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OBJECTIVE：To prepare chelerythrine nanoparticles（CHE-NPs），optimize their formulation ，and evaluate its drug release behavior in vitro and its inhibitory effect on melanoma. METHODS ：Using methoxy polyethylene glycol-poly （lactic-co- glycolic acid ）（mPEG-PLGA）as carrier ，CHE-NPs were prepared by the nano-precipitation method. HPLC method and dialysis bag method were used to determine entrapment efficiency and drug loading. The formulation of CHE-NPs was optimized by Box-Behnken response surface design using overall desirability （OD）of them as dependent variables ，CHE dosage ，mPEG-PLGA concentration and poloxamer 188（F68）concentration as independent variables. The particle size and Zeta potential of CHE-NPs prepared by the optimal formulation were detected ；the characteristics of drug release in vitro were investigated ；the effects of CHE and CHE-NPs on survival rate of mice B 16 melanoma cells were compared ，and median inhibition concentrations （IC50）of them were calculated. RESULTS ：The optimal formulation included CHE of 2 mg，mPEG-PLGA of 13 mg/mL，F68 of 1.8％. Average entrapment efficiency rate of CHE-NPs prepared by the optimal formulation was （80.18±1.11）％，average drug loading was （11.36±0.28）％，average OD value was 0.96±0.04 [the relative deviation from predicted value （0.90）of OD was 6.67％]； particle size was （113.1±1.40）nm，and Zeta potential was （－21.6±0.29）mV；polydispersity index was 0.07±0.01（n＝3）； accumulative release rates of CHE control and CHE-NPs were 90.87％ and 68.68％ within 8 h，and drug release behavior in vitro of the latter was in accordance with Weibull kinetic model. Inhibitory effect of CHE-NPs on B 16 melanoma cells was significantly stronger than that of CHE ；the 24 h IC 50 of CHE-NPs and CHEwere 69.35 and 107.36 μg/mL，respectively. CONCLUSIONS ：The prepared CHE-NPs show good sustained-effect and high capacity of drug loading ，and strengthen the inhibitory effect of CHE on melanoma.
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【Objective】 To investigate the mechanisms of chelerythrine on the treatment of breast cancer based on network pharmacology and molecular docking. 【Methods】 The targets corresponding to chelerythrine and breast cancer were obtained from Mala Cards and Swiss Target Prediction databases. Chelerythrine-related and breast cancer-related targets were found and then combined to get an intersection, which represented potential anti-breast cancer targets of chelerythrine. A protein-protein interaction (PPI) network was constructed from the STRING database and key genes were screened using the topological analysis. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of targets were conducted using metascape database. The relationship between the expressions of key target genes and the survival curve was analyzed using the Kaplan-Meier Plotter database. Molecular docking analysis was performed by AutoDock Vina to verify whether chelerythrine has a definite affinity with key targets. 【Results】 A total of 37 potential targets were obtained in chelerythrine against breast cancer. The result of the topology analysis included 8 key targets. The GO enrichment analysis included 317 GO items. The KEGG pathway analysis included 80 pathways, which were closely related to the PI3K/AKT signaling pathway, the ErbB signaling pathway, VEGF signaling pathway, and others. The results of the survival curve analysis showed that the expression levels of CHEK1, PIK3CA, mTOR and PTGS2 genes were related to the survival time of breast cancer patients. The results of molecular docking proved that the combined activity of chelerythrine with key targets was excellent. 【Conclusion】 Chelerythrine may play an anti-breast cancer role via the PI3K/AKT signaling pathway and has the potential to be developed into a clinical drug for breast cancer.
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To develop a fluorescence polarization (FP)-based high-throughput screening (HTS) assay to identify novel small-molecule antagonists targeting β-catenin/TCF4 (T-cell factor 4) interaction, recombinant human β-catenin was expressed in Escherichia coli Rosetta (DE3) cells and purified by HisTrapTM column. The bioactivity of purified β-catenin was further analyzed by enzyme-linked immunosorbent assay (ELISA). According to FP principle, the β-catenin/TCF4 binding model was performed, and fluorescence isothiocyanate (FITC) labeled TCF4 peptide (FITC-TCF4) served as the molecular probe of adaptor for binding to β-catenin. The FITC-TCF4 and β-catenin working concentration were optimized, and the binding conditions (complex stability and dimethylsulfoxide (DMSO) tolerance) have been investigated yet for further hits screening. The results showed that recombinant human β-catenin was successfully expressed and purified β-catenin exhibited favorable bioactivity in ELISA binding assay. Subsequently, the FP-based HTS assay was performed using 20 nmol·L-1 FITC-TCF4 and 100 nmol·L-1 β-catenin. Under these optimized conditions, a high Z´factor of 0.88 was achieved in a 384-well format and this FP-based HTS assay was very stable with regard to DMSO. Through screening of a natural-based product library (NBPL) using the established FP-based HTS assay, three hits (sanguinarine, chelerythrine, and compound S720) were identified as potential β-catenin/TCF4 interaction antagonists. Taken together, we have successfully developed a simple, robust and reliable FP-based HTS assay for screening of novel antagonists targeting β-catenin/TCF4 interaction.
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OBJECTIVE：To prepare Cheler ythrine （CHE） solid dispe rsion （SD），optimize the formulation technology ， characterize its preparation and investigate its in vitro antioxidant activity. METHODS ：The content of CHE in SD was determined by UV spectrophotometry. Based on single factor tests ，using the product yield as index ，using preparation method ，carrier material type，carrier material proportion （drug-carrier material mass ratio ）as factors ，the formulation technology of SD was optimized by L（9 34）orthogonal test and validated. Based on solubility and accumulative dissolution determination ，the product was characterized with thermal analyssis ，X-ray diffraction and scanning electron microscope. Using ascorbic acid as positive control ，in vitro antioxidant activity of the product was determined by DPPH method. RESULTS ：The linear range of CHE was 2.4-5.6 μg/mL； quantitation limit and detection limit were 0.066 9，0.022 1 μg/mL；RSDs of precision ，stability and reproducibility tests were all lower than 2％；recoveries were 97.50％-99.25％（RSD＜1％， n＝3）. The optimal preparation technology included using PEG 6000 as carrier material ，carrier material ratio of 1 ∶ 3， prepared by solvent method. Three batches of CHE-PEG-SD were prepared. Verification test results showed that the 话：0539-80311889。E-mail：zhenshengao@163.com accumulative dissolution of CHE-PEG-SD was （61.72 ± 0.67）％ at 15 min，and the yield was （99.04±0.83）％. The results of characterization showed that after CHE-PEG-SD prepared ， its solubility （3.725 mg/mL）and accumulative dissolution （61.25％，15 min）were higher than CHE raw material [ 0.098 mg/mL， 6.24％（180 min）]. The endothermic peak and crystal absorption peak moved or even disappeared compared with raw material and the carrier material ，and CHE was uniformly dispersed in the carrier material as an amorphous state. Results of in vitro antioxidation test showed that different concentration of CHE-PEG-SD showed certain ability of DPPH free radical scavenging ，and the IC 50 was 0.124 mg/mL，higher than 0.041 mg/mL of ascorbic acid. CONCLUSIONS ：Established content determination method is simple and accurate. The optimal SD formulation technology is stable and feasible. The solubility of prepared CHE-PEG-SD increases，and the dissolution in vitro increases，showing certain in vitro oxidation resistance.
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Objective: To observe the long-term toxicity of chelerythrine on the morphology of lung tissue and the expression level of nuclear factor-kappa B (NF-κB) in lung tissue of the rats, and to investigate the related mechanism which causing the lung tissue damage of rats. Methods: A total of 120 Wistar rats were divided into control group (given normal saline) and low (3. 7 mg middot; kg-1), moderate (5. 6 mg middot; kg-1), high (8. 4 mg middot; kg-1) dosages of chelerythrine groups (n=30). The general condition of rats in various groups was observed; HE staining was used to observe the morphology of lung tissue of the rats in various groups; the serum interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) levels of the rats in various groups were measured by ELISA method; real-time fluorescence quantitative PCR and Western blotting methods were used to detect the mRNA and protein levels of NF-κB and intercellular adhesion molecule-1 (ICAM-1). Results: The accumulative mortality of the rats in high dosage of chelerythrine group was the highest, followed by moderate and low dosages of chelerythrine groups. The body weights and food intakes of the rats in different dosages of chelerythrine groups were significantly lower than that in control group (P<0. 05), while the body weight and food intake of the rats in high dosage of chelerythrine group were lower than those in low and moderate dosages of chelerythrine groups (P<0. 05). Chelerythrine led to pulmonary congestion and bloody ascites of the rats. The HE staining results showed that the injuries of lung tissue in different dosages of chelerythrine groups were aggravated with the increasing of the dosage of chelerythrine. Compared with control group, the levels of IL-6, IL-8, and TNF-α in serum of the rats in different dosages of chelerythrine groups were increased significantly (P< 0.05); compared with low and moderate dosages of chelerythrine groups, the levels of IL-6, IL-8, and TNF-α in serum of the rats in high dosage of chelerythrine group were increased significantly (P<0. 05). Compared with control group, the expression levels of NF-κB and ICAM-1 mRNA and proteins in lung tissue of the the rats in different dosages of chelerythrine groups were increased significantly (P<0. 05) in a dose-dependent manner; compared with low and moderate dosages of chelerythrine groups, the expression levels of NF-κB and ICAM-1 mRNA and proteins in lung tissue of the rats in high dosage of chelerythrine group were increased (P<0. 05). Conclusion: Chelerythrine has a long-term toxic effect on lung tissue in a dose-dependent manner of the rats. Moderate and high dosages of chelerythrine may aggravate the toxic lung injury through activation of NF-κB and production of inflammatory cytokines.
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Objective To establish the fingerprint profiling of fruits of Macleaya cordata and study the method for its quality evaluation. Methods The fingerprint profiling of M. cordata fruits from different regions was established using high performance liquid chromatography (HPLC) based on the local standard which was the determination method of the content of protopine, allocryptopine, sanguinarine, and chelerythrine from Changsha of Hunan Province in 2009. The principal component analysis (PCA) and cluster analysis (CA) were performed to explore the correlation among the common fingerprint peaks, origins, and quality of M. cordata fruits. Results Eleven common fingerprint peaks were identified in the fingerprint profiling of chemical constituents of M. cordata fruits from different regions. M. cordata fruits produced from eight areas were classified into two classes by PCA and CA method, and there were five common peaks, including peak 5, 7, 8, 9, and 11 with significant contribution on the regional difference of the fingerprint. Also, common peak 6 was the right peak as the reference peak because of its less variation, appropriate retention time and intensity. Conclusion The fingerprint profiling of chemical constituents of M. cordata fruits established in this study has good precision, repeatability, and stability, which can be used to evaluate the quality of fruits of M. cordata from different producing areas.
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9-Acetoxycycloberberine (1) with a unique skeleton was first identified to display a potent antimicrobial profile against methicillin-resistant Staphylococcus aureus (MRSA) with MIC values of 1-16 μg·mL-1. Taking the compound as a lead, 14 target cycloberberine analogues with diverse structures, such as berberine and chelerythrine derivatives, were synthesized and evaluated for their anti-bacterial activities. Analysis of the structure-activity relationship revealed that:① ring E was essential for the activity; ② the removing of ring B decreased the activity against MRSA. However, the antimicrobial activity against vancomycin-resistant Enterococcus faecium (VRE) was improved; ③ the introduction of a suitable rigid substituent at the 9-position was beneficial for the activity. Among them, compound 9a showed the most potential activity against methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA isolates with MIC values of 0.5-1 μg·mL-1, suggesting a different mechanism from clinical drugs. It displayed higher stability in blood. Therefore, we consider 9a worthy of further investigation. The results provide key scientific evidence for development of such compounds into a new type of anti-MRSA candidates.
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OBJECTIVE: To develop a RP-HPLC method for simultaneous determination of hesperidin, nitidine chloride, ehelerythrine, and toddalolactone in Toddalia asiatica(L)Lam. Methods: The RP-HPLC system consisted of a Diamonsil C18 column (4.6 mm×250 mm, 5 μm) with the mobile phase of acetonitrile-water solution containing 75% glacial acetic acid. UV detector was used, and the detection wave length was 290 nm. Gradient elution was adopted at a flow rate of 1.0 mL · min-1 and the column temperature was 30°C. For different constituents, external standard method was used with the peak area at 290 nm as the quantitative index. RESULTS: The liner ranges of hesperidin, nitidine chloride, ehelerythrine and toddalolactone were 0.4874-2.4372 μg(r= 0.9997), 0.0303-0.1515 μg(r=0.9992), 0.0623-0.3117 μg(r=0.9993), and 0.0249-0.1246 μg(r=0.9999), respectively. The average recoveries(n=6) were 100.76%(RSD 2.75%), 97.98%(RSD 1.25%), 100.07%(RSD 3.24%), and 100.10% (RSD 3.83%), respectively. CONCLUSION: The method is accurate, simple, rapid, and reproducible for the determination of hesperidin, nitidine chloride, ehelerythrine, and toddalolactone in Toddalia asiatica(L)Lam. The determination results can be used as a reference for the rational medication, quality control, and further study of Toddalia asiatica(L.)Lam.
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Objective: To study the antitumor molecular mechanism started on sanguinarine (San) and chelerythrine (Che) from Macleaya cordata. Methods: Determining the IC50 values of San and Che against A-549, HCT-8, and Bel-7402 cell lines by using MTT to clarify whether these two alkaloids are the active components in M. cordata; studying the interaction between human telomeric DNA and two alkaloids respectively by using UV-Vis, FL, and CD methods. Results: San and Che are the active antitumor components of M. cordata; San could induce HT4 to form antiparallel G-quadruplex completely with Ka of 5×108 and Che could induce HT4 to form antiparallel G-quadruplex partially with Ka of 930. Conclusion: One of the antitumor molecular mechanisms of M. cordata is that the two active components could induce human telomeric DNA to form G-quadruplex.
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Objective To establish a mouse pancreatic exocrine secretion system,in order to study the effect of secretagogues,such as cholecystokinin (CCK) and carbachol (CCH),on the exocrine secretion of mouse pancreas,and use a protein kinase C (PKC) antagonist-chelerythrine(Che) to research the possible mechanism.Methods Prospective control study.Results CCK and CCH increased the secretion of amylase from the mouse pancreatic acini about two fold of that of the control (7.02%vs14.29%),and,CCK or CCH added Che,could increase the secretion of amylase to a level of about three fold(19.02%).Conclusion CCK or CCH stimulate the pancreatic secretion concerning the PKC cellular signal system;surprisingly,a PKC antagonist,chelerythrine,can reinforces their stimulation.
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Objective To establish the fingerprints of quaternary ammonium hydrate alkaloids in Radix Zanthoxyli nitidii by means of HPLC and to identify and evaluate the quality of different parts and commercial decoction pieces of Radix Zanthoxyli nitidii.Method The column of Zorbax Eclipse XDB-C_8(4.6?150mm,5?m)was selected.The mobile phase consisted of A:3 % glacial acetic acid-diethylamine(1000:7.8),B:methanol,and C:acetonitrile(non-lin- ear gradient elution).The elution speed was 0.8 mL?min~(-1),the detection wavelength was at 250 nm and 270 nm,and the column temperature was 20℃.Results The HPLC fingerprint of Radix Zanthoxyli nitidii consisted of 21 peaks which were chiefly composed by alkaloids such as Chelerythrine,Nitidine chloride,with a consistent peak-to-peak ratio.The constituents' distribution information provided quality information for assessing medicinal materials.Conclusion It showed that the alkaloids distributed mainly in the cortex of the roots,so the commercial decoction pieces of aged roots shed cortexes are inferior.The stems can not be used equivalently with the roots due to low content distribution of alkaloids.
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Objective: To determine the content of sanguinarine and chelerythrine in Macleaya microcarpa(Maxim) Fedde. Methods: RP-HPLC was adopted, kromasil C 18 column (150mm?4.6mm.5?m) with a mobile phase acetonitrile-0.1%(V/V) phosphoric acid solution(25∶75) by UV detector at 270nm. Results: The average recoveries of chelerythrine and sanguinarine were 90.2%,92.8%,RSD were 1.5%,2.4% respectively. Conclusion: A simple, rapid and sensitive method with good precision was established.
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AIM: To study the effects of protein kinase C (PKC) inhibitor, chelerythrine chloride (CH), on nociceptive response, nitric oxide synthase (NOS) expression and nitric oxide (NO) content in spinal cord of rats with inflammatory pain. METHODS: Inflammatory pain was induced by formalin injection into right hind paw. NADPH-d histochemistry was used to investigate the changes of NOS expression. Nitrate/nitrite (NO_2-/NO_3-) was assayed to represent NO content. RESULTS: Compared with the control group, the number of NADPH-d positive cells increased significantly in the superficial layer (LaminaeⅠ-Ⅱ) of the spinal cord dorsal horn and the grey matter surrounding the central canal (Laminae Ⅹ) in rats with inflammatory pain, the reactive degree of NADPH-d positive soma and fibers and NO content of the lumbar enlargement of spinal cord also increased significantly. Intrathecal injection of CH inhibited the spontaneous pain response in the second phase induced by formalin injection, and prevented the increases in the number and reactive degree of NADPH-d positive cells, as well as NO content of the lumbar enlargement of spinal cord. CONCLUSION: It is suggested that the activation of PKC promotes NOS expression and NO production in the nociceptive neurons of spinal cord during formalin-induced inflammatory pain.