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In order to reveal the distribution and population characteristics of endophytic fungi from Zanthoxylum nitidum and the antibacterial potential,this study performed molecular identification and analyzed the genetic diversity and antibacterial activity of endophytic fungi from Z. nitidum in Guangxi. Through culture and molecular identification,35 strains,belonging to 15 genera,12 families,10 orders,4 classes,and 2 phyla,were isolated from various tissues of Z. nitidum,of which Colletotrichum and Fusarium were the dominant genera,respectively accounting for 20% of total strains. The diversity of endophytic fungi was significantly different among roots,stems,and leaves,as manifested by the significantly higher Shannon index( H') in stems( 1. 678) than in roots( 0. 882 1) and leaves( 0. 515 4). The antimicrobial activity analysis showed that 14. 28% of endophytic fungi inhibited at least one indicator pathogen. Among them,Fusarium sp. ZN-34 and Fusarium sp. ZN-26 separately demonstrated the strongest inhibitory effect on Escherichia coli and Staphylococcus aureus. In general,Fusarium sp. ZN-26 and Phialemoniopsis plurioloculosa ZN-35 were advantageous in suppressing the two bacteria owing to the broad spectrum and strong efficacy. In summary,Z. nitidum in Guangxi boasts rich endophytic fungi with the majority showing strong antibacterial activity,which can be used as candidates for the extraction and separation of basic antibacterial substances and the development of natural antibacterial agents.
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Humans , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents , China , Colletotrichum , Endophytes/genetics , Fungi/genetics , Genetic Variation , Microbial Sensitivity Tests , ZanthoxylumABSTRACT
Objective:To analyze and identify the non-medicinal parts in Zanthoxylum nitidum husk by HPLC-LTQ/Orbitrap-MS, and study the antioxidant activity, in order to provide the scientific basis for further research and development of Z. nitidum. Method:Data is collected by HPLC-LTQ/Orbitrap-MS,and high-resolution MS and MS2 spectra of mass spectrogram of chromatographic peaks were analyzed and compared with the literature database. The structure of each chromatographic peak was calculated and confirmed. The anti-oxidative activity of the Z. nitidum husk was studied by DPPH scavenging free radical ability and ABTS free radical scavenging ability. Result:Twenty-five alkaloids were identified from Z. nitidum husk. The main constituents were isoquinoline alkaloids (nitidine,liriodenine,magnocurarine),pyrrolidine alkaloid (allocryptopine,oxymatrine,oxysophocarpine),quinoline alkaloid (magnoflorine,nitidine chloride),and organic amine alkaloids (γ-sanshool). Antioxygenic activity was studied by DPPH scavenging free radical ability and ABTS free radical scavenging ability. The results showed that they were within the measured concentration range, the antioxidant activity increased with the rise of the sample concentration, a good dose dependence was presented. Conclusion:In this paper,the chemical constituents and the activity Z. nitidum husk were studied. Studies have shown a variety of alkaloids, with a good antioxidant activity. This study provides a reference for further research and development of Z. nitidum.
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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.
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Ten compounds were isolated from the methanol extract of Zanthoxylum nitidum through silica gel, Sephadex LH-20, RP-18 and HPLC chromatography techniques. Their structures were elucidated by the MS and NMR spectra as zanthonitidine B(1), cyclo-(Leu-Leu-Leu-Leu-Ile)(2),6S-10-O-demethylbocconoline(3), liriodenine(4), isoplatydesmine(5), 5, 5'-dimethoxylariciresinol(6), syringaresinol (7), episyringaresinol (8), marmesin (9) and syringaldehyde (10). Among them,1 is a new alkaloid,2 is a cyclopentapeptide isolated from plant kingdom for the first time, and 3 is from the genus Zanthoxylum for the first time. Compounds 3 and 4 exhibited cytoxoxicity against three human cancer cell lines HT29, A549 and MDA-MB-231 with IC₅₈ values of 27.37, 24.10, 33.58 μmol·L⁻¹ and 9.12,6.05, 11.35 μmol·L⁻¹, respectively.
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Humans , Alkaloids , Chromatography, High Pressure Liquid , ZanthoxylumABSTRACT
Objective To optimize the technology conditions of ultrasonic-enzyme-assisted extraction for sanguinarine from Zanthoxylum nitidum.Methods Extraction rate of sanguinarine determined by HPLC was served as an index.The applicability of the extraction solvent added with acid and enzymatic hydrolysis pretreatment to the ultrasonic-enzyme-assisted extraction of Zanthoxylum nitidum was investigated.Ultrasonic power,extraction frequency and solvent volume were optimized by orthogonal experiment.Finally,ultrasonic extraction time was optimized in dynamic process.Results The optimal process was as follows:Zanthoxylum nitidum powder was extracted 3 times by ultrasonic-wave (250 W) with 40％ ethanol (0.2％hydrochloride) as solvent (extracted for 15 rmin with 6-fold solvent at the first time,then extracted for 12 min with 3-fold solvent at the second and the third time,respectively).The extraction rate of sanguinarine was 88.6％.Conclusion The process is economic,efficient,energy-and time-saving,and provides experimental basis for industrial production of sanguinarine.
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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.
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Objective To study whether crystal-8 exerts analgesic effect on the central nervous system,and to observe the relationship between crystal-8 and receptors. Methods A total of 32 SD rats were randomly divided into the 0.9% sodium chloride solution group, positive control group ( rotundine 2 mg·kg-1 ) , high dose of crystal-8 group ( 2 mg·kg-1 ) and low dose of crystal-8 group ( 1 mg · kg-1 ) . The changes of pain threshold were measured by the thermal stimulation test following intracerebroventricular (ICV) injection.The SD rats or mice were randomly divided into the 0.9% sodium chloride solution group, receptor tool medicine group ( including naloxone, reserpine, haloperidol, scopolamine) , crystal-8 group, receptor tool medicine plus crystal-8 group. The pain threshold was detected by the thermal stimulation test at 15, 30, 45, 60, 90, 120 min after dosing, then the influence of analgesic effect of crystal-8 on the neurotransmitter was observed. Results Compared with the 0.9% sodium chloride solution group, the pain threshold of rats was improved after taking the crystal-8 by intracerebroventricular (ICV) injection(P<0.01).Compared with the crystal-8 group, the naloxone plus crystal-8 group, the haloperidol plus crystal-8 group, the scopolamine plus crystal-8 group couldn't increase the pain threshold, there was no significant difference among these groups(P>0.05).However, Reserpine plus crystal-8 group could significantly decrease the pain threshold in rats compared with the crystal-8 group(P<0.01).The analgesic effect of crystal-8 was interfered by reserpine. Conclusion The analgesic effect of crystal-8 may be involved in the central mechanism,which relates to monoamine neurotransmitters, but has nothing to do with the opioid receptor, M receptor, and the inhibition of central DA receptor.
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Objective To study whether crystal-8 exerts analgesic effect on the central nervous system,and to observe the relationship between crystal-8 and receptors. Methods A total of 32 SD rats were randomly divided into the 0.9% sodium chloride solution group, positive control group ( rotundine 2 mg·kg-1 ) , high dose of crystal-8 group ( 2 mg·kg-1 ) and low dose of crystal-8 group ( 1 mg · kg-1 ) . The changes of pain threshold were measured by the thermal stimulation test following intracerebroventricular (ICV) injection.The SD rats or mice were randomly divided into the 0.9% sodium chloride solution group, receptor tool medicine group ( including naloxone, reserpine, haloperidol, scopolamine) , crystal-8 group, receptor tool medicine plus crystal-8 group. The pain threshold was detected by the thermal stimulation test at 15, 30, 45, 60, 90, 120 min after dosing, then the influence of analgesic effect of crystal-8 on the neurotransmitter was observed. Results Compared with the 0.9% sodium chloride solution group, the pain threshold of rats was improved after taking the crystal-8 by intracerebroventricular (ICV) injection(P<0.01).Compared with the crystal-8 group, the naloxone plus crystal-8 group, the haloperidol plus crystal-8 group, the scopolamine plus crystal-8 group couldn't increase the pain threshold, there was no significant difference among these groups(P>0.05).However, Reserpine plus crystal-8 group could significantly decrease the pain threshold in rats compared with the crystal-8 group(P<0.01).The analgesic effect of crystal-8 was interfered by reserpine. Conclusion The analgesic effect of crystal-8 may be involved in the central mechanism,which relates to monoamine neurotransmitters, but has nothing to do with the opioid receptor, M receptor, and the inhibition of central DA receptor.
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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.
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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.
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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.
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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.
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Objective To establish a simple method for determination the Alkaloids contents from Zanthoxylum nitidum. Methods Adopting nitidunechloride as reference,alkaloids in Zanthoxylum nitidum were by ultraviolet spectrophotometry at 329 nm. Results The liner arrange was 3～8 ?g/mL,regression equation:Y=97.75X -0.025 5,r =0.999 1 (n=6). The mean recovery of Nitidunechloride was 99.46%,RSD=0.93% (n =5). Conclusion The method performed is accurate and simple. The reproducibility and rate of extraction are also desirable.
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The chemical composition of essential oil of Zanthoxylum nitidum DC. from Thanh Hoa has been studied by combination of capilary GC and and found to contain 50 components, 40 of which have been identified. The major ones being beta-caryophyllene (24.6%), y- elemene (14.7%), bicyclogermacrene (12.9%). From the leaves of Zanthoxylum nitidum DC. were isolated stigmasterol and dihydrokaempferit. Its structure were elucidated by EI-MS, 1H-NMR, 13C-NMR
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Plants, Medicinal , Pharmaceutical PreparationsABSTRACT
Study on chemical coposition of essential oil from Zanthoxylum nitidum fruit collected at Me Linh district, Vinh Phuc province by GC/MS method. Results: the yield of essential oil from fruit was 0.35% by air-dry material, 27 constituents were identified, in which the main constituents were linalool L. (72.17%) and sabinen (11.24%)
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Chemistry , Oils, Volatile , ZanthoxylumABSTRACT
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.