Effect of Koumine on Proliferation and Apoptosis of Colorectal Cancer Cells via ROS/JNK/FoxO3a Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the effect and underlying mechanism of koumine （Kou） at different concentrations （0， 100， 200， 400 μmol·L^-1） on the proliferation and apoptosis of colorectal cancer HCT-116 cells. Method:After 24 hours of<italic> in vitro</italic> intervention with HCT-116 cells by Kou， cell counting kit-8 （CCK-8） assay was used to detect its effect on cell proliferation. Flow cytometry was used to detect cell cycle， apoptosis， and reactive oxygen species （ROS） expression. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of forkhead box O3a （FoxO3a）. Cells were transfected with small interfering ribonucleic acid （siRNA）. Western blot was employed to detect the protein expression of the FoxO3a target gene. Result:Compared with the conditions in the blank group， Kou treatment reduced the proliferation rate of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01） in a dose-dependent manner， caused cell cycle arrest in the G₀/G₁ phase， and induced the apoptosis of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01）， which was positively correlated with the concentration of Kou. FoxO3a siRNA interference reduced the expression of FoxO3a and its downstream target genes cyclin-dependent kinase inhibitor 1A （p21）， cyclin-dependent kinase inhibitor 1B （p27）， and Bcl-2 interacting mediator of cell death （Bim） （<italic>P</italic><0.01）. Kou treatment induced the activation of c-Jun <italic>N</italic>-terminal kinase （JNK） in HCT116 cells. SP600125 （JNK specific inhibitor） treatment inhibited the Kou-induced FoxO3a activation and the expression of its downstream target genes. <italic>N</italic>-acetyl cysteine （NAC） treatment reduced Kou-induced ROS levels （<italic>P</italic><0.01） and JNK signal activation. The above results were significantly different from those in the blank group （<italic>P</italic><0.01）. Conclusion:Kou can effectively inhibit the proliferation of HCT-116 cells and promote apoptosis， and the mechanism may be related to the regulation of the ROS/JNK/FoxO3a pathway.

Simultaneous Quantitative Analysis of Koumine, Gelsemine and Gelsenicine in Biological Samples by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a LC-MS/MS method which is accurate and sensitive for determination of koumine, gelsemine, and gelsenicine in biological samples and to verify the method.@*METHODS@#Strychnine was used as internal standard. Analytes in blood, urine and liver with 1% sodium hydroxide solution were extracted by ethyl acetate. Chromatographic separation was achieved on a ZORBAX SB-C₁₈ column （150 mm×2.1 mm, 5 μm）, and gradient elution was performed with the buffer solution of methanol-20 mmol/L ammonium acetate （including 0.1% formic acid and 5% acetonitrile） as mobile phase. Qualitative and quantitative analysis was performed in the multiple reaction monitoring mode coupled with an electrospray ionization source under positive ion mode（ESI⁺）.@*RESULTS@#The linearity of koumine, gelsemine and gelsenicine in blood, urine and liver was good within corresponding linear limitation and the correlation coefficients （r）>0.995 0. The limits of detection were 0.1 ng/mL （0.1 ng/g）, 0.1 ng/mL （0.1 ng/g） and 0.01 ng/mL （0.01 ng/g）, respectively. The extraction recovery and accuracy of the alkaloids ranged from 61.9% to 114.6% and 92.4% to 114.3%, respectively. The relative standard deviations of the intra-day and inter-day precisions were not more than 11.0%.@*CONCLUSIONS@#The method is selective, sensitive and suitable for simultaneous determination of koumine, gelsemine and gelsenicine in body fluids and tissues, which offering technical support for clinical diagnosis and treatment and forensic toxicological analysis of Gelsemium elegans poisoning.

Effect of koumine on the inflammation of RAW264.7 cells induced by LPS / 中国兽医学报

To reveal the anti-inflammation mechanism of koumine,we determined effects of different concentrations of koumine(100,200,400 mg/L) on the secretion of NO,IL-1β,IL-6 and TNF-a by nitrate reductase and ELISA as well as the mRNA of iNOS,IL-1β,IL-6 and TNF-α.Western blot was used to detect iNOS protein expression.The results showed that 100,200,400 mg/L of koumine can significantly inhibit the secretion of NO,IL-1β,IL-6 and TNF-α of RAW264.7 cell (P＜0.01).Koumine can dose-dependently down-regulate the mRNA of iNOS,IL-1β,IL-6 and TNF-α,meanwhile koumine can also significantly inhibit the protein expression of iNOS.The results indicated that koumine may play an anti-inflammation activity by mean of the reduction of NO and mediator of inflammation,down-regulating the mRNA expression of iNOS,IL-1β,IL-6 and TNF-a,decreasing protein expressions of iNOS.

Determination of gelsemine and koumine in human plasma by HPLC-UV / 中草药

Objective: To establish the methods for determination of gelsemine (GM) and koumine (KM) in human plasma. Methods :GM and KM in human plasma were extracted by SPE tubes and determined by HPLC-UV. The chromatographic conditions were as following: The column was Shim-Pack C18 (250 mm × 4.6 mm, 5 μm); The mobile phase consisted of methanol, water, and dibutylamine (58∶42∶0.01); The flow rate was 1.0 mL/min; The injection volume was 50 μL; Column temperature was 30℃; Detective wavelength was set at 263 nm. Results: GM and KM were in good linearity in 0.05-20 μg/mL (n = 6, r = 0.999 4) and 0.05-20 μg/mL (n = 6, r = 0.999 5), respectively. The limits of detection for GM and KM were both 50 ng/mL (S/N ≥ 10), respectively. The average recoveries of extraction for GM and KM were 90.88% and 87.84%, respectively. The average recoveries of method for GM and KM were 98.65% and 96.31%, respectively. Average inter-day RSD values for GM and KM were 9.81% and 10.63% as well as 7.79% and 8.24% in intra-day RSD, respectively. Conclusion: The established method for the determination of GM and KM in human plasma by SPE is sensitive, simple, accurate, reliable, and suitable for pharmacokinetics and toxicity study on Gelsemii Elegantis Herba.

An Experimental Study on Anti-stress Effect of Koumine on Mice / 广州中医药大学学报

【Objective】To observe the anti-stress effect of koumine(Kou)on mice.【Methods】Kunming mice were randomized into model group,low-,moderate-and high-dose koumine(1.2,2.4,and 4.8 mg?kg-1?d-1)groups.The treatment lasted 7 days.One hour after last administration,stress tests such as weight-bearing swimming,antihypoxia,high-temperature resistance and low-temperature resistance were carried out.The changes of serum superoxide dismutase(SOD)activity and malondialdehyde(MDA)content were also observed.【Results】During the anti-stress tests,the survival time was prolonged in koumine groups as compared with the model group(P0.05).【Conclusion】Koumine can increase the mice tolerance of weight-bearing swimming,cold and hypoxia,and its anti-stress mechanism may be related to the antilipid peroxidation.