The Therapeutic Potential of Four Main Compounds of Zanthoxylum nitidum (Roxb.) DC: A Comprehensive Study on Biological Processes, Anti-Inflammatory Effects, and Myocardial Toxicity.

Zanthoxylum nitidum (Roxb.) DC. (Z. nitidum) is a traditional Chinese medicinal plant that is indigenous to the southern regions of China. Previous research has provided evidence of the significant anti-inflammatory, antibacterial, and anticancer properties exhibited by Z. nitidum. The potential therapeutic effects and cardiac toxicity of Z. nitidum remain uncertain. The aim of this research was to investigate the potential therapeutic properties of the four main compounds of Z. nitidum in cardiovascular diseases, their impact on the electrical activity of cardiomyocytes, and the underlying mechanism of their anti-inflammatory effects. We selected the four compounds from Z. nitidum with a high concentration and specific biological activity: nitidine chloride (NC), chelerythrine chloride (CHE), magnoflorine chloride (MAG), and hesperidin (HE). A proteomic analysis was conducted on the myocardial tissues of beagle dogs following the administration of NC to investigate the role of NC in vivo and the associated biological processes. A bioinformatic analysis was used to predict the in vivo biological processes that MAG, CHE, and HE were involved in. Molecular docking was used to simulate the binding between compounds and their targets. The effect of the compounds on ion channels in cardiomyocytes was evaluated through a patch clamp experiment. Organ-on-a-chip (OOC) technology was developed to mimic the physiological conditions of the heart in vivo. Proteomic and bioinformatic analyses demonstrated that the four compounds of Z. nitidum are extensively involved in various cardiovascular-related biological pathways. The findings from the patch clamp experiments indicate that NC, CHE, MAG, and HE elicit a distinct activation or inhibition of the IK1 and ICa-L in cardiomyocytes. Finally, the anti-inflammatory effects of the compounds on cardiomyocytes were verified using OOC technology. NC, CHE, MAG, and HE demonstrate anti-inflammatory effects through their specific interactions with prostaglandin-endoperoxide synthase 2 (PTGS2) and significantly influence ion channels in cardiomyocytes. Our study provides a foundation for utilizing NC, CHE, MAG, and HE in the treatment of cardiovascular diseases.

A new phenylpropanoic acid congener from Zanthoxylum nitidum.

One new phenylpropanoic acid congener, 2R-(5'-methoxy) pandanusphenol B (1), along with 26 known isolates, were isolated from Zanthoxylum nitidum. Their structures were elucidated by comprehensive spectroscopic data and circular dichroism analyses. All compounds, except 4, 7-10, 15, 17, 19 and 25, were reported from Z. nitidum for the first time. Among them, 16 compounds (1-3, 5-6, 12-14, 16, 20-24 and 26-27) were discovered from genus Zanthoxylum for the first time, while 15 compounds (1-3, 5-6, 12-14, 20-24 and 26-27) were isolated from the Rutaceae family for the first time. All isolates were evaluated for their cytotoxicity against five human cancer cell lines and the results showed that compound 27 exhibited significant cytotoxicity toward HepG2 and T24, with IC50 values of 2.49 and 7.0 µM, respectively.

Structurally diverse isobutylamides from Zanthoxylum nitidum.

Five pairs of new racemic alkamides (1a/1b and 4a/4b-7a/7b) and two new achiral derivatives (2-3), as well as five known ones (8-12), were purified from the 95% EtOH extract of Zanthoxylum nitidum. Their structures were elucidated based on spectroscopic analyses (NMR and HR-ESI-MS), electronic circular dichroism (ECD) and NMR calculations. The enantiomeric separation was successfully achieved by chiral-phase HPLC-ECD measurements. Among all the isolates, compounds 2, 3, and 10 showed inhibitory effects against five human cancer cell lines, with IC50 values in range of 18.51-48.03 µM.

Undescribed isoquinolines from Zanthoxylum nitidum and their antiproliferative effects against human cancer cell lines.

Eleven previously undescribed alkaloids, including three pairs of enantiomers nitidumalkaloids A-C, a pair of scalemic mixtures nitidumalkaloid D and three optically pure or achiral alkaloids, nitidumalkaloids E-G, along with 20 known alkaloids, were isolated from an ethanolic extract of the whole Zanthoxylum nitidum (Roxb.) DC plant. The chemical structures of the alkaloids were elucidated using a combination of comprehensive nuclear magnetic resonance (NMR) and high-resolution electro-spray ionization mass spectrometry (HR-ESI-MS) analyses. The configuration of the stereogenic centers of all undescribed compounds was precisely established based on single-crystal X-ray diffraction and electronic circular dichroism (ECD) calculations. Racemic mixtures of nitidumalkaloids A-D were purified, and their enantiomers were analyzed via chiral-phase high-performance liquid chromatography with electrochemical detection measurements (HPLC-ECD). Twelve compounds exhibited significant antiproliferative activities against a panel of cancer cell lines. Further studies were designed to investigate the underlying molecular mechanism of (1'S, 6R)-nitidumalkaloid B, which was the most active antiproliferative agent against human cancer A549 cells. G2/M cell cycle arrest, induction of apoptosis, and suppression of the Wnt/ß-catenin signaling pathway were in part associated with the antiproliferative activity of (1'S, 6R)-nitidumalkaloid B. Moreover, (1'S, 6R)-nitidumalkaloid B inhibited cell migration by downregulating the epithelial-mesenchymal transition process in A549 cells. These data suggest that the antiproliferation activity of (1'S, 6R)-nitidumalkaloid B was correlated with the stereoselectivity of the stereoisomers, and (1'S, 6R)-nitidumalkaloid B was prioritized as a potential leading compound for the management of aggressive human non-small-cell lung cancer (NSCLC) from natural products.

Nitidine chloride induces cardiac hypertrophy in mice by targeting autophagy-related 4B cysteine peptidase.

Nitidine chloride (NC) is a standard active component from the traditional Chinese medicine Zanthoxylum nitidum (Roxb.) DC. (ZN). NC has shown a variety of pharmacological activities including anti-tumor activity. As a number of anti-tumor drugs cause cardiotoxicity, herein we investigated whether NC exerted a cardiotoxic effect and the underlying mechanism. Aqueous extract of ZN (ZNE) was intraperitoneally injected into rats, while NC was injected into beagles and mice once daily for 4 weeks. Cardiac function was assessed using echocardiography. We showed that both ZNE administered in rats and NC administered in mice induced dose-dependent cardiac hypertrophy and dysfunction, whereas administration of NC at the middle and high dose caused death in Beagles. Consistently, we observed a reduction of cardiac autophagy levels in NC-treated mice and neonatal mouse cardiomyocytes. Furthermore, we demonstrated that autophagy-related 4B cysteine peptidase (ATG4B) may be a potential target of NC, since overexpression of ATG4B reversed the cardiac hypertrophy and reduced autophagy levels observed in NC-treated mice. We conclude that NC induces cardiac hypertrophy via ATG4B-mediated downregulation of autophagy in mice. Thus, this study provides guidance for the safe clinical application of ZN and the use of NC as an anti-tumor drug.

Spectrum-Effect Relationship Analysis of Bioactive Compounds in Zanthoxylum nitidum (Roxb.) DC. by Ultra-High Performance Liquid Chromatography Mass Spectrometry Coupled With Comprehensive Filtering Approaches.

Zanthoxylum nitidum (Roxb.) DC. (ZN), with strong effects of anti-inflammation and antioxidant activities is treated as a core herb in traditional Chinese medicine (TCM) preparation for treating stomachache, toothache, and rheumatoid arthritis. However, the active ingredients of ZN are not fully clarified due to its chemical complexity. In the present study, a double spectrum-effect analysis strategy was developed and applied to explore the bioactive components in herbs, and ZN was used as an example. Here, the chemical components in ZN were rapidly and comprehensively profiled based on the mass defect filtering-based structure classification (MDFSC) and diagnostic fragment-ion-based extension approaches. Furthermore, the fingerprints of 20 batches of ZN samples were analyzed by high-performance liquid chromatography, and the anti-inflammatory and antioxidant activities of the 20 batches of ZN samples were studied. Finally, the partial least squares regression (PLSR), gray relational analysis models, and Spearman's rank correlation coefficient (SRCC) were applied to discover the bioactive compounds in ZN. As a result, a total of 48 compounds were identified or tentatively characterized in ZN, including 35 alkaloids, seven coumarins, three phenolic acids, two flavonoids, and one lignan. The results achieved by three prediction models indicated that peaks 4, 12, and 17 were the potential anti-inflammatory compounds in ZN, whereas peaks 3, 5, 7, 12, and 13 were involved in the antioxidant activity. Among them, peaks 4, 5, 7, and 12 were identified as nitidine, chelerythrine, hesperidin, and oxynitidine by comparison with the standards and other references. The data in the current study achieved by double spectrum-effect analysis strategy had great importance to improve the quality standardization of ZN, and the method might be an efficiency tool for the discovery of active components in a complex system, such as TCMs.

Rapid identification and pharmacokinetic studies of multiple active alkaloids in rat plasma through UPLC-Q-TOF-MS and UPLC-MS/MS after the oral administration of Zanthoxylum nitidum extract.

Zanthoxylum nitidum (Roxb.) DC. (ZN) belongs to the genus Zanthoxylum of Rutaceae and has various chemical ingredients and pharmacologic effects. Alkaloids are its main active constituents responsible for diverse pharmacologic effects, such as anti-tumor, anti-bacterial, anti-inflammatory, and analgesic activities. The chemical and pharmacological effects of ZN are well reported, but the in vivo pharmacokinetic profiles of its main active alkaloids are poorly investigated. This study aims to elucidate the absorbed constituents and pharmacokinetic behavior of main active ingredients in rat plasma after the oral administration of ZN extract. The absorbed constituents in rat plasma were qualitatively analyzed using ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination and pharmacokinetic studies of dihydrochelerythrine (DHCHE), nitidine chloride (NIT), chelerythrine (CHE), sanguinarine (SAN), liriodenine (LIR), skimmianine (SKI), γ-fagarine (FAG), and dictamnine (DIC) in rat plasma. Eighteen prototypes and metabolites were identified according to exact mass, characteristic diagnostic fragment ions, and reference standards. The established UPLC-MS/MS quantitative method met the requirements of FDA for biological analysis methods. Method validation showed that this method has good linearity (r ≥ 0.9910), precision (RSD ≤ 18.63 %), accuracy (88.11 %-117.50 %), and stability. The limit of detection (LOD) could reach 1 ng/mL, and the limit of quantitation could reach 2 ng/mL. The plasma drug concentration of benzophenanthridine alkaloids, such as NIT, CHE, and DHCHE, were still low even after dose differences were deducted. For the furan quinoline alkaloids (such as SKI, FAG, and DIC), only SKI showed high plasma drug concentration, although SKI content comprised only approximately 1/6 of benzophenanthridine alkaloids. This study is the first to simultaneously determine the above-mentioned active alkaloids in rat plasma and would contribute to the comprehensive understanding of in vivo pharmacokinetic behavior on active alkaloids in ZN extract.

Insight into the inhibitory effects of Zanthoxylum nitidum against Helicobacter pylori urease and jack bean urease: Kinetics and mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum nitidum (Roxb.) DC. is a traditional Chinese medicine characterised by anti-inflammatory and anti-Helicobacter pylori, which is widely used to treat H. pylori-induced gastric disease in China. However, the underlying mechanism related to its anti-H. pylori activity remains unclear. Urease plays a crucial role in the colonisation and survival of H. pylori. AIM OF THE STUDY: The root aqueous extract of Z. nitidum against H. pylori urease (HPU) and jack bean urease (JBU) was investigated to illuminate the inhibitory potency, kinetics and potential mechanism. MATERIALS AND METHODS: Z. nitidum components were determined by UPLC. The enzyme inhibitory effects of Z. nitidum were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. Urease inhibition kinetics were determined by Lineweaver-Burk plots. Sulfhydryl group reagents and Ni2+-binding inhibitors were used in the mechanism study. Moreover, the molecular docking technique was used to investigate the binding conformations of the main compounds of Z. nitidum on Urease. RESULTS: According to UPLC results, the major components of Z. nitidum were magnoflorine, sanguinarine, nitidine chloride, chelerythrine, skimmianine and L-Sesamin. Z. nitidum has higher enzyme inhibitory activity on HPU (IC50 = 1.29 ± 0.10 mg/mL) than on JBU (IC50 = 2.04 ± 0.27 mg/mL). Enzyme inhibitory kinetic analysis revealed that the type of Z. nitidum inhibition against HPU was a slow-binding and mixed-type, whereas a slow-binding and non-competitive type inhibited JBU. Further mechanism study indicated that the active site of sulfhydryl group might be the target of inhibition by Z. nitidum. The molecular docking study indicated that the above six main components of Z. nitidum exhibited stronger affinity to HPU than to JBU through interacting with the key amino acid residues located on the mobile flap or interacting with the active site Ni2+. Results indicated that these components are potential active ingredients directed against urease. CONCLUSIONS: Z. nitidum inactivated urease in a concentration-dependent manner through slow-binding inhibition and binding to the urease active site sulfhydryl group. Our investigation might provide experimental evidence for the traditional application of Z. nitidum in the treatment of H. pylori-associated gastric disorders.

Targets prediction and mechanism of Zanthoxylum nitidum in intervention of inflammation based on network pharmacology / 中草药

Objective: To construct the “active components-inflammatory target-anti-inflammatory pathway” network of Zanthoxylum nitidum intervened in inflammation, and predict the target of Z. nitidum intervened in inflammation and its anti-inflammatory mechanism. Methods: Using domestic and foreign literatures, TCMSP database, Pharmmapper server, oral availability (OB), and pharmacodynamics (DL) as the limiting conditions, the components of Z. nitidum were screened and the relative targets were predicted and collected. OMIM database was used to screen inflammation-related genes and protein targets; The STRING database was used to construct the interactive network between inflammatory targets; The network file of “active ingredient-predictive target-inflammatory target” was obtained by PPI analysis and imported into Cytoscape 3.5.1 software to construct the network of “active ingredient- inflammatory target”, so as to obtain the targets directly related to the anti-inflammatory effects of Z. nitidum. DAVID database was used to enrich the KEGG pathway of the selected targets, and then ClueGO plug-in was used to analyze the biological function of the target involved. Finally, the “active component-inflammatory target-anti-inflammatory pathway” network was constructed by combining the above relationships. Results: Twenty-three active ingredients were screened, and nine core anti-inflammatory targets were identified as COX-2, iNOS, PPARG, COX1, MAPK-14, JUN, NR3C1 and so on; The most critical pathways included TNF TRLs signaling pathways. Conclusion: It is preliminarily revealed that the anti-inflammatory effect of Z. nitidum is achieved through the interaction of multiple components and multiple targets, regulating the joint intervention of multiple pathways. However, the key targets and specific regulatory mechanisms need to be explored and verified by further experimental studies.

Determination and Pharmacokinetic Study of Nitidine Chloride in Rat Plasma after Intragastrical Administration by LC-ESI-MS/MS Method / 中草药·英文版

Objective To study the pharmacokinetics of nitidine chloride (NC) in rat plasma after intragastrical (i.g.) administration. Methods A liquid chromatography-electrospray ionization-mass/mass sprectrometry (LC-ESI-MS/MS) was used and carbamazepine was used as an intermal standard (I.S.). The rat plasma samples were deproteinized with acetonitrile and the resultant supernatant was assayed on an analytical Diamonsil™ ODS C18 column (2.1 mm × 150 mm) equipped with a C18 guard column (4 mm × 20 mm) with a mobile phase of acetonitrile–10 mM ammonium acetate buffer–formic acid (35: 65: 0.2, v/v/v) at the flow rate of 0.25 mL/min. The LC–MS was carried out on a triple-quadrupole mass spectrometry equipped with an ESI and positive selected-ion monitoring. Target ions were monitored at [M-Cl]+ m/z 348.2 for NC and [M + H]+ m/z 237.2 for I.S., respectively. Results The simple one step deproteinize and rapid analysis method were successfully used in pharmacokinetic study on NC after i.g. administration. The linear relationship was good over the range of 2.5 – 1000.0 ng/ml (r2 = 0.999 2) in rat plasma. The lower limit of quantification and detection were 2.5 and 1.6 ng/ml, respectively. The extraction recovery was in the range of 86.54 – 98.60%. The intra- and inter-day precisions (relative standard deviation) were less than 6.00%, with accuracies deviation between 89.40 to 95.57%. A two-compartment pharmacokinetic open model was proposed and validated to explain the apparent biphasic disposition of NC in rat plasma after i.g. administration. Conclusion This study was successfully applied to a pharmacokinetic study of NC in rats plasma following i.g. administration and could be used for preclinical and clinical pharmacokinetic evaluation of NC.

Study on pollen viability and morphological characteristics of Zanthoxylum nitidum / 中草药

Objective: To investigate the pollen morphological and physiological characteristics of Zanthoxylum nitidum. Methods: The pollen grain viability was studied using an in vitro method and the optimal concentration of sucrose, H3BO3, and CaCl2 on pollen germination were investigated by 3414 test. At the same time it contrasted with the I2-KI and TTC methods to find the proper determination method for Z. nitidum pollen vitality. And then It was further studied that different storage time and methods had an effect on pollen grain viability by having established method. Pollen morphology was studied by scanning electron microscopy (SEM). Results: The I2-KI and TTC methods were not fit for determining the mature pollen vitality of Z. nitidum. The optimal medium was 10% sucrose + 0.1 mg/mL H3BO3 + 0.1 mg/mL CaCl2 on pollen germination in vitro and the pollen germination ratio was up to 66.67%. Pollen viability was the highest in 24 h and it could keep about 15 d in 4 ℃ environments. The pollen grains were 3-colpate prolate, with reticulate ornamentation. Conclusion: The determination and storage of pollen grain viability is established preliminarily to provide reliable basis of knowing physiological characteristics, breeding and taxonomy.

Cytotoxicity of benzophenanthridine alkaloids from the roots of Zanthoxylum nitidum (Roxb.) DC. var. fastuosum How ex Huang.

This work aimed to investigate benzophenanthridine from the roots of Zanthoxylum nitidum (Roxb.) DC. var. fastuosum How ex Huang for the first time. Thirteen benzophenanthridines were isolated, and our results of the cytotoxic activities indicated that compound 6 exhibited the best potency against A549, Hela, SMMC-7721 and EJ, with the IC50 values of 27.50, 37.50, 16.95 and 60.42 µM, respectively. Compounds 7 and 11 also showed strong cytotoxicity when tested against the four human cancer cell lines (A549, Hela, SMMC-7721 and EJ), while only compounds 12 and 13 displayed cytotoxicity in inhibiting BALL-1 proliferation among all the compounds. These results suggested that benzophenanthridines may become a valid alternative of potential basis for new anti-proliferative agents.

Antibacterial constituents from roots of Zanthoxylum nitidum / 中草药

Objective: To isolate and identify the antibacterial constituents from the roots of Zanthoxylum nitidum. Methods: Bioassay-guided fractionation led to the isolation of compounds from the roots of Z. nitidum by using various chromatographic techniques such as silica gel, alumina, preparative TLC, and HPLC, and their chemical structures were then elucidated on the basis of spectroscopic data, including NMR, MS analysis, and their physicochemical properties. Results: Eleven compounds were isolated from the bioactive extracts in the roots of Z. nitidum and then were identified as skimmianine (1), oxychelerythrine (2), 8-methoxy-dihydrochelerythrine (3), β-sitosterol (4), L-sesamin (5), 8-methoxy-9-demethoxyldihydrochelerythrine (6), 4-hydroxy-N- methylproline (7), liriodenine (8), avicine (9), nitidine (10), and isobutyl benzoate (11), respectively. Compounds 1, 3, 6, 8, and 10 showed the potential inhibition on Staphylococcus aureus. Compound 8 showed the most potential inhibitory activity with MIC value of 31.3 μg/mL; Further studies demonstrated that compound 8 inhibited the clinical multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) activity with MIC value of 93.8 μg/mL, significantly. Conclusion: A series of bioactive alkaloids with the anti-staphylococcal activities were identified from the roots of Z. nitidum. Compounds 7, 9, and 11 are obtained from this plant for the first time, and the potential anti-staphylococcal activity of compound 8 against MRSA has been demonstrated, which has provided the chemical template as a new anti-bacterial agent against clinical multidrug-resistant MRSA infection.

Simultaneous determination of five effective components in zanthozylum nitidum (Roxb) DC.f. fastuosum how ex Huang by RP-HPLC / 中国药学杂志

OBJECTIVE: To develop a RP-HPLC method for simultaneous determination of magnoflorine, hesperidin, nitidine chloride, ethoxychelerythrine and toddaloactone in Zanthozylum nitidum (Roxb) DC.f. fastuosum How ex Huang. METHODS: The RP-HPLC system consisted of a Diamonsil C18 column (4.6 mm×250 mm, 5μm) with the mobile phase of acetonitrile solution-water solution (containing 0.2% phosphoric acid and 0.2% triethylamine) for gradient elution. DAD detector was used and the detection wave lengths were 273, 283 and 328 nm. The flow rate was 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 the maximum absorption wavelength as the quantitative index. RESULTS: The liner ranges of magnoflorine, hesperidin, nitidine chloride, ethoxychelerythrine and toddaloactone were 0.0957-1.3391 μg (r=0.9995), 0.3189-2.1260 μg(r=0.9998),0.0397-0.2648 μg (r=0.9995), 0.1004-1.0040 μg(r=0.9999), and 0.1080-2.1600 μg (r=0.9999), respectively. The average recoveries (n=6) were 100.2%, 99.8%, 97.1%, 98.8% and 101.6% (n=6) Respectively. CONCLUSION: The method is accurate, simple, rapid, and reproducible for the determination of magnoflorine, hesperidin, nitidine chloride, ethoxychelerythrine and toddaloactone in Zanthoxylum nitidum (Roxb) DC.f. fastuosum How ex Huang. The determination result can be used as a reference for the reasonable medication, quality control and further study of Zanthoxylum nitidum (Roxb) DC.f. fastuosum How ex Huang.

Anti-hepatoma activity of nitidine chloride and its effect on topoisomerase / 中国药理学通报

Aim To investigate the anti-tumor effect of nitidine chloride(NC)on human HepG2 hepatocellular transplanted tumor in nude mice and its effect on topoisomerase.Methods The subcutaneous transplantable tumor model of human liver cancer in nude mice was established and the anti-tumor effect of NC was calculated.The effects of NC on TopoⅠ/Ⅱ mediated-pBR322 DNA relaxation were measured by using agarose gel electrophoresis.Results NC inhibited significantly the growth of hepatoma,The inhibitory rate at the dose of 2.5,5,10 mg·kg~(-1) was 12.06%,35.63% and 60.91% respectively.At the concentration of 6.25 μmol·L~(-1),NC completely inhibited the pBR322 DNA cleavage mediated by TopoⅠ;at the concentration of 25 μmol·L~(-1),NC completely inhibited the pBR322 DNA cleavage mediated by Topo Ⅱ.Conclusion Nitidine Chloride can inhibit hepatic carcinoma growth in nude mice,The anti-tumor mechanism is probably related to the inhibitory effect on Topo.

Two new alkaloids from roots of Zanthoxylum nitidum / 中草药

Objective To study the chemical constituents from the roots of Zanthoxylum nitidum.Methods The constituents were isolated and purified by column chromatography.Their structures were identified on the basis of physiochemical properties and spectral data.Results Two benzophenanthridine alkaloids,1,3-bis(8-dihydronitidinyl)-acetone(1)and 8-acetonyldihydrofagaridine(2),have been iso-lated from the roots of Z.nitidum.Conclusion Compounds 1 and 2 are new compounds named nitidumtone A and nitidumtone B.