Mechanism of nitidine chloride inducing apoptosis of cervical cancer cells by inhibiting p53 ubiquitination degradation / 中国药理学通报

Aim To investigate the effect of nitidine chloride (NC) on the apoptosis of cervical cancer cells and its mechanism. Methods Cervical cancer cell lines HeLa and SiHa were selected as subjects. The cytotoxicity of NC and its inhibitory effect on cell growth were detected by CCK-8 assay and clone formation assay. The effect of NC on the apoptosis of cervical cancer cells was detected by TUNEL assay, and the expression of apoptosis-related proteins was detected by Western blot. The effects of NC on the interaction between p53 and E6AP protein, the level of p53 ubiquitination modification and the stability of p53 protein in cervical cancer cells were analyzed by immunoprecipi-tation assay, ubiquitination assay and Western blot assay. Results NC could significantly inhibit the proliferation and induce apoptosis of cervical cancer cells. NC could inhibit the interaction between tumor suppressor protein p53 and E6AP in cervical cancer cells, reduce the level of p53 ubiquitination modification, delay the degradation of p53 and increase the expression level of p53 protein. Conclusions NC can inhibit the ubiquitination and degradation of p53, improve the expression level of p53 protein, restore its tumor suppressor function, and thus play an anti -cervical cancer role.

Apoptosis of small cell lung cancer cells H1688 and H446 induced by nitidine chloride through PI3K/Akt/Bcl-2/caspase-3/PARP pathway / 中国药理学通报

Aim To explore the apoptosis of small eell lung eancer ( SCLC ) eells HI688 and H446 induced by nitidine chloride and its possible mechanism.Methods The effect of nitidine chloride or cisplatin ( DDP ) on the activity of SCLC cells was detected by j J MTT method; the morphological changes of cells trea¬ted with nitidine chloride or DDP were observed by in- verted fluorescence microscope and HE staining; the effect of nitidine chloride or DDP on apoptosis was de¬tected by flow cytometry; the effect of apoptosis inhibi¬tor Z-VAD-FMK on apoptosis induced by nitidine chlo¬ride or DDP was detected by MTT method.The expres¬sions of Bax , Bcl-2, caspase-3 , PARP, p-PI3K and p- Akt in the cells treated with nitidine chloride or DDP were detected by Western blot.Results MTT results showed that the viability of SCLC cells was significantly reduced after 48 hours of treatment with nitidine chlo¬ ride; compared with DDP, nitidine chloride could in¬hibit SCLC cells with less IC50; inverted fluorescence microscope and HE staining showed that nitidine chlo¬ride could induce apoptosis in SCLC cells, similar to DDP; flow cytometry showed that nitidine chloride J J could induce apoptosis in SCLC cells.The results of MTT assay showed that the inhibitory effect of nitidine chloride on apoptosis of SCLC cells could be partially antagonized by apoptosis inhibitor Z-VAD-FMK.West¬ern blot results showed that, similar to DDP, nitidine chloride could inhibit the expression of PI3K and Akt, increase Bax, inhibit Be 1-2, and promote the cleavage of caspase-3 and PAH P.Conclusion Nitidine chlo¬ride can induce apoptosis of SCLC cells by inhibiting the activation of P13K and Akt.

Research advances on the chemical components and pharmacological activities of Zanthoxyli Radix / 药学学报

Zanthoxyli Radix is a traditional Chinese medicine. It can be used for the treatment of wind-cold-dampness arthralgia, muscle and bone pain, fall fracture, hernia, sore throat, toothache and other diseases. Due to possessing many excellent and mild pharmacological properties, there are lots of reports about Zanthoxyli Radix worldwide. At present, more than 100 bioactive components have been extracted and purified from Zanthoxyli Radix. Nitidine chloride (NC), one of the most important alkaloids in Zanthoxyli Radix, has the activities of anti-tumor, anti-inflammation, anti-bacteria, etc. In this review, we summarize the chemical components of Zanthoxyli Radix, pharmacological activity and mechanism of action of NC to provide references for further research and utilization of Zanthoxyli Radix.

Accumulation of nitidine chloride in rat heart and the underlying mechanism / 药学学报

Nitidine chloride (NC) is a compound with prominent anti-tumor activity. To determine potential cardiotoxicity of NC, this study was designed to investigate the distribution of NC in rat heart and the underlying mechanism. The animal studies were approved by Institutional Animal Care and Use Committee of Zhejiang University Medical Center (2015-380-01) and complied with the standards of animal welfare in China. At 0.25, 0.5 and 2 h after a single intravenous injection (iv) of 5 mg·kg-1 NC, the concentrations of NC in rat heart were 47.7, 71.1 and 63.2 μg·g-1 respectively, which were 576, 1 352 and 1 212 folds of that in plasma. This study also revealed that the NC concentration in heart was 458.5 μg·g-1 (7 336 folds of that in plasma) at 2 h after the last dose in rats, after daily iv administration of NC at 5 mg·kg-1·day-1 for successive 20 days. Further studies showed that the accumulations of NC in MDCK-hOCT1 and MDCK-hOCT3 cells were 16.1 and 4.99 folds higher than that of the mock cells, respectively. There is no significant difference between the accumulations of NC in MDCK cells transfected with hOCTN1, hOCTN2 or hPMAT and the mock cells. Additionally, quinidine, L-tetrahydropalmatine and Decynium 22, the inhibitors of OCTs, clearly reduced the accumulations of NC in primary cardiomyocytes and cardiac fibroblasts from neonatal rats. MTT assay showed that the LC50 of NC on cardiomyocytes and cardiac fibroblasts were 10.9 and 10.4 μmol·L-1, respectively. Moreover, treatment of the primary cardiomyocytes and cardiac fibroblasts with NC (1～15 μmol·L-1) for 48 h resulted in significantly increased LDH enzyme leakage. These results indicated that NC can be highly accumulated in the heart, and accumulation is mediated by OCT1 and OCT3, but not by OCTN1, OCTN2 and PMAT. The accumulated NC has potential cytotoxicity as shown in the results from primary cardiomyocytes and cardiac fibroblasts.

Inhibitory effect and mechanism of nitidine chloride on human esophageal cancer Eca109 cells / 中草药

Objective: To investigate the inhibitory effect of nitidine chloride (NC) on human esophageal carcinoma cell line Eca109 and the molecular mechanism of its induction. Methods: CCK-8 method was used to detect the inhibition rate of human esophageal cancer Eca109 cells with different concentrations of NC and different intervention time. According to the result of CCK-8 method, the experiment was divided into four groups, and the concentrations of NC in each group were 0, 5, 10, and 15 μmol/L, respectively, and the drug action time was 48 h. The apoptotic rate was detected by flow cytometry with Annexin V-FITC/PI double staining. The mRNA expressions of Caspase-3, Caspase-9, and Noxa were detected by qRT-PCR. The expressions of cleaved Caspase-3 and Bcl-2, p53, and Noxa protein levels were detected by Western blotting. Results: NC had inhibitory effect on Eca109 cells in a certain range of time and dose-dependent manner. Flow cytometry showed that NC at 5 μmol/L mainly induced early apoptosis (P < 0.01); NCs at 10 and 15 μmol/L mainly induced late apoptosis (P < 0.01). qRT-PCR results showed that the mRNA expression of Caspase-3, Caspase-9 and Noxa was increased with the increase of NC concentration, of which 10 and 15 μmol/L group increased significantly. The results of Western blotting showed that the protein levels of cleaved Caspase-3, p53, and Noxa were both increased with the increase of NC concentration (P < 0.01), but the increase of Noxa was not significant in 5 μmol/L group (P < 0.01). The expression of Bcl-2 protein was decreased with the increase of NC concentration, and which was significantly higher in 10 and 15 μmol/L groups (P < 0.05, P < 0.01). Conclusion: The inhibitory effect of NC on human esophageal carcinoma Eca109 cells is mainly through apoptosis. The apoptosis of NC induced of Eca109 cells is associated with increased expression of p53 and Noxa, downregulation of Bcl-2, and activation of Caspase-3.

Optimization of purification process for total alkaloids from Longzuan Tongbi Recipe with macroporous resin by central composite design and response surface method / 中草药

bjective To optimize the optimal purification technology for total alkaloids from Longzuan Tongbi Recipe (LTR). Methods Five types of macroporous resins were used to adsorb and desorb the total alkaloids in LTR, and the adsorption capacities, adsorption ratios and desorption ratios of total alkaloids were regarded as the indexes to screen out the suitable resin. The purification technology for total alkaloids was optimized from LTR by single factor investigation and central composite design and response surface method. Results The best purification technology of total alkaloid from LTR were determined as follows: the concentration of sample solution of 0.17 g/mL (crude drug) with pH 1.7, sample flow rate at 1 mL/min, washing impurity with 9-column volume of 80% ethanol at a flow rate of 1 mL/min. The mass scores of total alkaloids was 22.23% and the yield of that was 173.27 mg/g. Conclusion HPD100 resin has good purification effect on total alkaloids from LTR, which could provide reference for the further study of pharmacodynamics.

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.

Simultaneous determination of four effective components in Toddalia asiafica(L.)Lam by RP-HPLC / 中国药学杂志

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.

Determination of Nitidine Chloride, 5-Ethoxychelerythrine, Bergeninum and Ardisiacrispin A in Shangtong Tinctures by HPLC-DAD-ELSD / 中国药师

Objective:To develop an HPLC-DAD-ELSD method for the determination of nitidine chloride, 5-ethoxychelerythrine, bergeninum and ardisiacrispin A in Shangtong tinctures ( STD) . Methods: A Hypersil C18 column was used as the chromatographic column, the flow rate was 0. 8 ml·min-1 . For nitidine chloride and 5-ethoxychelerythrine, the mobile phase A consisted of acetoni-trile,the mobile phase B consisted of 0. 1% formic acid-triethylamine (pH 4. 5),and the DAD detection wavelength was at 273 nm. For bergeninum and ardisiacrispin A, the mobile phase consisted of methanol-water(25∶75), the temperature of drift tube was set at 95℃, and the gas flow (N2) was set at 2. 5 SLPM·min-1. Results:There was a good linear relationship between the concentration and peak area for nitidine chloride and 5-ethoxychelerythrine within the range of 0. 021-0. 426 μg (r=0. 999 5) and 0. 075-1. 494 μg (r=0. 999 8), respectively. The average recovery was 99. 22%(RSD=0. 64%) and 98. 61%(RSD=0. 46%), respectively. There was a good linear relationship between the concentration and peak area for bergeninum and ardisiacrispin A within the range of 0. 215-4. 304 μg(r=0. 999 3) and 0. 286-5. 728 μg(r=0. 999 7), respectively. The average recovery was 99. 15%(RSD=0. 77%) and 99. 25%(RSD=0. 56%) accordingly. Conclusion:The method is accurate, sensitive and reproducible, and can be used in the de-termination of nitidine chloride, 5-ethoxychelerythrine, bergeninum and ardisiacrispin A in STD.

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.

Reaction between antineoplastic nitidine chloride and sodium hydroxide solution / 第二军医大学学报

Objective: To challenge the previously proposed anticancer mechanism of nitidine chloride, which suggest there exist anti tumor C(6)=N(5) double bond structure. Methods: An antitumor alkaloid, nitidine chloride, was treated in aqueous sodium hydroxide solution without the presence of certain kinds of amino acids and oxygen. Results and Conclusion: It had been revealed that virtually equal amount of 5, 6 dihydronitidine and oxynitidine was produced under such circumstances. The results suggest the anticancer mechanism of nitidine chloride need further investigation. [

HPLC Fingerprinting Analysis and Qaulity Assessment of Herbal Drug Radix Zanthoxyli nitidii / 中药新药与临床药理

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.

Protection in vitro of aFGF on human liver cell L-02 and human kidney cell 293 caused by nitidine chloride / 中成药

AIM: To detect the inhibition effect of nitidine chloride on proliferation of human liver cell L-02 and human kidney cell 293 and the protective effect of acidic fibroblast growth factor(aFGF) on human liver cell L-02 and human kidney cell 293 damaged by nitidine chloride in vitro. METHODS: The MTT assay was used to assess the proliferation of human liver cell L-02 and human kidney cell 293 treated with nitidine chloride.The contents of SOD and MDA and LDH in cultural supernate were measured by ultraviolet spectrophotometry. RESULTS: Nitidine chloride inhibited the proliferation of human liver cell L-02 and human kidney cell 293 in a dose-dependent manner. CONCLUSION: Nitidine chloride has certain toxicity on human liver cell L-02 and human kidney cell 293.aFGF could protect human liver cell L-02 and human kidney cell 293 damaged by nitidine chloride.