Qing-Kai-Ling oral liquid alleviated pneumonia via regulation of intestinal flora and metabolites in rats.

Background: Qing-Kai-Ling (QKL) oral liquid, evolving from a classical Chinese formula known as An-Gong-Niu-Huang pills, is a well-established treatment for pneumonia with its mechanism remaining muddled. Studies have shown that the regulation of both intestinal flora and host-microbiota co-metabolism may contribute to preventing and treating pneumonia. The study aimed to investigate the potential mechanism by which QKL alleviates pneumonia from the perspective of 'microbiota-metabolites-host' interaction. Methods: We evaluated the therapeutic effects of QKL on lipopolysaccharide (LPS)-induced pneumonia rats. To explore the protective mechanism of QKL treatment, a multi-omics analysis that included 16S rDNA sequencing for disclosing the key intestinal flora, the fecal metabolome to discover the differential metabolites, and whole transcriptome sequencing of lung tissue to obtain the differentially expressed genes was carried out. Then, a Spearman correlation was employed to investigate the association between the intestinal flora, the fecal metabolome and inflammation-related indices. Results: The study demonstrated that pneumonia symptoms were significantly attenuated in QKL-treated rats, including decreased TNF-α, NO levels and increased SOD level. Furthermore, QKL was effective in alleviating pneumonia and provided protection equivalent to that of the positive drug dexamethasone. Compared with the Model group, QKL treatment significantly increased the richness and αlpha diversity of intestinal flora, and restored multiple intestinal genera (e.g., Bifidobacterium, Ruminococcus_torques_group, Dorea, Mucispirillum, and Staphylococcus) that were correlated with inflammation-related indices. Interestingly, the intestinal flora demonstrated a strong correlation with several metabolites impacted by QKL. Furthermore, metabolome and transcriptome analyses showed that enrichment of several host-microbiota co-metabolites [arachidonic acid, 8,11,14-eicosatrienoic acid, LysoPC (20:0/0:0), LysoPA (18:0e/0:0), cholic acid, 7-ketodeoxycholic acid and 12-ketodeoxycholic acid] levels and varying lung gene (Pla2g2a, Pla2g5, Alox12e, Cyp4a8, Ccl19, and Ccl21) expression were observed in the QKL group. Moreover, these metabolites and genes were involved in arachidonic acid metabolism and inflammation-related pathways. Conclusion: Our findings indicated that QKL could potentially modulate intestinal flora dysbiosis, improve host-microbiota co-metabolism dysregulation and regulate gene expression in the lungs, thereby mitigating LPS-induced pneumonia in rats. The study may provide new ideas for the clinical application and further development of QKL.

["Component-target-efficacy" network analysis and experimental verification of Qingkailing Oral Preparation].

This study aims to explore the mechanism of Qingkailing(QKL) Oral Preparation's heat-clearing, detoxifying, mind-tranquilizing effects based on "component-target-efficacy" network. To be specific, the potential targets of the 23 major components in QKL Oral Preparation were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. The target genes were obtained based on UniProt. OmicsBean and STRING 10 were used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. Cytoscape 3.8.2 was employed for visualization and construction of "component-target-pathway-pharmacological effect-efficacy" network, followed by molecular docking between the 23 main active components and 15 key targets. Finally, the lipopolysaccharide(LPS)-induced RAW264.7 cells were adopted to verify the anti-inflammatory effect of six monomer components in QKL Oral Preparation. It was found that the 23 compounds affected 33 key signaling pathways through 236 related targets, such as arachidonic acid metabolism, tumor necrosis factor α(TNF-α) signaling pathway, inflammatory mediator regulation of TRP channels, cAMP signaling pathway, cGMP-PKG signaling pathway, Th17 cell differentiation, interleukin-17(IL-17) signaling pathway, neuroactive ligand-receptor intera-ction, calcium signaling pathway, and GABAergic synapse. They were involved in the anti-inflammation, immune regulation, antipyretic effect, and anti-convulsion of the prescription. The "component-target-pathway-pharmacological effect-efficacy" network of QKL Oral Preparation was constructed. Molecular docking showed that the main active components had high binding affinity to the key targets. In vitro cell experiment indicated that the six components in the prescription(hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide) can reduce the expression of nitric oxide(NO), TNF-α, and interleukin-6(IL-6) in cell supernatant(P<0.05). Thus, the above six components may be the key pharmacodynamic substances of QKL Oral Preparation. The major components in QKL Oral Prescription, including hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide, cholic acid, isochlorogenic acid A, and γ-aminobutyric acid, may interfere with multiple biological processes related to inflammation, immune regulation, fever, and convulsion by acting on the key protein targets such as IL-6, TNF, prostaglandin-endoperoxide synthase 2(PTGS2), arachidonate 5-lipoxygenase(ALOX5), vascular cell adhesion molecule 1(VCAM1), nitric oxide synthase 2(NOS2), prostaglandin E2 receptor EP2 subtype(PTGER2), gamma-aminobutyric acid receptor subunit alpha(GABRA), gamma-aminobutyric acid type B receptor subunit 1(GABBR1), and 4-aminobutyrate aminotransferase(ABAT). This study reveals the effective components and mechanism of QKL Oral Prescription.

Simultaneous Quantification of Limonin, Two Indolequinazoline Alkaloids, and Four Quinolone Alkaloids in Evodia rutaecarpa (Juss.) Benth by HPLC-DAD Method.

A simple and efficient HPLC-DAD (225 nm) method was developed and validated for the simultaneous determination of limonin and six key alkaloids (evodiamine, rutaecarpine, 1-methyl-2-undecyl-4(1H)-quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine) in Evodia rutaecarpa (Juss.) Benth, which has been widely used as one of the Traditional Chinese Medicines. The chromatographic separation was carried out on a Hypersil BDS C18 column, and gradient elution was employed with a mobile phase containing acetonitrile and water. Contents of the analytes in 18 batches of samples were analyzed by ultrasonic extraction with ethanol and water mixture (80 : 20, v/v) followed by HPLC analysis. Separation of the seven analytes was achieved within 60 min with good linearity (r > 0.999). The RSD of both the intraday and interday precision was below 1.85%. The accuracy at different concentrations was within the range of 97.91 to 100.49%. Hierarchical clustering analysis was performed to differentiate and classify the samples based on the contents of the seven constituents. This study indicated that the quality control of E. rutaecarpa could be simplified to the measurement of four constituents, and that limonin, 1-methyl-2-undecyl-4(1H)-quinolone, and dihydroevocarpine should also be served as the chemical markers together with evodiamine for the quality control of Evodia rutaecarpa (Juss.) Benth.

Evaluation of the levofloxacin release characters from a rabbit foldable capsular vitreous body.

The authors have manufactured a novel rabbit foldable capsular vitreous body (FCVB). The aim of this study was to determine whether this rabbit FCVB can release levofloxacin in vitro and in vivo, and to evaluate the release characteristics. In vitro, the rabbit FCVB with levofloxacin 500 µg/mL was immersed in cups of modified Franz diffusion cells. Following this, 200 µL of liquid was aspirated at intervals from 10 minutes to 24 hours. In vivo, the FCVB with levofloxacin was implanted into the right eyes of five rabbits. After implantation, the aqueous humor was aspirated on days 1, 7, 14, 28, and 56. The levofloxacin concentrations in the cups and aqueous humor samples were detected by high-performance liquid chromatography-tandem mass spectrometry. The FCVB was observed under a scanning electron microscope. The results showed that the released levofloxacin was stabilized at 20 ng/mL at time points from 10 minutes to 24 hours in vitro. In vivo, levofloxacin concentrations in the aqueous humor were 132, 50, 39, 11, and 15 ng/mL on days 1, 7, 14, 28, and 56, respectively. In the FCVB capsules, 300 nm apertures were observed. These results suggest the rabbit FCVB released levofloxacin stably in vitro and sustainably in vivo. This study provides a novel combined approach, with the FCVB as a vitreous substitute and drug delivery system for the treatment of bacterial endophthalmitis.

Evaluation of levofloxacin release characteristics from a human foldable capsular vitreous body in vitro.

PURPOSE: We have manufactured a novel human foldable capsular vitreous body (FCVB). In this article, we determine whether the human FCVB releases levofloxacin in vitro and evaluate the release characteristics. METHODS: Levofloxacin at dosages of 100, 250, and 500 µg/mL in 4.0 mL H2O was injected into the novel human FCVB; then, the capsules were immersed in cups of modified Franz diffusion cells. Two hundred microliters of liquid was aspirated at intervals of 10, 20, 40, 60, 120, 180, 240, 300, and 360 min. The levofloxacin concentrations in the cups were detected using the liquid chromatographic-tandem mass spectrometry (HPLC-MS/MS) method. Five hundred µg/mL levofloxacin liquor at volumes of 4.0, 5.0, and 6.0 mL was injected into the novel human FCVB, and the release characteristics were detected for each. The capsules of the FCVB were observed under scanning electron microscopy before and after the release study. RESULTS: The novel human FCVB released levofloxacin in a time-dependent manner under 100, 250, and 500 µg/mL dosages from 10 to 360 min. Particularly in the 250 µg/mL levofloxacin group, the concentration (Q) had a good linear relationship with time (t(1/2)), Q=1.3381t(1/2)+10.2818 (r=0.9954, Higuchi equation). The human FCVB released levofloxacin in a dose-dependent manner under 100, 250, and 500 µg/mL dosages from 10 to 360 min in the total tendency, but the 500 µg/mL group released less levofloxacin than the 250 group at 300 and 360 min. The human FCVB did not release levofloxacin in a volume-dependent manner with 4.0, 5.0, and 6.0 mL volume. The 4.0-mL volume group released more levofloxacin than the other 2 volume groups. Numerous 300-nm-mili apertures were observed in the capsule of the FCVB at the beginning and end of this observation. CONCLUSIONS: A human FCVB can sustainably, in vitro, mechanically release levofloxacin in both a time-dependent and dose-dependent manner, but not in a volume-dependent manner. This study sets forth a novel combined research and therapeutic strategy as a vitreous substitute and intravitreal drug delivery system for the treatment of bacterial endophthalmitis.

Rapid simultaneous determination of codeine and morphine in plasma using LC-ESI-MS/MS: application to a clinical pharmacokinetic study.

A rapid and sensitive high-performance LC-MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid-liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C(18 )column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H](+ )ions, mass-to-charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2-100/0.5-250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC-MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.

Simultaneous determination of berberine, palmatine and jatrorrhizine by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of coptis-evodia herb couple.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine berberine, palmatine and jatrorrhizine in rat plasma. After mixing with the internal standard (IS) tetrahydropalmatine, plasma samples were pretreated by protein precipitation with acetonitrile-methanol (1:2, v/v). Chromatographic separation was carried out on a C18 column using a mixture of water (containing 0.1% formic acid) and acetonitrile (30:70, v/v) as mobile phase. The detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source operating in the positive ionization mode. The method was linear over the concentration range of 1.0-250.0 ng/mL for all components. The intra- and inter-day precision values were less than 14.6% and the deviations were within +/-4.0%. The fully validated LC-MS/MS method has been successfully applied to the pharmacokinetic study of berberine, palmatine and jatrorrhizine in rat plasma after oral administration of coptis-evodia herb couple. Three peaks were observed in both individual and mean plasma-concentration curves of berberine, palmatine and jatrorrhizine, which may be attributed to distribution re-absorption and enterohepatic circulation.