Mechanisms of Cyclovirobuxine D on APD Prolongation in Rat Ventricular Myocytes

Aim To study the effects of cyclovirobuxine D on inward rectifier K^- current(I_(k1) ) > transient outward K^+ current (I_(to)), L-type Ca^(2+) current (I_(Ca-L)), and actionpotential duration (APD) in isolated rat ventricular myocytes. Methods The whole cell patch-clamptechniques were used to study the changes of I_(k1), I_(to), I_(Ca-L) and APD in rat ventricularmyocytes. Results Cyclovirobuxine D (1-10 μmol·L^(-1)) significantly prolonged APD_(50) andAPD_(90) in isolated rat ventricular myocytes. Resting potential (RP) was decreased by 10μmol·L^(-1) of cyclovirobuxine D. Cyclovirobuxine D significantly decreased both inward andoutward components of I_(k1) . At - 100 mV, 1 and 10 μmol·L^(-1) of cyclovirobuxine D decreasedI_(k1), density from (-8.0+- 1.1) pA/pF to ( - 4.1 +- 0.7) pA/pF and ( - 3.4 +- 0.8) pA/pF,respectively, whereas at - 30 mV, I-(k1) density was decreased from (1.10 +-0.24) pA/pF to (0.61+-0.18) pA/pF and (0.36+- 0.11) pA/pF, respectively. 1_(to) was markedly inhibited bycyclovirobuxine D from the test potential of 0 mV to + 60 mV. At + 40 mV, 1 and 10μmol·L^(-1) ofcyclovirobuxine D decreased I_(to) density from (8.9+- 2.0) pA/pF to (5.5 +- 1.2) pA/pF and (4.9+-0.9) pA/pF, respectively. Cyclovirobuxine D inhibited I_(Ca-L) in a concentration-dependentmanner. At 10 mV, 1 and 10μmol·L^(-1) of cyclovirobuxine D decreased I_(Ca-L) density from ( - 9.9+- 1.8) pA/pF to ( - 6.4 +- 1.4) pA/pF and (-4.2+-0.6) pA/pF, respectively. ConclusionCyclovirobuxine D significantly prolonged APD and inhibited I_(k1), I_(to), and I_(Ca-L) in ratventricular myocytes. The inhibitory effects of cyclovirobuxine D on _(k1) and I_(to) are majormolecular mechanisms of APD prolongation in rat.

Effect of cyclovirobuxine D on human growth-associated protein 43 and neurocan expression in ischemic brain tissue of stroke-prone renovascular hypertensive rats

BACKGROUND： Experimental data indicate that human growth-associated protein 43 mRNA expression coincides with axonal growth during nerve ganglion development; while neurocan, secreted from astrocytes, can inhibit sprouting and elongation of the axonal growth cone. OBJECTIVE： To verify regulatory effects of cyclovirobuxine D （CVB-D） extracted from Chinese box branchlet on human growth-associated protein 43 （GAP-43）, and neurocan expression in brain tissue of stroke-prone renovascular hypertensive （RHRSP） rats, at different time points after cerebral ischemia/reperfusion. DESIGN： Randomized grouping design and controlled animal study. SETTING： This study was performed at the Center of Guangdong Hospital of Traditional Chinese Medicine （a national key laboratory） from March 2003 to September 2006. MATERIALS： 100 healthy male Sprague-Dawley rats, aged 2 3 months and weighing 90-120 g, were selected for this study. CVB-D was provided by Nanjing Xiaoying Pharmaceutical Factory （Batch number： 307701）. METHODS： The initial tip of renal arteries was clamped bilaterally for 10 weeks to establish the RHRSP model. 100 RHRSP rats were randomly divided into 4 groups： naive group （n = 10）, sham surgery group （n = 10）, CVB-D group （n = 40）, and lesion group （n = 40）. Rats in the naive group did not undergo any treatment, and cervical vessels of rats in the sham surgery group were exposed, but not blocked. The right middle cerebral artery of rats in the CVB-D group and lesion group were occluded to establish cerebral ischemia. Rats in the CVB-D group were intraperitoneally injected with CVB-D （6.48 mg/kg） every day and with saline （1.5 mL/injection） twice a day. Rats in the lesion group were intraperitoneally injected with saline （2 mL/injection）. MAIN OUTCOME MEASURES： Immunohistochemistry was applied to detect GAP-43 and neurocan expression in the ischemic penumbra region of CVB-D and lesion brains at 2 hours post-cerebral ischemia and at 1, 7, 14, and 30 days post-perfusion （n = 10 at each time point）. Similarly, GAP-43 and neurocan expression was detected in the right hemisphere of naive and sham-operated animals. The results were expressed as positive cells. RESULTS： A total of 100 rats were included in the final analysis. The number of GAP-43 positive cells increased in the CVB-D group 1, 7, 14, and 30 days post-cerebral ischemia/perfusion compared to the lesion group, as indicated by a significant difference between the CVB-D and lesion group （P 〈 0.054）.01）. The number of neurocan-positive cells decreased in the CVB-D group on the first day compared to the model group; however, there was no significant difference between the two groups （P 〉 0.05）. On post-ischemia days 7, 14, and 30, the number of neurocan-positive cells in the CVB-D group was significantly less than in the lesion group （P 〈 0.05）. Both, GAP-43 and neurocan expression was not detectable in brains of naive and sham-operated animals. CONCLUSION： CVB-D treatment up-regulated GAP-43 expression and down-regulated neurocan expression in the ischemic region of RHRSP rats.

A new horizon for the steroidal alkaloid cyclovirobuxine D(huangyangning)and analogues:Anticancer activities and mechanism of action

The steroidal alkaloid cyclovirobuxine D(Cvb-D)is the active principle of the oral drug huangyangning used for many years in China for the treatment of cardiovascular and cerebrovascular diseases.The drug is listed in the Chinese pharmacopeia.Recent studies have revealed that this unsung alkaloid also displays anticancer properties in vitro and in vivo.The drug activates several signaling pathways,and notably represses phosphorylation of proteins EGFR,ERK,Akt,mTOR.Thereby,Cvb-D exerts antiproliferative and antimetastatic activities.In the present review,the anticancer effects of Cvb-D and related natural products isolated from Buxus species have been analyzed.The molecular targets of Cvb-D are unknown at present,but hypotheses are formulated based on the signaling pathways modulated by the drug and the analogy with other compounds.Proteins EGFR and CTHRC1,implicated in the antiproliferative action of Cvb-D,could be considered as upstream targets.A bolder assumption is also formulated with the metastasis-associated protein S100A4 as a potential co-target for Cvb-D.This review aims to shed light on the anticancer properties of Cvb-D and to encourage further mechanistic studies with this drug with a good safety profile and a recognized anti-cardiovascular efficacy.

Cyclovirobuxine D inhibits blood coagulation and venous thrombosis

Cyclovirobuxine D （CVB-D） is a compound extracted from Chinese traditional plant Buxus microphylla, which has been used for treating arrhythmia and myocardial ischemia in China. In this study, we investigated its effect on blood coagulation and thrombotic formation in mouse and rat models. The doses of CVB-D used in this study （5-20 mg/kg） prolonged clotting time （CT） in a dose-dependent manner （P〈0.01）. It also significantly prolonged thrombin time （TT）, prothrombin time （PT） and activated partial thromboplast time （aPTT） （P〈0.05 or P〈0.01） at the doses of 10-20 mg/kg. CVB-D did not affect the bleeding time （BT） compared with the control group, while warfarin significantly prolonged the bleeding time. CVB-D at the doses of 5-20 mg/kg reduced wet weight of thrombosis （P〈0.01）. This study demonstrated the anti-coagulation effect and anti-thrombosis effect of orally administered CVB-D without substantially increasing bleeding. These findings suggest that CVB-D probably can be used as an oral anti-coagulant in addition to its current applications.

Cyclovirobuxine D inhibits dengue virus replication by impeding the complete autophagy in a cholesterol-dependent manner

Dengue virus(DENV)is the most common mosquito-borne flavivirus,and it affects millions of people globally every year.Currently,there are no approved drugs for the treatment of dengue infection.By screening a natural product library,we identified a novel compound,cyclovirobuxine D(Cvb D),that displays anti-DENV activity.Cvb D inhibits DENV replication in vitro in a dose-dependent manner and protects suckling mice against lethal DENV infection.Mechanistically,Cvb D regulates the expression of genes related to the cellular cholesterol pathway.As a result,Cvb D increases cellular cholesterol synthesis and accumulation,activates mTOR,and inhibits viral-dependent autophagy.Cvb D does not suppress autophagy initiation but impedes the nuclear translocation of the lysosome transcription factor TFEB.In addition,Cvb D restricts the replication of other positive-strand RNA viruses such as Zika virus and Coxsackievirus B3.We speculate that Cvb D could be a broad-spectrum antiviral drug candidate for use against positive-strand RNA viruses that require autophagy for optimal replication.

Determination of cyclovirobuxine D in human plasma by liquid chromatography tandem mass spectrometry and application in a pharmacokinetic study

A sensitive and reliable method based on liquid chromatography tandem mass spectrometry(LC–MS/MS)for the quantitation of cyclovirobuxine D in human plasma has been developed and validated.Sample preparation by solid phase extraction was followed by separation on a CN column with a mobile phase of methanol–water(95:5,v/v)containing 0.2%formic acid.Mass spectrometric detection in the positive ion mode was carried out by selected reaction monitoring(SRM)of the transitions at m/z 403.0→372.0 for cyclovirobuxine D and m/z 325.0→234.0 for citalopram(internal standard).The method was linear in the range 10–200 ng/L with LLOQ of 10 ng/L,recovery >85%,and no significant matrix effects.Intra-and inter-day precisions were all <9% with accuracies of 94.0–104.8%.The method was successfully applied to a pharmacokinetic study involving a single oral administration of a 2 mg cyclovirobuxine D tablet to twenty-two healthy Chinese volunteers.

黄杨宁片溶出度测定方法研究及溶出行为评价

目的:建立黄杨宁片的溶出度方法,并对不同厂家样品进行溶出曲线评价。方法:采用反相HPLC法,使用Agela Durashell RP C18色谱柱(4.6 mm×250 mm,5μm)分离,以0.01 mol·L^-1庚烷磺酸钠与0.01mol·L^-1磷酸二氢钾等量混合水溶液(含0.2%三乙胺,用磷酸调节pH至3.5)-乙腈(77∶23)为流动相,流速1.0 mL·min^-1,检测波长206 nm;溶出度试验采用小杯法,以150 mL的0.1 mol·L^-1盐酸溶液为溶出介质,转速75 r·min^-1。结果:环维黄杨星D进样量在0.025 5~4.084 8μg范围内线性关系良好(r=1.000);平均回收率为97.6%(n=6);不同厂家黄杨宁片的溶出行为有较大差异。结论:建立的HPLC溶出度测定方法简单灵敏可靠,能较好地体现不同厂家样品间的溶出差异。