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Objective To discuss the proliferation inhibition and apoptosis induction of human pancreatic cancer cell line SW1900 by dauricine and its possible mechanism. Methods The MTT colorimetric method was used to detect the inhibitory effects of cell viability. The apoptosis rate was tested by the Annexin Ⅴ-FITC / PI fluorescent staining of flow cytometric method . The expressions of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and B-cell lymphoma-2 (Bcl-2) were detected by Real-time PCR and Western blotting assay. Results MTT assay showed that dauricine significantly inhibited the proliferation of SW1900 cells and the inhibitory effect was enhanced with the increasing of dauricine concentration, F = 783. 7, P < 0. 001. The apoptosis of 3 groups cells were (4. 34 ± 1. 30) % (0 mg / L dauricine), (14. 94±1. 94) % (6 mg / L dauricine) and (22. 68±3. 61) % (12 mg / L dauricine) . The mean difference was statistically significant among the three groups (F = 58. 52, P < 0. 001) . Dauricine could significantly induce apoptosis human pancreatic cancer cells with dose-dependent manner. Real-time PCR showed that the gene expressions of PI3K, Akt and Bcl-2 were lower obviously (PI3K mRNA, F = 101, P = 0. 01; Akt mRNA, F = 1666, P < 0. 01; Bcl-2 mRNA, F = 753, P<0. 001) with dose-dependent manner. Western blotting assay also showed that the protein expression of PI3K, Akt and Bcl-2 was down-regulated with dose-dependent manner. Conclusion Dauricine has proliferation inhibition and apoptosis inducement effect on human pancreatic cancer cells line SW1900. This function may be concerned with the regulation of PI3K / Akt signal pathway and lower Bcl-2 expression.
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Objective To study the effect of dauricine on the proliferation and apoptosis of hepatoma Huh7 cells, and explore its anti-tumor mechanism and its relationship with Hedgehog signaling pathway. Methods The effects of different concentrations of dauricine (2, 4, 8 μg/mL) on the proliferation of Huh7 cells were detected by MTT assay. Apoptosis of Huh7 cells was analyzed by flow cytometry. Real-time PCR and Western blotting were used to detect the levels of Hedgehog signaling pathway-related genes and proteins. Results With the increase of the concentration of dauricine and the duration of action, the inhibition rate of Huh7 cell proliferation was increased. Among them, 8 μg/mL dauricine had the highest inhibition rate (48.8%) at 48 h. Dauricine induced the apoptosis in Huh7 cells. With the increase of the concentration of dauricine, the apoptotic rate of cells was increased significantly (P < 0.05, 0.01). The mRNA and protein expression levels of PTCH1, GLi1, SMO and SHH genes in Hedgehog signaling pathway were significantly decreased, while the level of cleaved Caspase-3 protein was significantly increased, accompany with the decreased expression of Bcl-2 in dauricine concentration-dependent pattern (P < 0.05, 0.01) in dauricine group compared with the control group. Conclusion Dauricine could significantly inhibit the proliferation and promote apoptosis of Huh7 cells, which may play a role by blocking Hedgehog signaling pathway.
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Objective To establish an HPLC method to determine the contents of dauricine in Menispermi Rhizoma from different producing areas. Methods C18 was set as chromatographic column filler, with acetonitrile-water-triethylamine (45:55:0.1) as the mobile phase, 284 nm as the ultraviolet wavelength detection, 1 mL/min as the flow rate, 30 ℃ as the column temperature. HPLC chromatograms of eight different batches of Menispermi Rhizoma were established. Results HPLC testing conditions of Menispermi Rhizoma was established. Within 20-100 μg/mL, there was a good linear relationship between the injection volume of the reference substance and the peak area (r=0.9995). The average recovery of dauricine was 100.30%, RSD=1.000%. The contents of dauricine in Menispermi Rhizoma from different producing areas were different. Conclusion The HPLC method is with sensitivity, accuracy, precision, good reproducibility and simple operation, which can be used as detection method to determine the content of dauricine in Menispermi Rhizoma.
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Objective The objective of this study was to investigate the effect of dauricine on the sensi-tivity of 5-fluorouracil(5-FU)in human breast cancer MCF-7 cells.Methods MCF-7 cells were treated with 2.5 μg/mL of dauricine,50 μg/mL of 5-FU,or 2.5 μg/mL of dauricine with 50 μg/mL of 5-FU,the cell proliferation was measured by MTT assay.The cell migration was determined by Transwell assay;The cell apopto-sis was detected by DAPI staining;The expression of cyclin D1 and Bcl-2 gene was examined by Western blot. Results The results showed that the combination of subthreshold concentration of dauricine enhanced the inhibi-tory effect of 5-FU on proliferation in MCF-7 cells.The combined use of subcutaneous concentration of dau-ricine further aggravated the inhibitory effect of 5-FU on cell migration.The combination of subcapsular dau-ricine enhanced the induction of apoptosis by 5-FU.The combination of dauricine with 5-FU could inhibit the expression of cyclin D1 and Bcl-2 protein in MCF-7 cells.Conclusion Dauricine can effectively enhance the sensitivity of 5-FU in human breast cancer MCF-7 cells.
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Objective The objective of this study was to investigate the effect of dauricine on the sensi-tivity of 5-fluorouracil(5-FU)in human breast cancer MCF-7 cells.Methods MCF-7 cells were treated with 2.5 μg/mL of dauricine,50 μg/mL of 5-FU,or 2.5 μg/mL of dauricine with 50 μg/mL of 5-FU,the cell proliferation was measured by MTT assay.The cell migration was determined by Transwell assay;The cell apopto-sis was detected by DAPI staining;The expression of cyclin D1 and Bcl-2 gene was examined by Western blot. Results The results showed that the combination of subthreshold concentration of dauricine enhanced the inhibi-tory effect of 5-FU on proliferation in MCF-7 cells.The combined use of subcutaneous concentration of dau-ricine further aggravated the inhibitory effect of 5-FU on cell migration.The combination of subcapsular dau-ricine enhanced the induction of apoptosis by 5-FU.The combination of dauricine with 5-FU could inhibit the expression of cyclin D1 and Bcl-2 protein in MCF-7 cells.Conclusion Dauricine can effectively enhance the sensitivity of 5-FU in human breast cancer MCF-7 cells.
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OBJECTIVE:To study relative bioavailability of dauricine self-microemulsifying drug delivery system (SMEDDS) in rats. METHODS:12 rats were randomly divided into dauricine SMEDDS group (20 mg/kg) and dauricine solution group (50 mg/kg),6 rats in each group. They were given relevant medicine intragastrically. Then,0.3 ml plasma was collected from orbital venous plexus before medication and 0.167,0.333,0.5,0.75,1,2,4,8,12,24,36 h after medication. The plasma concentration of da- uricine was determined by HPLC-MS/MS,and DAS 3.0 was used to calculate pharmacokinetic parameters and evaluate the relative bioavailability of dauricine with dauricine SMEDDS. RESULTS:The linear range of dauricine in plasma were 2.12-424 ng/ml (r=0.999 9);RSDs of intra-day and inter-day were all lower than 10%. Pharmacokinetic parameters of dauricine solution and dau-ricine SMEDDS were that cmax were(126.3±37.4)ng/ml and(179.6±51.5)ng/ml;t1/2 were(11.48±4.58)and(21.79±6.59)h;AUC0-t were (1 963.5±638.3)ng·h/ml and(2 535.8±739.5)ng·h/ml;AUC0-∞ were(2 256.3±703.5)ng·h/ml and(2 854.6± 768.7)ng·h/ml,respectively. The relative bioavailability of dauricine SMEDDS were 323% and 316% by calculating with AUC0-t and AUC0-∞,respectively. CONCLUSIONS:Intragastric administration of dauricine SMEDDS can improve relative bioavailability of dauricine significantly.
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OBJECTIVE: To investigate the absorption characteristics of dauricine in rat intestine. METHODS: In situ single-pass perfusion model was used and the concentrations of dauricine in perfusate were determined by HPLC. The effects of perfusion rates, intestinal segments and drug concentrations on the intestinal absorption of dauricine were studied. RESULTS: The absorption rate and absorption degree of dauricine increased with the perfusion rates(0.1, 0.2 and 0.4 mL · min-1)(P0.05); at high, middle and low concentrations, the drug absorption rate constants(Ka) were (2.36±0.0073) × 10-2, (3.73 ± 0.0052) × 10-2 and(5.62 ± 0.0136) × 10-2 min-1, respectively, the apparent permeation coefficients(P.,) were(2.02±0.0002) × 103, (3.10±0.0007) × 10-3 and (5.31±0.0010) × 10-3 cm · min-1, respectively, the absorption percentages(P%) were 8.66%, 10.17% and 19.06%, respectively, and the accumulate absorption amount and accumulate absorption percentages of different concentrations at different time were very low. CONCLUSION: The absorption degree of dauricine increases with perfusion rates; there is no specific absorption site in the whole rat intestinal tract; the absorption of dauricine is very poor and the active transport is involved in the absorption mechanism of dauricine.
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To study the prevention of dauricine (Dau) on bradykinin (BK) induced alteration of intracellular calcium homeostasis and tau phosphorylation, fluorescence spectrophotometer with dual excitation was utilized to measure the intracellular calcium concentration ([Ca2+]i), MTT to detect cell viability and immuncytochemistry to examine tau phosphorylation. The results showed (1) cells treated with BK 1 μmol/L induced a transit increase in [Ca2+]i in all the cell lines detected, among them, the sustained increase of [Ca2+]i level was only seen in PS1Δ9/APPswe cell at 2 h and 24 h after the treatment. Dau (3μmol/L or 6 μmol/L) prevented BK-induced transit and sustained elevation and fluctuation of [Ca2+]i;(2) BK treatment decreased the cell metabolism detected at 2 h in PS1Δ9/APPswe and Dau antagonized the effect; (3) BK induces Alzheimer-like tau hyperphosphorylation at tau-1 epitope and Dau partially antagonized this effect. In conclusion,Dau inhibits BK-induced disturbance in intracellular calcium homeostasis and tau hyperphosphorylation at tau-1 sites.
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Objective To investigate the inhibitory effect of dauricine on the growth of human bladder cancer T-24 cell and its mechanism.Methods The T-24 cells were treated with dauricine(Dau) at different concentrations.The proliferation of T-24 cells was assayed with MTT colorimetric method.The apoptosis of T-24 cells induced by Dau was studied by fluorescent staining,flow cytometry(FCM) and DNA agarose gel electrophoresis.Results Dau effectively inhibited the proliferation of T-24 cells in a concentration dependent manner.A characteristic DNA ladder was observed in Dau treated groups.Dau at the concentration of 5-30 ?g/ml induced the apoptosis of T-24 cells and the apoptosis rate increased in a time-dependeut manner.Cell cycle was arrested at G_0/G_1 phase.The expression of bcl-2 was inhibited and the expression of bax was up-regulated.Conclusion Dauricine significantly inhibits the growth of bladder cancer cells.Cell cycle arrest and apoptosis might be the functional mechanisms.
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AIM: To investigate the possibility of dauricine (DAU) to inhibit the irreversible platelet aggregation of patients with acute myocardial infarction (AMI). METHODS: Glycoprotein IV (GP IV) and thrombospondin(TSP) levels on the membrane surface of the stationary platelet or platelet activated by thrombin(50,100, 500, 1 000 U·L-1) in 12 patients with AMI were measured with a washed platelet flowcytometric assay and compared with those of the healthy (n = 14). RESULTS: The GP IV, TSP level of stationary platelet and activated platelet in patients with AMI were higher than those in the healthy significantly (P<0.05, 0.01), DAU (50 μmol·L-1) inhibited the GPIV membrane redistribution and the release of TSP from intracellular αgranules induced by thrombin (50 U·L-1, 100 U·L-1, 500 U·L-1) apparently (P<0.05, 0.01), inhibitory effect of DAU was not found in platelets activated with high concentraction of thrombin (1 000 U·L-1). CONCLUSION: The activity and reactivity to thrombin of platelets increased in patients with AMI and DAU may have a blocking effect on the consolidation of platelet aggregation in AMI.
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To establish the determination method of dauricine (Dau) concentration in rats' blood and other biological samples, a reverse-phase HPLC method was adopted. Under the given condition, dauricine could be well separated. The retention time (tR) of Dau and its internal standard,daurisoline were 9.2 and 6.1 respectively. The detection limit was 10-2 mg/ml. The absolute recoveries of all kinds of samples were above 70%, and the relative ones were over 85%. A good liner relationship has been obtained over the entire range of 0.030 to 3.000 mg/L in blood samples and 0.050 to 5.000 mg/L in other tissue samples. The intraday and interday coefficients of variation were below 10%. The results showed that the method can be used for detecting Dau in all kinds of biological samples.
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To establish the determination method of dauricine (Dau) concentration in rats' blood and other biological samples, a reverse-phase HPLC method was adopted. Under the given condition, dauricine could be well separated. The retention time (tR) of Dau and its internal standard,daurisoline were 9.2 and 6.1 respectively. The detection limit was 10-2 mg/ml. The absolute recoveries of all kinds of samples were above 70%, and the relative ones were over 85%. A good liner relationship has been obtained over the entire range of 0.030 to 3.000 mg/L in blood samples and 0.050 to 5.000 mg/L in other tissue samples. The intraday and interday coefficients of variation were below 10%. The results showed that the method can be used for detecting Dau in all kinds of biological samples.
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K21. The mean t1/2(?) was (2.7?0.4) h, Vd was about 11.18 L?kg-1.The C-T profile conformed to two compartment open model. The plasma Dau concentration-time curves showed a double-peak phenomenon in all dosages of all dogswhen dauricine was given by intragastric was.The tpeak(1) was (0.8?0.6) ～(1.2?0.5) h,tpeak(2) was (5.2?3.2) ～(6.5?1.9)h,Cmax(2) 0.05) and the AUC was increased in proportion.The drug is eliminated non-linearly when the dosage is above 50 mg?kg-1, the parameters t1/2(el),CL, AUC/X0 shows great difference (P
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Dauricine ( Dau ) significantly prolonged monophasic action potential duration of 50% and 90% repolarization ( MAPD50 and MAPD90 ) and functional refractory period (FRP)of the hearts in anesthetized cats in dose-dependent manner. At 9 mg/kg of Dau, MAPD50 was increased from 170?19 ms to 197?20 ms; MAPD90 from 216?16 ms to 249?18 ms; FRP from 177 ? 15 ms to 238 ? 20 ms.Dau 5 mg/kg iv followed by constant perfusion for 30 min could prevent the shortening of MAPD50 in the ischemic boundary area ( BA ) and increased FRP in the non-ischemic area ( NA ) , ischemic central area ( CA ) and BA, and markedly reduced the extent of dispersion of FRP of hearts in anesthetized cats. The results suggest that the antagonizing effect of Dau on the arrhythmias caused by acute myocardial ischemia and reperfusion might be related to its action of prolonging FRP and decreasing the dispersion of FRP in ischemic hearts.
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Five min coronary artery occlusion followed by reperfusion in rats could produce arrhythmias which were rapid in onset & of short duration. The incidences of VT & VF being 100% & 72% respectively. Quinidine & dauricine reduced the incidence & severity of VT significantly; and abolished VF. Tetrandrine could reduce the incidence of VF significantly, but it had no significant effect on the incidence of VT.
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The effect of dauricine on level of TXA2 and cAMP in the rabbi platan-elet was studied. The results showed that dauricine protected signific-tly mice against the sudden death induced by intravenous arachidonic acid in vivo, and that dauricine inhibited the platelet aggregation ind uced by arachi- donic acid, inhibited the production of TXA2 in the plateletand did not change the basic cAMP level in the platelet invitro. These suggested that one of the action mechanism of the inhibition of the platelet aggregation by dauricine was possibly related to the inhibition of the production of TXA2.
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Aim To investigate the relation between pharmacokinetics and pharmacodynamics of dauricine in dogs with the combined PK-PD model. Methods The plasma drug concentration was determined by a validated reversed-phase HPLC method that entailed ultraviolet detector and the effects on cardiac electrophsiology; blood pressure and hemodynamics were recorded by polygraph. Results The main pharmacokinetic parameters T_(1/2?),T_(1/2?),V_d, and AUC were (0.049?0.016) h, (2.7?0.6) h, (15.8?3.5) L?kg~(-1),and (1.48?0.17) mg?h?L~(-1),respectively.The maximal increase in Q-Tc intervals was(25.5?9.4)%, whereas the maximal decrease in SBP,DBP,?(dp/dt)_(max) were (23.0?4.9)%,(21.9?5.9)%,(42.8?6.6)%, and(39.0?17.1)%, respectively.The peak effects were detected approximately 10～15 min later than the plasma concentration. Relation between effects and effect compartments was analyzed with the sigmoid-E_(max) model. Conclusion The relation between plasma concentrations,time and effects is established in beagle dogs.
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AIM To investigate the protective effects of dauricine on anoxia and acute cerebral ischemia-reperfusion in mice. METHODS In this study, anoxia was induced by close normobaric hypoxia to search the effects of dauricine on the survival time of mice. The activities of SOD, GSH-Px and levels of MDA in cortex or mitochondria, the activities of cortex LDH and mitochondria ATPase were all measured by ELISA-Reader and spectrophotography. RESULTS ① Dauricine prolonged the survival time of mice in the close normobaric hypoxia, among all doses, Dauricine 60 mg?kg -1 and 120 mg?kg -1 had significant most effects (P
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Dauricine ( Dau ) was studied on the pharmacokinetics in experimental acute renal failure (ARF ) induced by HgCl2 or acute liver injury ( ALI ) induced by CC14 in rabbits.After iv administration of 5 mg/kg in normal rabbits the main pharmacokinetic parameters of Dau were as follows:t1/2=0.18?0.12 (h), t1/2?=3.18?0.95 ( h ) , Vd = 26.7l? 3.61 ( L/kg ) and Cl = 6.91 ? 1.74 ( L/h?kg-1 ). The pharmacokinetic parameters of Dau in ARF were not significantly different from that in normal. Cl (3.31?1.88 L/h?kg-1 ) in ALI was significantly lower than that in normal and t1/2? ( 8.85?4.57 h ) in ALI was considerably longer than that in normal.The results indicated that the pharmacokiaetics of Dau had no change in rabbits with ARF and its elimination was prolonged in rabbits with ALI.
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The effect of dauricine on platelet aggregation and its mechan-ism have been studied in the patients and in rabbits. Dauricine inhibited platelet aggregation induced by ADP and generation of thromboxane B2 significantly, but had no effect on plasma 6-keto-PGF1a concentrations in the patients. It did not influence the levels of cAMP of platelets and plasma in rabbits. Its property of antipla-telet aggregation may relate to the influence on arachidonic acid metabolism.