGA) in initiating oxidative stress injury triggered by the utilization of injections of Chinese herbs and the underlying mechanism. A total of 90 male Wistar rats were raised for varying periods by using Shuang-Huang-Lian (SHL) injection or CGA in diverse dosages. Western blot analysis examined the expression of nicotinamide adenine dinucleotide phosphate oxidase subunits, spectrophotometry was used to examine the activity taken by catalase, ELISA was used to examine the concentrations of inflammatory factors in serum, and intravital microscopy was employed to examine the microcirculation. The results showed that the excessive peroxide production induced by CGA in high-dose or SHL in the venule walls may well be through nicotinamide adenine dinucleotide phosphate oxidase along with a decline in the activity of catalase, and led to imbalance of basal levels of pro-(TNF-α) and anti-(IL-10) inflammatory cytokines. On the basis of the aforementioned results, the mechanism hidden behind the adverse effects of CGA induced by irrational use of Chinese herbal injection can be identified from a deeper perspective.

Antiviral effects of Shuanghuanglian injection powder against influenza A virus H5N1 in vitro and in vivo

The current study was to identify a protective role of Shuanghuanglian (SHL) injection powder in vitro and in vivo after H5N1 viral infection. Immunofluorescent staining was used to determine the susceptibility of rat intestinal mucosa microvascular endothelial cells (RIM-MVECs) to the H5N1 virus. Viral replication of RIM-MVECs was measured by transmission electron microscopy (TEM) a hemagglutination assay and real-time quantitative PCR. H5N1 virally infected RIM-MVECs, and BALB/c mice were treated with SHL to investigate its therapeutic effect. Animal survival and the weight of H5N1 virally infected BALB/c mice after SHL treatment was noted, and histology and real-time PCR applied to mouse lungs were used to confirm the anti-H5N1 viral effects of SHL. RIM-MVECs supported replication of the H5N1 virus in vitro. SHL treatment reduced viral titers in H5N1 virally infected RIM-MVECs and mouse lungs. SHL -treated mice survived compared to controls. Mild pathological changes, reduced inflammatory cell infiltration and fewer viral antigens were observed in the lungs of SHL-treated mice at days 3 and 6 post-infection. In conclusion, SHL may have the antiviral activity against the H5N1 virus infection by inhibiting viral replication and alleviating lung injury.

Shuang-Huang-Lian prevents basophilic granulocyte activation to suppress Th2 immunity

BackgroundBasophilic granulocytes (BGs) not only initiate the induction of Th2 cell differentiation, but also amplify the ongoing Th2 response. Shuang-Huang-Lian (SHL) is clinically used for relieving type I hypersensitivity by continuous treatment for several weeks.MethodsELISA, flow cytometry, magnetic activated cell sorting, isoelectric precipitation, hybridoma technique, transfection and luciferase reporter assay were used in this study. The statistical analysis was performed using a one-way ANOVA.ResultsOur recently published study demonstrated that SHL exerted a remarkable effect on mast cell stabilization. Herein, we sought to elucidate the effect of SHL on shrimp tropomyosin (ST)-induced Th2 immunity and its underlying mechanisms. The obtained data showed that continuous treatment with SHL significantly suppressed ST-stimulated Th2-cytokines release and IgE synthesis. A mechanistic study indicated that SHL not only reduced BG early IL-4 release before ST-specific IgE (sIgE) production, but also inhibited BG activation in the presence of sIgE, including suppressing CD200R surface expression and decreasing IL-4 production. Moreover, SHL markedly decreased the cytosolic Ca2+ (Ca2+[c]) level and inhibited the nuclear factor of activated T cells (NFAT) activation in RBL-2H3 cells.ConclusionsCollectively, SHL potently reduces ST-induced Th2 immunity by inhibiting the BG Ca2+-NFAT pathway and, thus, suppressing the early IL-4 release before sIgE synthesis and inhibiting BG activation in the presence of sIgE. This study provides the pharmacological basis for the clinical use of SHL to relieve type I hypersensitivity by a successive dose regimen.

Simultaneous Determination of Multiple Classes of Hydrophilic and Lipophilic Components in Shuang-Huang-Lian Oral Liquid Formulations by UPLC-Triple Quadrupole Linear Ion Trap Mass Spectrometry.

The Shuang-Huang-Lian (SHL) oral liquid is a combined herbal prescription used in the treatment of acute upper respiratory tract infection, acute bronchitis and pneumonia. Multiple constituents are considered to be responsible for the therapeutic effects of SHL. However, the quantitation of the multi-components from multiple classes is still unsatisfactory because of the high complexity of constituents in SHL. In this study, an accurate, rapid, and specific UPLC-MS/MS method was established for simultaneous quantification of 18 compounds from multiple classes in SHL oral liquid formulations. Chromatographic separation was performed on a HSS T3 (1.8 μm, 2.1 mm × 100 mm) column, using a gradient mobile phase system of 0.1% formic acid in acetonitrile and 0.1% formic acid in water at a flow rate of 0.2 mL·min−1; the run time was 23 min. The MS was operated in negative electrospray ionization (ESI−) for analysis of 18 compounds using multiple reaction monitoring (MRM) mode. UPLC-ESI−-MRM-MS/MS method showed good linear relationships (R2 > 0.999), repeatability (RSD < 3%), precisions (RSD < 3%) and recovery (84.03–101.62%). The validated method was successfully used to determine multiple classes of hydrophilic and lipophilic components in the SHL oral liquids. Finally, principal component analysis (PCA) was used to classify and differentiate SHL oral liquid samples attributed to different manufacturers of China. The proposed UPLC-ESI−-MRM-MS/MS coupled with PCA has been elucidated to be a simple and reliable method for quality evaluation of SHL oral liquids.