A comprehensive quality evaluation strategy for ReDuNing injection by integrating UPLC-Orbitrap MS/MS profile and biological potency combined with multivariate statistical analysis.

Reduning injection (RDN) is a traditional chinese medicine injection widely used in clinical practice. In this study, qualitative and quantitative analysis of RDN were conducted by UPLC-Orbitrap MS/MS simultaneously. Totally 118 compounds were identified and 34 compounds were quantified in RDN. The method with completed method validation proved the high sensitivity and efficiency of the method and it was applied to quantify compounds in RDN. Multivariate statistical analysis method selected 11 key variables that affect the content consistency of RDN. 20 batches with high biological potency were screened by cox-2 enzyme activity assay. Spectrum-effect relationship analysis and multivariate statistical analysis showed that 7 batches were high-quality selected after comprehensive quality evaluation and 9 compounds were key indicators for screening it. This strategy including fingerprint, qualitative analysis and multiple-component quantification could be well applied to modern quality evaluation of RDN, which could be valuable for the further quality control of more other traditional Chinese medicines.

The pulmonary biopharmaceutics and anti-inflammatory effects after intratracheal and intravenous administration of Re-Du-Ning injection.

BACKGROUND: Re-Du-Ning injection (RDN) is a renowned heat-clearing traditional Chinese medicine for the treatment of respiratory diseases owing to its anti-inflammatory effects. However, very little is known about the pulmonary distribution and lung exposure-efficacy relationships. This study aimed to investigate the pulmonary distribution and biopharmaceutics concerning lung penetrability and affinity and the local anti-inflammatory effects after intravenous and pulmonary administration of RDN. METHODS: Two iridoids and seven phenolic acid components were selected as the chemical markers in RDN. The in vitro pulmonary distribution and biopharmaceutics were conducted by evaluating the binding and disassociation kinetics of chemical markers in lung tissue explants whereas the in vivo evaluation was performed by determining the time-dependent concentrations of chemical markers in plasma, lung epithelial lining fluid (ELF), lung tissues and immune cells in the ELF after intratracheal and intravenous administrations of RDN. The inhibitory effects on tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production were used to evaluate the anti-inflammatory effect of RDN on lung tissues in vitro and on mice with LPS-induced lung inflammation. RESULTS: The chemical markers of RDN exhibited excellent lung penetrability but poor lung affinity in vitro and in vivo. After intravenous administration, the chemical markers appeared to rapidly penetrate through the lung tissue to reach the ELF, leading to markedly higher drug exposure to ELF and immune cells in the ELF than to lung tissues. Compared to intravenous injection, the intratracheal instillation of RDN increased drug exposure to lung tissue and immune cells in the ELF by up to > 80-fold, leading to improved anti-inflammatory potency and prolonged duration of action. CONCLUSION: The drug exposure to immune cells in the ELF was correlated with the lung-targeted anti-inflammatory effects of RDN and pulmonary delivery has the potential to replace intravenous injection of RDN for the treatment of respiratory diseases.

Re-Du-Ning injection ameliorates LPS-induced lung injury through inhibiting neutrophil extracellular traps formation.

BACKGROUND: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening diseases and could occur in severe COVID-19 patients. Re-Du-Ning injection (RDN) is a tradition Chinese medicine preparation which has been clinically used for treatment of respiratory diseases including COVID-19. PURPOSE: To elucidate the potential mechanisms of RDN for the treatment of ALI. METHODS: Female C57BL/6J mice were used to establish ALI model by intraperitoneal injection 10 mg/kg LPS, and RDN injection was intraperitoneally administered with the dose of 5 and 10 ml/kg. The cytokines were measured by ELISA and qPCR. The data related to NETs were analyzed by ELISA, immunofluorescence, Western blotting and network pharmacological approach. RESULTS: RDN robustly alleviated LPS-induced ALI. Meanwhile, RDN downregulated the expression of pro-inflammatory cytokines, such as IL-1ß, IL-6 and TNF-α. Specifically, RDN treatment inhibited the formation of neutrophil extracellular traps (NETs) and remarkably suppressed the protein of PAD4. The active compound from RDN decreased the phosphorylation of ERK1/2. CONCLUSION: These findings demonstrate that RDN ameliorates LPS-induced ALI through suppressing MAPK pathway to inhibit the formation of NETs.

Efficacy and safety of Re-Du-Ning injection in the treatment of seasonal influenza: results from a randomized, double-blinded, multicenter, oseltamivir-controlled trial.

OBJECTIVE: To assess the efficacy and safety of RDNI in the treatment of seasonal influenza. RESULTS: 1575 participants were screened and 229 completed the study and had a RT-PCR laboratory confirmation of influenza virus infection. Fever alleviation time was 2 and 6 hours, and fever clearance time was 27 and 47 in RDNI and oseltamivir, with significant difference between two groups. Total scores of influenza symptoms descended more in RDNI than oseltamivir on day 2 and day 3. Single symptom such as fever, aversion to cold, sore throat and nasal obstruction score descended more in RDNI than oseltamivir on different days. 20 subjects used aspirin during the trial, and there was no significant difference between two groups. MATERIALS AND METHODS: We conducted a randomized, double-blind, double-dummy, oseltamivir controlled clinical trial. Patients with a positive influenza rapid test diagnosis were enrolled and randomized to receive RDNI or oseltamivir. Primary outcome was the median fever alleviation and clearance time. Secondary outcomes were total 8 influenza symptom scores, the single influenza symptom score, and the frequency of aspirin usage. CONCLUSIONS: The effect of RDNI was not worse than oseltamivir on the alleviation of influenza symptoms. RDNI was well tolerated, with no serious adverse events noted during the study period.

Direct inhibition of Re Du Ning Injection and its active compounds on human liver cytochrome P450 enzymes by a cocktail method.

The aim of this study was to investigate the direct inhibitory effects of Re Du Ning Injection (RDN) and its active compounds on the major cytochrome P450 enzyme (CYP) isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomes by 'a cocktail method'. The activity of each CYP isform was represented as the formation rate of the specific metabolite from relevant substrate. Then a sensitive and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously analyze the seven metabolites. RDN (0.035-2.26 mg/mL) showed a strong inhibitiory effect on CYP2C8, followed by CYP2C9, CYP2B6, CYP2C19, CYP1A2 and CYP3A4. The IC50 value for each enzyme was 0.19, 0.66, 0.72, 1.27, 1.66 and 2.13 mg/mL, respectively. RDN competitively inhibited the activities of CYP1A2 (Ki = 1.22 mg/mL), CYP2B6 (Ki = 0.65 mg/mL) and CYP3A4 (Ki = 0.88 mg/mL); it also exhibited mixed inhibition of CYP2C8, CYP2C9 and CYP2C19 with a Ki value of 0.26, 0.64 and 0.82 mg/mL, respectively. However, the activity of CYP2D6 was not significantly inhibited even by 2.26 mg/mL RDN. Moreover, the data of nine active compounds on the CYPs showed that cryptochlorogenin acid, sochlorogenic acid B and sochlorogenic acid C were the major contributors to the inhibitory effect of RDN on CYP2C8, while the inhibitory effect of RDN on CYP2C9 might be caused by sochlorogenic acid A and sochlorogenic acid C. Moreover, neochlorogenic acid might be the major contributor to the inhibitory effect on CYP2B6. All of the findings suggested that drug-drug interactions may occur and great caution should be taken when RDN is combined with drugs metabolized by these CYPs.

Study on Extraction Process Trajectory for In-line Monitoring of Extraction Process of Lonicera Japonica of Re-Du-Ning Injection / 世界科学技术-中医药现代化

This study was aimed to develop in-line monitoring of extraction process of Lonicerajaponica in the extraction process trajectory of Re-Du-Ning (RDN) injection.Several batches of near-infrared (NIR) spectrum dates were collected and multivariate statistical process control (MSPC) method was in conjunction with.The results showed that using score,Hotelling T2 and DModX control chart,various normal and abnormal behaviors of the test batches were detected in time by comparison with the extraction process trajectory.It was concluded that the process trajectory for in-line quality control based on NIR spectrum dates and MSPC could indicate the changes of process.It was a feasible technology tool of the total process quality control during traditional Chinese medicine (TCM) manufacturing process.

Research on the change of chemical composition in productive process of Re Du Ning injection by HPLC/Q-TOF MS.

A high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) was developed for the analysis of chemical composition change in the production process of Re Du Ning injection, a Chinese medicine preparation with a combination of Lonicera japonica Thunb., Gardenia jasminoides Ellis and Artemisia annua L. A total of 90 compounds from raw materials-intermediates-Re Du Ning injection were detected; among them, 55 compounds were identified or tentatively characterized, and the characteristic ions of different types of compounds were described. Based on these studies, the different types of compounds in the various process routes were analyzed. A total of 28 compounds, including seven iridoid glycosides and six monoterpenes from G. jasminoides Ellis, five iridoid glycosides, nine phenolic acids and one unknown compound from L. japonica Thunb., were transferred to Re Du Ning injection, and two unknown compounds were generated in the production process of Re Du Ning injection. The results indicated that the Chinese Medicine Pharmaceutical process control is very important. This method could provide some reference for other Chinese medicine preparations.

Research on chemical constituents from Re-Du-Ning Injection (III) / 中草药

Objective: To study the antiviral constituents from the active fraction of Re-Du-Ning (RDN) Injection. Methods: In this study, the active fraction of RDN Injection was screened by the mice model loaded with restraint stress infected with influenza virus. The chemical constituents were isolated by chromatography on silica gel, ODS, Sephadex LH-20 & Toyopearl HW-40 columns, and reverse phase MPLC & HPLC repeatedly. Their structures were identified by spectral data and physicochemical property. Results: The 95% ethanol eluate of RDN Injection on the macroporous adsorption resin column was proved to be the antivirus active fraction of RDN Injection. Fourteen compounds were isolated and identified as (2E,6S)-8-[α-L-arabinopyranosyl-(1″-6')-β-D-glucopyranosyloxy]- 2,6-dimethylct-2-eno-1,2″-lactone (1), lyoniresinol (2), 5'-methoxyisolariciresinol (3), ent-isolariciresinol (4), (7R,8R)-4,7,9,9'- tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan (5), (7S,8R)-4,7,9,9'-tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan (6), ceplignan (7), 5'-methoxyceplignan (8), (-)-dihydrodehydrodiconiferyl alcohol (9), (7S,8R)-3,3',5-trimethoxy-4',7-epoxy-8,5'-neolignan-4,9,9'-triol (10), (2-cis, 4-trans)-abscisic acid (11), (2-trans, 4-trans)-abscisic acid (12), (1S,3R,4R,5S,7R,9R)-decane-6-carboxylic acid (13), and (1S,3R,4S,5S,7R,9R)-decane-6-carboxylic acid (14); Among them, compound 1 exhibited the antivirus activity against Dengue virus. Conclusion: Compound 8 is a new compound and the other isolated compounds are reported from RDN Injection for the first time, and compound 1 shows the anti-virus activity against Dengue virus.

Influences of Re Du Ning Injection, a traditional Chinese medicine injection, on the CYP450 activities in rats using a cocktail method.

ETHNOPHARMACOLOGICAL RELEVANCE: Re Du Ning Injection (RDN), a traditional Chinese medicine injection, is made from the extracts of Lonicerae japonicae flos (LJF), Artemisiae annuae herba (AAH) and Gardeniae fructus (GF). Since last decade, RDN has been widely used in China for the treatment of fever, inflammation, allergy and viral infection. AIM OF THE STUDY: To elucidate the potential influences of RDN on the activities of four cytochrome P450 (CYP) isozymes in rats (CYP1A2, CYP2C11, CYP2D1 and CYP3A1/2) by "cocktail method". MATERIALS AND METHODS: A sensitive and specific LC-MS method capable of simultaneous quantification of four substrates and their metabolites in rat plasma was developed and validated. Relative activity estimation of four isozymes was carried out by comparing plasma pharmacokinetics of substrates and their metabolites (phenacetin/ paracetamol for CYP1A2, tolbutamide/4-hydroxytolbutamide for CYP2C11, dextromethorphan/ dextrorphan for CYP2D1 and dapsone/N-acetyl dapsone for CYP3A1/2) between control and RDN treatment groups. RESULTS: Compared with control group, RDN at different levels could increase the total amount of tolbutamide, dextromethorphan and dapsone absorbed into blood, while decrease the total amount of phenacetin absorbed into blood at low and medium dosage and increase it at high dosage. CONCLUSIONS: RDN could inhibit the activities of CYP2C11, CYP2D1 and CYP3A1/2, induce the activity of CYP1A2 at low and medium dosage but inhibit it at high dosage. The results indicated that drug co-administrated with RDN may need dose adjustment.

Study on Antibacterial Activity and Immunomodulation Effect ofRe-Du-Ning Injection / 世界科学技术-中医药现代化

This study was aimed to discuss the antibacterial and immunomodulation effect ofRe-Du-Ning (RDN) injection. Mice were randomly divided into the normal group, model group,Shuang-Huang-Lian (SHL) group (7.8 mL·kg-1), RDN high dose group (20.30 g·kg-1), middle dose group (10.15 g·kg-1), and low dose group (5.08 g·kg-1), in order to observe the death protective effect of mice with bacterial infection on antibacterial experimentin vivo. Mice were randomly divided into the normal group, model group,Xiang-Gu Duo-Tang (XGDT) group (0.19 mg·kg-1), RDN high dose group (20.30 g·kg-1), middle dose group (10.15 g·kg-1), and low dose group (5.08 g·kg-1). The 2, 4-dinitrochlorobenzene was used to induce delayed hypersensitivity. Immunomodulation was observed by the content of serum hemolysin and the carbon particle clearance index. The results showed that the RDN high dose group and middle dose group had antibacterial effect, which reduced the mortality of mice. The RDN high dose, middle dose and low dose group can enhance the phagocytosis of macrophage in immunosuppressive mice, increase the formation of hemolysin, and strengthen delayed hypersensitivity reaction among immunocompromised mice. It was concluded that RDN injection had antibacterial effect. Its immunomodulation effect was through the enhancing of non-specific immunity, humoral immunity and cellular immunity of mice.

Anti-inflammatory and Immunization Properties ofRe-Du-Ning Injection in Treatment of AECOPD with Phlegm-heat Stagnated in the Lung Syndrome / 世界科学技术-中医药现代化

This study was aimed to discuss the anti-inflammatory and immunization properties ofRe-Du-Ning (RDN) injection in the treatment of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) with phlegm-heat stagnated in the lung syndrome. A total of 110 in-patients were collected from January 2012 to December 2013. Cases were randomly divided into the treatment group (basic treatment plan + 20 ml RDN injection + 250 mL of 5% GS injection, once a day, intravenous injection) and the control group (basic treatment plan + 20 ml of 0.9% NS injection + 250 mL of 5% GS injection, once a day, intravenous injection), with 55 cases in each group. The treatment course was 14 days. Observations were made on traditional Chinese medicine (TCM) symptom score, clinical effect, blood routine examination, blood gas analysis and T lymphocyte subgroups before and after the treatment in both groups. The results showed that in the aspects of TCM symptom score and clinical effect, the posttreatment TCM symptom score and total integration in the treatment group were obviously improved compared to the control group except for shortness of breath, dry mouth and thirsty, flushing (P < 0.05). The total effective rate was obviously better than that of the control group. In the aspect of anti-inflammation, the total white blood cell (WBC) count and the normal neutrophil percentage of both pretreatment and posttreatment in the treatment group were obviously decreased compared to pretreatment (P < 0.05). However, there were no statistical differences on WBC count and normal neutrophil percentage compared to posttreatment in the control group. In the aspect of blood gas analysis, the posttreatment PaCO2 and PaO2 of the treatment group were obviously better than the control group (P < 0.05). In the aspect of immune regulation, the posttreatment T lymphocyte subgroups CD3+ and CD4+ of the treatment group were higher than the control group. The expression inhibition / cytotoxic lymphocyte (CD8+) was obviously lower than the control group (P < 0.05). The CD4+ / CD8+ was back to the normal reference level. It was concluded that in the treatment of AECOPD with phlegm-heat stagnated in the lung syndrome, on the basis of western medicine symptomatic treatment plan, RDN injection was assisted to clear heat, relieve toxin and remove phlegm. It can obviously improve patient’s clinical symptoms and increase the clinical therapeutic effects. The treatment was especially targeted to infection-induced respiratory failure patients combined low immunity with possible identified therapeutic effects.

Influence on Pulmonary Function and Immune Function in Children with Capillary Bronchitis byRe-Du-NingInjection Combined with Acupoint Sticking / 世界科学技术-中医药现代化

This study was aimed to observe the influence on immune function in children with capillary bronchitis byRe-Du-Ning(RDN) injection combined with acupoint sticking, in order to explore the clinical application value of RDN injection combined with acupoint sticking. A total of 64 children with capillary bronchitis treated in the Henan Province Hospital of Traditional Chinese Medicine from October 2012 to August 2014 were randomly divided into two groups. In the control group, conventional treatment, including sputum excretion, oxygen therapy, cough-relieving and phlegm-dissolving, anti-inflammation, antifebrile, panting-relieving and atomization inhalation, was used. In the observation group, RDN injection combined with acupoint sticking of Ru-Yi Jin-Huang (RYJH) powder was used on the basis of treatment of the control group. Observations were made on the clinical therapeutic effects as well as the recovery condition of pulmonary function and immune function in both groups. The results showed that the asthmatic suffocating relieving time, asthmatic suffocating, cough, antifebrile, dry rale and wet rale disappearing time, hospitalization days of the observation group were less than those of the control group with significant inter-group difference (t = 4.346, 5.301, 4.445, 2.238, 4.116, 3.733, 3.681,P 0.05). There were no statistical differences on the pretreatment RR, VT, FRC, VPEF/VE, tPTEF/tE, Reff in both groups (P > 0.05);the posttreatment RR, VT, VPEF/VE, tPTEF/tE were obviously higher than those of the pretreatment; FRC and Reff were obviously decreased compared with those of the pretreatment (P 0.05). There were no significant differences on the pretreatment T lymphocyte subsets, which included CD3+, CD4+ and CD8+ T lymphocyte (P > 0.05). The posttreatment CD3+ and CD4+ were higher than those of pretreatment; CD8+ was lower than that of pretreatment in both groups with significant difference (P 0.05). The posttreatment IgG, IgA, IgM and IGF-1 were higher than those of the pretreatment; IgE was lower than that of the pretreatment with significant difference (P < 0.05). The posttreatment IgG, IgA, IgM and IGF-1 of the observation group were higher than those of the control group;IgE was lower than that of the control group with significant inter-group difference (P < 0.05). It was concluded that on the basis of conventional modern medicine treatment, the RDN injection combined with acupoint sticking can improve the clinical symptoms and pulmonary function, boost immune function in children with capillary bronchitis.

Study on chemical constituents from Re-Du-Ning Injection (II) / 中草药

Objective: To investigate the chemical constituents from Re-Du-Ning Injection (RDN). Methods: The chemical constituents were isolated by chromatography on silica gel, ODS, Sephadex LH-20, and Toyopearl HW-40 columns and reverse phase MPLC and HPLC repeatedly. Their structures were identified by spectral data and physicochemical property. Results: Sixteen compounds were isolated and identified as 5-O-caffeoylquinic acid (1), 5-O-caffeoylquinic acid methyl ester (2), 4-O-caffeoylquinic acid (3), 5-O-caffeoylquinic acid methyl ester (4), 4,5-di-O-caffeoylquinic acid (5), 4,5-di-O-caffeoylquinic acid methyl ester (6), 3,5- di-O-caffeoylquinic acid (7), 3,5-di-O-caffeoylquinic acid methyl ester (8), 3,4-di-O-caffeoylquinic acid (9), 3,4-di-O-caffeoylquinic acid methyl ester (10), secologanic acid (11), vogeloside (12), 7-epi-vogeloside (13), E-aldosecologanin (14), Z-aldosecologanin (15), and 5H,8H-pyrano[4,3-d] thiazolo[3,2-a] pyridine-3-carboxylic acid (16). Compounds 1-10 showed high efficiency and low toxicity with antivirus activity against RSV. Conclusion: All the isolated compounds are reported from RDN Injection for the first time, and caffeoylquinic acids may be one of antivirus pharmacodynamic material bases of RDN.

Study on Analgesic and Antipyretics Effect of Re-Du-Ning Injection / 世界科学技术-中医药现代化

This article was aimed to study the analgesic and antipyretic effect of Re-Du-Ning (RDN) Injection. Heating model was building on SD rats after subcutaneous injection of yeast. And then, RDN Injection was intravenous injected at the dose of 10.16 g·kg-1, 5.08 g·kg-1, and 2.54 g·kg-1, respectively. Observation was made on changes of rats' temperature. RDN Injection was intravenous injected into ICR mice at the dose of 20.30 g·kg-1, 10.15 g·kg-1, 5.08 g·kg-1 for 7 consecutive days. The methods of mice twist, hot plate and jaw pain were used in the testing of analgesic action of RDN Injection. The results showed that RDN Injection at high and middle dose can significantly reduce the temperature of heating rats models (P < 0.05 or P < 0.01). It can also significantly reduce the twisting times of mice by acetic acid (P< 0.05 or P< 0.01). It can also significantly increase the pain threshold of mice by hot plate and jaw pain (P< 0.05 or P< 0.01). It was concluded that RDN Injection had a good analgesic and antipyretic effect in mice.

Research on antiviral constituents in Re-Du-Ning Injection (I) / 中草药

Objective: To study the antiviral constituents from the active fraction of Re-Du-Ning (RDN) Injection. Methods: In this study, the active fraction of RDN Injection was screened by the mice model loaded with restraint stress infected with influenza virus. The investigation on this fraction led to the isolation and identification of compounds through various chromatographic techniques and spectroscopic methods. In addition, the in vitro activity on influenza virus A (A/PR/8/34 H1N1) of the flavonoids was evaluated in vitro by the method for the detection of anti-influenza virus neuraminidase activity. Results: Through the macroporous adsorption resin, 95% ethanol eluate of RDN Injection was proved to be the antivirus active fraction of RDN Injection. Twenty compounds were obtained and characterized as syringic acid (1), ferulic acid (2), benzoic acid (3), caffeic acid (4), p-hydroxy benzaldehyde (5), vanillin (6), 4-hydroxy-3-methoxy styrene acrylic acid (7), trans-p-hydroxy cinnamic acid (8), trans-o-hydroxy cinnamic acid (9), trans-cinnamic acid (10), 7-hydroxy-6-methoxy coumarin (11), 7-hydroxy-6, 8-dimethoxy coumarin (12), coumarin (13), 3-hydroxy-1, 2-bis (4-hydroxy-3-methoxyphenyl)-1-propanone (14), isorhamnetin (15), quercetin (16), luteolin (17), rutin (18), hyperoside (19), and luteolin-7-O-β-D-glucoside (20). Among them, luteolin exhibited the antivirus activity against Flu A virus. Conclusion: All the isolated compounds are reported from RDN Injection for the first time, and luteolin exhibits the most potential activity against H1N1.