A bacteriophage against Citrobacter braakii and its synergistic effect with antibiotics.

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2 nm long and 72.9 ± 4.2 nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80 min, optimum living temperature of 30-40 °C, and living pH of 5-10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests.

Elucidation of the mechanism of Yiqi Tongluo Granule against cerebral ischemia/reperfusion injury based on a combined strategy of network pharmacology, multi-omics and molecular biology.

BACKGROUND: Ischemic stroke is caused by local lesions of the central nervous system and is a severe cerebrovascular disease. A traditional Chinese medicine, Yiqi Tongluo Granule (YQTL), shows valuable therapeutic effects. However, the substances and mechanisms remain unclear. PURPOSE: We combined network pharmacology, multi-omics, and molecular biology to elucidate the mechanisms by which YQTL protects against CIRI. STUDY DESIGN: We innovatively created a combined strategy of network pharmacology, transcriptomics, proteomics and molecular biology to study the active ingredients and mechanisms of YQTL. We performed a network pharmacology study of active ingredients absorbed by the brain to explore the targets, biological processes and pathways of YQTL against CIRI. We also conducted further mechanistic analyses at the gene and protein levels using transcriptomics, proteomics, and molecular biology techniques. RESULTS: YQTL significantly decreased the infarction volume percentage and improved the neurological function of mice with CIRI, inhibited hippocampal neuronal death, and suppressed apoptosis. Fifteen active ingredients of YQTL were detected in the brains of rats. Network pharmacology combined with multi-omics revealed that the 15 ingredients regulated 19 pathways via 82 targets. Further analysis suggested that YQTL protected against CIRI via the PI3K-Akt signaling pathway, MAPK signaling pathway, and cAMP signaling pathway. CONCLUSION: We confirmed that YQTL protected against CIRI by inhibiting nerve cell apoptosis enhanced by the PI3K-Akt signaling pathway.

Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway.

BACKGROUND: A proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized. OBJECTIVE: To explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels. METHODS: Chemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro. RESULTS: Seven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo. CONCLUSIONS: DDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.

[Identification of chemical components and network pharmacology of Huanglian Decoction based on UPLC-Q-TOF-MS/MS technology].

The chemical components of Huanglian Decoction were identified by ultra-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS) technology. The gradient elution was conducted in Agilent ZORBAX Extend-C_(18) column(2.1 mm×100 mm, 1.8 µm) with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B) at a flow rate of 0.3 mL·min~(-1) and the column temperature of 35 ℃. The MS adopted the positive and negative ion mode of electrospray ionization(ESI), and the MS data were collected under the scanning range of m/z 100-1 500. Through high-resolution MS data analysis, combined with literature comparison and confirmation of reference substances, this paper identified 134 chemical components in Huanglian Decoction, including 12 alkaloids, 23 flavonoids, 22 terpenes and saponins, 12 phenols, 7 coumarins, 12 amino acids, 23 organic acids, and 23 other compounds, and the medicinal sources of the compounds were ascribed. Based on the previous studies, 7 components were selected as the index components. Combined with the network pharmacology research and analysis me-thods, the protein and protein interaction(PPI) network information of the intersection targets was obtained through the STRING 11.0 database, and 20 core targets of efficacy were screened out. In this study, UPLC-Q-TOF-MS/MS technology was successfully used to comprehensively analyze and identify the chemical components of Huanglian Decoction, and the core targets of its efficacy were discussed in combination with network pharmacology, which laid the foundation for clarifying the material basis and quality control of Huanglian Decoction.

Cerebral iron deficiency may induce depression through downregulation of the hippocampal glucocorticoid-glucocorticoid receptor signaling pathway.

BACKGROUND: Iron is a trace essential element to sustain the normal neurological function of human. Many researches had reported the involvement of iron deficiency (ID) in neural development and cognitive functions. However, the role of ID in pathogenesis of depression and its underlying mechanism are still unclear. METHODS: In this study, we first used chronic unpredicted mild stress (CUMS) and iron deprivation mouse models to clarify the pathogenesis role of cerebral ID in depression. Then the role of hippocampal glucocorticoid (GC)-glucocorticoid receptor (GR) pathway in cerebral ID induced depression were elucidated in iron deprivation mice and iron deficiency anemia patients. RESULTS: Our results revealed that both CUMS and iron deprivation could induce cerebral ID in mice, and combination of iron deprivation and CUMS could accelerate the onset and aggravate the symptoms of depression in mice. In hippocampus, ID led to neuronal injury and neurogenesis decrease, which might be related to downregulation of GC-GR signaling pathway caused GR dysfunction, thereby inhibiting the negative feedback regulation function of hippocampus on hypothalamic-pituitary-adrenal (HPA) axis. Moreover, the overactivity of HPA axis in iron deprivation mice and iron deficiency anemia patients also confirmed GR dysfunction. LIMITATIONS: Iron deprivation led to food and water intake decrease of mice, which may affect the behavioral test. In addition, we mainly evaluated the role of hippocampal ID in depression, and the number of iron deficiency anemia patients was limited. CONCLUSIONS: Our results identified that cerebral iron homeostasis was a key factor for maintaining mental stability.

Interaction between Changan Granule and its main components in the plasma and CYP450 enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: Changan Granule (CAG) is a Chinese patent drug developed based on an empirical prescription in accordance with the formulation theory of Traditional Chinese Medicine. The prescription is composed of eight herbal drugs which have been traditionally used by Chinese people for a long history. It has effects of invigorating spleen and supplementing qi, as well as regulating liver and ceasing diarrhea, and is indicated for the treatment of irritable bowel syndrome (IBS). AIM OF THE STUDY: This study was aimed to investigate the interaction between CAG and its main components and cytochrome P450 (CYP450) enzymes so as to characterize the major metabolites and metabolic enzymes and evaluate the safety concerns to its clinical use. MATERIALS AND METHODS: Both in vivo and in vitro experiments using such as diarrhea-predominant IBS (IBS-D) rat model, HepG2 cells, and human liver microsomes (HLM) were carried out to investigate the interaction between CAG and its main components and CYP450 enzymes. Real-time quantitative PCR (qPCR), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and cocktail probes were employed to qualitatively or quantitatively measure the metabolites and metabolic enzymes. RESULTS: CAG inhibited the enzyme activities of CYP1A2, CYP2E1, CYP2D6, CYP2C9, and CYP3A4 and the mRNA expressions of CYP2E1, CYP2C9, CYP3A4, and CYP2D6 in vitro. CAG down-regulated the increased expression of CYP1A2 and up-regulated the decreased expression of CYP3A1 in vivo. Twenty-two metabolites were characterized from the main components of CAG after incubation with HLM in vitro. CYP2D6, CYP2E1, CYP3A4 and CYP2C9 were identified as the characteristic metabolic enzymes. CONCLUSIONS: This study provides a reference for clinical application of CAG in safety. CAG and CYP450 enzymes are interacted. CAG is mainly metabolized by CYP2E1 and CYP2D6. The expression of CYP2E1 and CYP2D6 are more susceptible to be influenced by CAG in comparison with that of CYP3A4, CYP2C9 and CYP1A2. It implies the potential risk of interaction when CAG is taken together with the drugs metabolized by CYP2E1 and CYP2D6.

[Analysis on quality value transmitting of Qinggusan reference sample of classical prescription based on quantitative fingerprint technology].

Qinggusan is the 69th prescription in the first batch of "Catalogue of Ancient Chinese Classic Formulas". In modern clinical practice， Qinggusan is mainly used to treat noninfectious fever. However， because few studies on Qinggusan reference samples and their quality value transfer are available， the development and promotion of its compound preparations are restricted. Therefore， establishing an accurate and comprehensive quality control method to clarify the critical quality attributes of Qinggusan reference samples is of great importance. In this study， 15 batches of Qinggusan reference samples were processed to determine the range of their dry extract ratios. Quantitative high-performance liquid chromatography （HPLC） fingerprint analysis was performed using a Waters Symmetry Shield RP18 column （250 mm×4.6 mm， 5 µm） with acetonitrile-0.1% （v/v） formic acid aqueous solution as the mobile phase in gradient elution mode. The flow rate was 1.0 mL/min， the column temperature was 30 ℃， and the detection wavelength was 254 nm. The HPLC fingerprints of the Qinggusan reference samples were established under these conditions to evaluate their similarity. The established method was systematically validated and found to demonstrate good precision， repeatability， and sample stability. Subsequently， characteristic peaks were identified and attributed by HPLC-quadrupole-time-of-flight-mass spectrometry （HPLC-Q-TOF-MS） analysis. MS was performed in electrospray ionization mode， the data were collected in both positive- and negative-ion modes， and the detection range was m/z 50-2000. The contents and transfer rate ranges of the index components， namely， gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid， were determined to analyze the quality value transfer of the samples. The results demonstrated that the dry extract rate of the 15 batches of Qinggusan reference samples ranged from 24.10% to 26.88% and that their fingerprint similarities were generally greater than 0.95. Twelve common peaks were identified by reference identification， literature comparison， and high-resolution MS analysis. Twelve compounds， including six iridoid glycosides， two flavonoids， one alkaloid， one triterpenoid saponin， and two others. Among them， L-picein， androsin， picroside Ⅳ， picroside Ⅱ and picroside Ⅰ were from Picrorhizae Rhizoma， loganin acid， swertiamarin and gentiopicrin were from Gentianae Macrophyllae Radix， neomangiferin and mangiferin were from Anemarrhenae Rhizoma， dichotomine B was from Stellariae Radix， and glycyrrhizic acid was from Glycyrrhizae Radix et Rhizoma. The five key components presented good linear relationships in their respective linear ranges， and all correlation coefficients were higher than 0.999. The relative standard deviations （RSDs） of precision， stability， and repeatability were less than 1.3%. The average recoveries varied between 95.92% and 102.5%， with RSDs less than 3.9%； these values meet the requirements defined in the 2020 edition of the Chinese Pharmacopoeia. The contents of gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid in the 15 batches of reference samples were in the range of 17.92-27.55， 1.83-4.42， 23.08-36.44， 8.43-15.04， and 0.94-2.39 mg/g， respectively， and their transfer rates from the decoction pieces to the reference samples were 47.91%-63.95%， 22.96%-59.39%， 60.82%-77.82%， 64.25%-99.53%， and 15.30%-39.30%， respectively. In this study， the chemical components of Qinggusan reference samples were comprehensively identified and their quality value transfer was studied through the combination of HPLC fingerprinting and MS. Clarification of the critical quality attributes of Qinggusan reference samples could provide a basis for the quality control of Qinggusan compound preparations.

Overcoming Suppressive Tumor Microenvironment by Vaccines in Solid Tumor.

Conventional vaccines are widely used to boost human natural ability to defend against foreign invaders, such as bacteria and viruses. Recently, therapeutic cancer vaccines attracted the most attention for anti-cancer therapy. According to the main components, it can be divided into five types: cell, DNA, RNA, peptide, and virus-based vaccines. They mainly perform through two rationales: (1) it trains the host immune system to protect itself and effectively eradicate cancer cells; (2) these vaccines expose the immune system to molecules associated with cancer that enable the immune system to recognize and destroy cancer cells. In this review, we thoroughly summarized the potential strategies and technologies for developing cancer vaccines, which may provide critical achievements for overcoming the suppressive tumor microenvironment through vaccines in solid tumors.

[Identification of chemical components based on UPLC-Q-TOF-MS/MS and network pharmacology of Zhuru Decoction].

This study analyzed the main chemical components of Zhuru Decoction via ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS), and then predicted the mechanism of Zhuru Decoction in clearing heat, resolving phlegm, detoxifying, and treating vomiting and alcohol-related vomiting caused by heat in stomach based on network pharmacology. The gradient elution was conducted in Agilent ZORBAX extend-C_(18) column(2.1 mm×100 mm, 1.8 µm) with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B) at a flow rate of 0.3 mL·min~(-1) and the column temperature of 35 ℃. The MS adopted the positive and negative ion mode of electrospray ionization(ESI), and the data were collected in the scanning range of m/z 100-1 500. A total of 98 compounds in Zhuru Decoction were identified via BATMAN, SYMMAP, TCMSP, and relevant literature, including 36 flavonoids, 7 triterpenoids, 8 gingerols, 20 organic acids, 5 amino acids, and 22 other compounds. On the basis of the available studies, 9 components were selected as index components, and the protein-protein interaction(PPI) network of the common targets was established with STRING 11.0. Finally, 10 core targets associated with the pharmacodynamic effect were screened out. This study established the UPLC-Q-TOF-MS/MS method for identifying the chemical components in the classic prescription Zhuru Decoction, and employed network pharmacology to explore the core targets of its efficacy, which provided a refe-rence for the quality control and the research of the pharmacodynamic substances of Zhuru Decoction.

PA-MSHA induces inflamed tumor microenvironment and sensitizes tumor to anti-PD-1 therapy.

A low response rate to immune checkpoint inhibitor (ICI) therapy has impeded its clinical use. As reported previously, an inflamed tumor microenvironment (TME) was directly correlated with patients' response to immune checkpoint blockade (ICB). Thus, restoring the cytotoxic effect of immune cells in the TME is a promising way to improve the efficacy of ICB and overcome primary resistance to immunotherapy. The effect of Pseudomonas aeruginosa mannose-sensitive-hemagglutinin (PA-MSHA) in facilitating T cell activation was determined in vitro and in vivo. Subsets of immune cells were analyzed by flow cytometry. Proteomics was carried out to comprehensively analyze the discriminated cellular kinases and transcription factors. The combinational efficacy of PA-MSHA and αPD-1 therapy was studied in vivo. In this study we demonstrated that PA-MSHA, which is a clinically used immune adjuvant, effectively induced the anti-tumor immune response and suppressed the growth of non-small cell lung cancer (NSCLC) cells. PA-MSHA showed great potential to sensitize refractory "cold" tumors to immunotherapy. It effectively enhanced macrophage M1 polarization and induced T cell activation. In vivo, in combination with αPD-1, PA-MSHA suppressed tumor growth and prolonged the survival time of allograft model mice. These results indicate that PA-MSHA is a potent agent to stimulate immune cells infiltration into the TME and consequently induces inflammation in tumors. The combination of PA-MSHA with αPD-1 is a potential strategy to enhance the clinical response rate to ICI therapy.

[Study on characteristic chromatogram and content determination of Wuzhuyu Decoction reference sample].

In this study, the critical quality attributes of Wuzhuyu Decoction reference sample were explored by using characteristic chromatogram, index component content and dry extract rate as indexes.The dissemination relationship of quantity value between medicinal materials-decoction pieces-reference sample was investigated to preliminarily formulate the quality standard of the reference sample.The characteristic chromatogram of 15 batches of Wuzhuyu Decoction was established by high performance liquid chromatography(HPLC) and the similarity analysis was conducted.Common peaks were demarcated and assigned to medicinal materials.Moreover, quantitative determination of limonin, evodiamine, rutaecarpine and ginsenoside Rb_1 of Wuzhuyu Decoction were performed.The dissemination of quantity value was explored combined with dry extract rate, similarity of characteristic chromatogram and transfer rate of index component content.A total of 18 common peaks were identified in the corresponding materials of Wuzhuyu Decoction reference sample, with the similarity of characteristic chromatogram greater than 0.9, and Fructus Evodiae, Radix Ginseng, Rhizoma Zingiberis Recens and Fructus Jujubae contributed 9, 5, 8 and 2 chromatographic peaks, respectively.The index component content of corresponding materials and the transfer rates of medicinal materials-decoction pieces and decoction pieces-reference sample of different batches of Wuzhuyu Decoction reference sample were as follows: the content of limonin was 0.16%-0.51%, and the transfer rates were 83.66%-115.60% and 38.54%-54.58%, respectively; the content of evodiamine was 0.01%-0.11%, the transfer rated were 80.80%-116.15% and 3.23%-12.93%, respectively; the content of rutaecarpine was 0.01%-0.05%, the transfer rates were 84.33%-134.53% and 5.72%-21.24%, respectively; the content of ginsenoside Rb_1 was 0.06%-0.11%, and the transfer rates were 90.00%-96.92% and 32.45%-67.24%, respectively.The dry extract rate of the whole prescription was 22.58%-29.89%.In this experiment, the dissemination of quantity value of Wuzhuyu Decoction reference sample was analyzed by the combination of characteristic chromatogram, index component content and dry extract rate.A scientific and stable quality evaluation method of the reference sample was preliminarily established, which provided basis for the subsequent development of Wuzhuyu Decoction and the quality control of related preparations.

Bacteria-based nanodrug for anticancer therapy.

Bacteria-based immunotherapy has become a promising strategy to induce innate and adaptive responses for fighting cancer. The advantages of bacteriolytic tumor therapy mainly lie in stimulation of innate immunity and colonization of some bacteria targeting the tumor microenvironment (TME). These bacteria have cytotoxic proteins and immune modulating factors that can effectively restrain tumor growth. However, cancer is a multifactorial disease and single therapy is typically unable to eradicate tumors. Rapid progress has been made in combining bacteria with nanotechnology. Using the nanomolecular properties of bacterial products for tumor treatment preserves many features from the original bacteria while providing some unique advantages. Nano-bacterial therapy can enhance permeability and retention of drugs, increase the tolerability of the targeted drugs, promote the release of immune cell mediators, and induce immunogenic cell death pathways. In addition, combining nano-bacterial mediated antitumor therapeutic systems with modern therapy is an effective strategy for overcoming existing barriers in antitumor treatment and can achieve satisfactory therapeutic efficacy. Overall, exploring the immune antitumor characteristics of adjuvant clinical treatment with bacteria can provide potential efficacious treatment strategies for combatting cancer.

Simulated digestion and in vitro fermentation of a polysaccharide from lotus (Nelumbo nucifera Gaertn.) root residue by the human gut microbiota.

Lotus root polysaccharide (LRP) is an active water-soluble polysaccharide with average molecular weight of 1.24 × 104. It was composed of (1 â†’ 4)-α-D-glucan backbone with α-D-glycopyranosyl moieties connected to C-6 positions of the glucose residues as side chains approximately every six residues. However, little information is available for its digestion and fermentation characteristics in vitro. The results showed that the levels of reducing sugars were increased slightly, and the molecular weight was also reduced slightly, in simulated gastric and small intestinal juices. During in vitro fermentation, the total sugar, reducing sugar and glucose contents decreased gradually with increasing fermentation time. The molecular of LRP was degraded and to metabolize into a variety the short-chain fatty acids (SCFAs) such as acetic, propionic, and butyric acids. Furthermore, LRP fermentation decreased the pH of the fermentation broth and increased its absorbance. Meanwhile, LRP modulated the gut microbiota by altering the Firmicutes/Bacteroidetes ratio and increasing the relative abundance of Bifidobacterium. The findings from this study showed that LRP could be developed as potential prebiotic to regulate the composition of gut microbiota, thereby promote the production of SCFAs.

[Quality value transmitting of substance benchmarks of Zhuru Decoction].

A total of 18 batches of Zhuru Decoction samples were prepared. Chromatographic fingerprints were established for Zhuru Decoction and single decoction pieces, the content of which was then determined. The extraction rate ranges, content, and transfer rate ranges of puerarin, liquiritin, and glycyrrhizic acid, together with the common peaks and the similarity range of the fingerprints, were determined to clarify key quality attributes of Zhuru Decoction. The 18 batches of Zhuru Decoction samples had 25 common peaks and the fingerprint similarity higher than 0.95. Puerariae Lobatae Radix, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens had 21, 3, and 1 characteristic peaks, respectively. The 18 batches of samples showed the extraction rates within the range of 18.45%-25.29%. Puerarin had the content of 2.20%-3.07% and the transfer rate of 38.5%-45.9%; liquiritin had the content of 0.24%-0.85% and the transfer rate of 15.9%-37.5%; glycyrrhizic acid had the content of 0.39%-1.87% and the transfer rate of 16.2%-32.8%. In this paper, the quality value transmitting of substance benchmarks of Zhuru Decoction was analyzed based on chromatographic fingerprints, extraction rate, and the content of index components. A scientific and stable method was preliminarily established, which provided a scientific basis for the quality control and formulation development of Zhuru Decoction.

[Quality value transfer of substance benchmarks in Huanglian Decoction].

Following the preparation of substance benchmarks in Huanglian Decoction from 18 batches, the method for detecting their characteristic spectra was established to identify the similarity range and peak attribution. The content and transfer rate ranges of the index components coptisine, palmatine, berberine, liquiritin, glycyrrhizic acid, 6-gingerol, and cinnamaldehyde and the extraction amount were combined for analyzing the quality value transfer from the Chinese medicinal pieces to substance benchmarks and clarifying the key quality attributes of substance benchmarks in Huanglian Decoction. The results showed that the substance benchmarks in Huang-lian Decoction of 18 batches exhibited good similarity in characteristic spectra(all greater than 0.98). There were 17 characteristic peaks identified in the substance benchmarks of Huanglian Decoction, including 10 from Coptidis Rhizoma, 3 from Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle(processed with water), 1 from Zingiberis Rhizoma, and 3 from Cinnamomi Ramulus. The contents and average transfer rates of the index components were listed as follows: coptisine 2.20-6.46 mg·g~(-1) and 18.50%±2.93%; palmatine 3.03-8.13 mg·g~(-1) and 26.56%±4.69%; berberine 7.71-22.29 mg·g~(-1) and 17.34%±3.00%; liquiritin 0.88-2.18 mg·g~(-1) and 9.88%±4.88%; glycyrrhizic acid 1.83-4.44 mg·g~(-1) and 8.50%±3.72%; 6-gingerol 0.56-1.43 mg·g~(-1) and 11.36%±2.37%; cinnamaldehyde 1.55-3.48 mg·g~(-1) and 19.02%±4.36%. The extraction amount of the substance benchmarks from the 18 batches was controlled at 10.65%-13.88%. In this paper, the quality value transfer of substance benchmarks in Huanglian Decoction was analyzed based on the characteristic spectra, the index component contents and the extraction amount, which has provided a basis for the subsequent development of Huanglian Decoction and the quality control of its related preparations.

Chemical composition and protective effect of cerebrospinal fluid of Dan-Deng-Tong-Nao capsules on brain microvascular endothelial cells injured by OGD/R.

ETHNOPHARMACOLOGICAL RELEVANCE: Dan-Deng-Tong-Nao Capsules (DDTNC) is a Chinese patent medicine and has been used in treating cerebral ischemic stroke (IS) for a long time in China, protection of brain microvascular endothelial cells (BMECs) is the main treatment strategy. But the holistic chemical information and potential bioactive components of DDTNC on protecting BMECs and its underlying mechanism is still unclear. AIM OF THE STUDY: To identify the active ingredients of DDTNC and to explore the protective effects of DDTNC on BMECs associated with Wnt/ß-catenin pathway. MATERIALS AND METHODS: The components of DDTNC and cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF) were detected by High performance liquid chromatography combined with Diode array detector (HPLC-DAD) and Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), respectively. The experiment rat model was established with middle cerebral artery occlusion (MCAO), the therapeutic effects of DDTNC were assessed by Longa assay and TTC staining. The cerebral micro vessel density was determined by immunofluorescence staining. The injured BMECs caused by oxygen-glucose deprivation and reperfusion (OGD/R) was used to evaluate the protective effect of cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF). The cell survival rate was detected by the method of CCK-8, the intracellular Ca2+ and reactive oxygen species (ROS) was estimated by Fluo-3. Moreover, the proteins of Bax, Bcl-2, Wnt, ß-catenin, GSK-3ß was determined by Western blotting. RESULTS: The RSD values of all methodological studies were less than 3.0%. A total of 20 compounds were detected under the optimized HPLC-DAD chromatographic condition. In the UPLC-Q-TOF-MS negative mode, peak 1 and peak 2 were detecteted in DDTNC-CSF and was identified as Danshensu and Puerarin, respectively. In the UPLC-Q-TOF-MS positive mode, peak 1 and peak 3 were detecteted in DDTNC-CSF and was identified as Danshensu and Scutellarin, respectively. DDTNC significantly decreased the Longa values and infarct volume and significantly increased the cerebral microvessel density of the MCAO rats. The accumulation of intracellular Ca2+ and ROS in BMECs injured by OGD/R decreased significantly in DDTNC-CSF group. The expression of Bcl-2, ß-catenin, wnt-1 was upregulated by DDTNC-CSF and the level of Bax and GSK3ß could be downregulated by DDTNC-CSF. CONCLUSION: The present study provided a scientific basis for revealing the mechanism of DDTNC in the treatment of IS and DDTNC is expected to be an effective drug for the treatment of IS.

Senkyunolide H protects PC12 cells from OGD/R-induced injury via cAMP-PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Senkyunolide H (SNH) is a bioactive phthalide isolated from Ligusticum chuanxiong Hort rhizome and was reported to have multiple pharmacological effects. AIM OF THE STUDY: The study was performed to verify the potency of SNH protecting PC12 cells from oxygen glucose deprivation/reperfusion (OGD/R)-induced injury and to elucidate the underlying mechanisms. MATERIALS AND METHODS: OGD/R model was established in PC12 cells and the cell viability was measured by MTT assay. The cell morphology was observed using scanning electron microscope (SEM). The potential targets of SNH and related targets of OGD/R were screened, and a merged protein-protein interaction (PPI) network of SNH and OGD/R was constructed based on the network pharmacology analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway analysis. Intracellular cAMP level and the protein expression levels were measured to elucidate the underlying mechanisms. RESULTS: SNH pretreatment protected PC12 cells against OGD/R-induced cell death. SNH also significantly protected the cell protrusion. A merged PPI network was constructed and the shared candidate targets significantly enriched in cAMP signaling pathway. The level of intracellular cAMP and the protein level of p-CREB, p-AKT, p-PDK1 and PKA protein were up-regulated after the treatment of SNH compared with OGD/R modeling. CONCLUSIONS: The present study indicated that SNH protected PC12 cells from OGD/R-induced injury via cAMP-PI3K/AKT signaling pathway.

Antidiarrheal Effect of Sechang-Zhixie-San on Acute Diarrhea Mice and Network Pharmacology Deciphering Its Characteristics and Potential Mechanisms.

Sechang-Zhixie-San (SCZX) is an ancient prescription used for pediatric diarrhea by the Yi people in China, which consists of Rodgersia sambucifolia Hemsley (known as Yantuo and abbreviated as YT) and Bentonite (BN). Now, it is also a Chinese patent medicine used in the clinic to treat infantile diarrhea. Besides evaluating the antidiarrheal effect of SCZX on diarrhea mice induced by Folium Sennae, the purpose of this study is to outline the characteristics of the antidiarrheal effect and reveal the potential mechanisms of SCZX through the analysis of the mechanism and active components of YT via network pharmacology and molecular docking, combined with the research progress of BN obtained from the literature. SCZX (3.12 and 12.48 g/kg) effectively inhibited diarrhea in mice, significantly lowering the loose stool rate (LSR), loose stool level (LSL), and loose stool index (LSI). Using network pharmacology, the "herb-compound-target-pathway-pharmacological action" network was mapped to indicate the antidiarrheal mechanism of YT. And the docking results revealed that 4 components of YT including quercetin, geranyl-1-O-α-L-arabinopyranosyl-(1 ⟶ 6)-ß-D-glucopyranoside, 3α-O-(E)-p-hydroxy-cinnamoyl-olean-12-en-27-oic acid, and daucosterol showed significant docking activities with STAT3, EGFR, and SLC10A2, involving 11 pathways such as Th17 cell differentiation, Jak-STAT signaling pathway, ErbB signaling pathway, and HIF-1 signaling pathway. According to our research results and literature reports, the antidiarrheal could be summarized into five aspects: inhibiting intestinal inflammation, acting as a barrier to the intestinal mucosal, regulating water and ion transport, involving the purification of intestinal microorganisms, and intestinal transmission, which might be dependent on multiple proteins and intervention in multiple pathways.

Effect of Ultrafine Powderization and Solid Dispersion Formation via Hot-Melt Extrusion on Antioxidant, Anti-Inflammatory, and the Human Kv1.3 Channel Inhibitory Activities of Angelica gigas Nakai.

Angelica gigas Nakai (AGN) was first processed by ultrafine grinding technology and hot-melt extrusion (HME). The potential antioxidant and anti-inflammatory activities of AGN with a different process were compared, and the effect on the human Kv1.3 potassium channel was detected. The process of ultrafine powderization on AGN significantly increased the total phenolic and flavonoid contents, antioxidant activity, and DNA damage protective effect. On the contrary, AGN solid dispersion (AGN-SD) based on Soluplus® showed the highest inhibitory effect on NO production and the human Kv1.3 channel. In addition, AGN-SD inhibited the production of prostaglandin E2 and intracellular reactive oxygen species and the mRNA expression of inducible nitric oxide synthase, cyclooxygenase-2, interleukin 1ß, and interleukin 6. Taken together, these results suggest that ultrafine powderization and solid dispersion formation via HME can significantly improve the biological activities of AGN. The results also suggested that ultrafine powderization and HME may be developed and applied in the pharmaceutical industry.

Current strategies against COVID-19.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently was declared a pandemic by world health organization (WHO) Due to sudden outbreaks, currently, no completely effective vaccine or drug is clinically approved. Several therapeutic strategies can be envisaged to prevent further mortality and morbidity. Based on the past contribution of traditional Chinese medicines (TCM) and immune-based therapies as a treatment option in crucial pathogen outbreaks, we aimed to summarize potential therapeutic strategies that could be helpful to stop further spread of SARS-CoV-2 by effecting its structural components or modulation of immune responses. Several TCM with or without modification could be effective against the structural protein, enzymes, and nucleic acid should be tested from available libraries or to identify their immune-stimulatory activities to enhance several antiviral biological agents for effective elimination of SARS-CoV-2 from the host. TCM is not only effective in the direct inhibition of virus attachment and internalization in a cell but can also prevent their replication and can also help to boost up host immune response. Immune-modulatory effects of TCMs may lead to new medications and can guide us for the scientific validity of drug development. Besides, we also summarized the effective therapies in clinical for controlling inflammation. This review will be not only helpful for the current situation of COVID-19, but can also play a major role in such epidemics in the future.