Permeability of rhynchophylline across human intestinal cell in vitro

Rhynchophylline (Rhy) is the major component of Uncaria species, which is used in Chinese traditional medicine for the treatment of central nervous system disorders. However, its oral bioavailability has not been known. This study aims to investigate the intestinal permeability and related mechanisms of Rhy using cultured human epithelial Caco-2 cells. The cytotoxicity of Rhy on Caco-2 cells was evaluated with MTT assay. The effect of Rhy on the integrity of Caco-2 cell monolayer was assayed with transepithelial electrical resistance. The permeability of Rhy across cell monolayer was assayed by measuring Rhy quantity in received side with HPLC. The effect of Rhy on the expression of P-glycoprotein and MDR1 was detected with Western blot and flow cytometry, respectively. In the concentration of Rhy, which did not produce toxicity on cell viability and integrity of Caco-2 cell monolayer, Rhy crossed the monolayer with velocity 2.76~5.57×10^-6 cm/sec and 10.68~15.66×10^-6 cm/sec from apical to basolateral side and from basolateral to apical side, respectively. The permeability of Rhy was increased by verapamil, a P-glycoprotein inhibitor, or rhodamine123, a P-glycoprotein substrate. Rhy revealed an induction effect on P-glycoprotein expression in Caco-2 cells. These results demonstrate the low permeability of Rhy in intro, and suggest that P-glycoprotein may underlie the mechanism.     

候钩藤总碱含量测定及其药理作用的初步研究

目的检测候钩藤的总碱含量,探讨其中枢药理作用,比较其与中药钩藤的差异。方法采用紫外分光光度法检测候钩藤总碱的含量;采用小鼠自主活动实验及戊巴比妥钠诱导催眠实验,观察候钩藤的镇静催眠作用;通过士的宁致小鼠惊厥实验,观察候钩藤的抗惊厥作用。结果紫外分光光度法检测到候钩藤带钩茎枝中总生物碱的含量为0.038%(g/g),钩藤的带钩茎枝的总碱含量为0.207%(g/g)。药理实验结果表明,候钩藤提取物能减少小鼠自主活动次数,增加戊巴比妥钠诱导小鼠入睡个数,并延长小鼠睡眠时间,减少士的宁致小鼠惊厥死亡数。候钩藤高剂量(20 g/kg)组的中枢抑制作用较显著,与钩藤组(15 g/kg)无明显差异(P>0.05),但候钩藤中、低剂量组的药理作用与钩藤组有显著差异(P<0.05或P<0.01)。结论候钩藤总碱含量较钩藤低。候钩藤具有一定程度的镇静催眠和抗惊厥作用,其作用强度与剂量有一定关系。     

钩藤碱对缺血再灌注诱导大鼠星形胶质细胞损伤的作用

目的:以原代培养的大鼠大脑星形胶质细胞进行缺血再灌注损伤处理,观察钩藤碱(Rsy)对星形胶质细胞损伤的保护作用.方法:1～3 d龄SD大鼠乳鼠大脑星形胶质细胞原代培养,含连二亚硫酸钠的无糖Earle's液进行缺血缺氧处理造模,MTT法测定细胞存活率,Hoechst 33258荧光显微镜观察细胞形态学变化,流式细胞仪检测细胞坏死、凋亡率,用LDH试剂盒测定LDH漏出率.结果:与模型组比较,钩藤碱0.02、0.2 mg·mL-1均能显著提高细胞存活率,显著降低细胞坏死率和细胞凋亡率,也能显著降低细胞LDH漏出率.结论:钩藤碱对缺血再灌注损伤后星形胶质细胞的坏死凋亡具有显著抑制作用,提示钩藤碱可能通过抑制星形胶质细胞凋亡和坏死而对脑缺血损伤产生保护作用.     

HPLC法测定中药钩藤中钩藤碱和异钩藤碱的含量

目的：建立高效液相色谱法测定中药钩藤中有效成分钩藤碱和异钩藤碱的含量。方法：色谱柱Lichrospher C18（4.0＆#215;250 mm,5μm）,流动相：甲醇：水（含10 mmol/L的三乙胺,冰醋酸调节pH值为7.4-7.6）=55：45,检测波长为254 nm,加入1%盐酸10μL作为改性剂。温度为25℃,测定钩藤碱和异钩藤碱的线性关系方程。结果：钩藤碱与异钩藤碱的回归方程分别为Y=9.2×10^4-0.281×10^4,Y=7.6×10^4-0.375×10^4,线性范围分别为0.100-1.60μg/mL（r=0.9999）,0.211-1.379μg/mL（r=0.9998）,钩藤碱的平均回收率为99.154%,异钩藤碱的平均回收率为99.40%。结论：HPLC法测定中药钩藤中两种成分的含量,方法简单、快速,精密度及稳定性好,可以用于控制中药材钩藤的品质。     

复方连翘口服液中钩藤碱的鉴定研究

目的鉴定钩藤碱为复方连翘口服液中的主要成分。方法用碱化醚提取法提取复方连翘口服液中的主要成分钩藤碱与生药钩藤之钩藤碱。再用熔点测定、光谱分析等鉴定两钩藤碱的特征。结果与结论两碱性的理化检测鉴定结果显示:从性状、熔点、薄层板析斑点、与碘化铋钾的反应、光谱分析的特征峰及化学结构均呈现完全一致。     

钩藤碱和异钩藤碱对豚鼠心房的负性变时和变力作用

钩藤碱(Rhy)及异钩藤碱(Isorhy)呈剂量依赖性减慢右心房自发节律,且不被Atr阻断,二药非竞争性地拮抗Iso和组胺正性变时作用,其pD_2值分别为Iso 5.45和5.57,H 5.28和5.48,并能取消CaCl_2的正性变时效应,Rhy和Isorhy还剂量依赖性地抑制心房收缩力,在1μmol·L~(-1)普萘洛尔存在下,二药对苯肾上腺素正性变力作用的量效曲线是非竞争性拮抗,其pD_2值分别为43.6和4.43.Rhy和Isorhy 0、3mmol·L~(-1)能显著抑制左心房静息后增强效应和阶梯现象.结果提示,Rhy和Isorhy的负性变时和变力作用,似与二药抑制心肌细胞膜Ca~(2+)转运有关。     

Celastrol and Rhynchophylline in the mitigation of simulated muscle atrophy under in vitro

BackgroundMuscular atrophy (MA) is a disease of various origins, i.e., genetic or the most common, caused by mechanical injury. So far, there is no universal therapeutic model because this disease is often progressive with numerous manifested symptoms. Moreover, there is no safe and low-risk therapy dedicated to muscle atrophy. For this reason, our research focuses on finding an alternative method using natural compounds to treat MA. This study proposes implementing natural substances such as celastrol and Rhynchophylline on the cellular level, using a simulated and controlled atrophy process. Methods: Celastrol and Rhynchophylline were used as natural compounds against simulated atrophy in C2C12 cells. Skeletal muscle C2C12 cells were stimulated for the differentiation process. Atrophic conditions were obtained by the exposure to the low concertation of doxorubicin and validated by FoxO3 and MAFbx. The protective and regenerative effect of drugs on cell proliferation was determined by the MTT assay and MT-CO1, VDAC1, and prohibitin expression. Results: The obtained results revealed that both natural substances reduced atrophic symptoms. Rhynchophylline and celastrol attenuated atrophic cells in the viability studies, morphology analysis by diameter measurements, modulated prohibitin VDAC, and MT-CO1 expression. Conclusions: The obtained results revealed that celastrol and Rhynchophylline could be effectively used as a supportive treatment in atrophy-related disorders. Thus, natural drugs seem promising for muscle regeneration.     

潜阳降压胶囊的鉴别及含量测定研究

目的：探讨潜阳降压胶囊的质控标准。方法：用沉淀显色法鉴别方中钩藤碱及钙盐；用薄层色谱法鉴别白芍及玄参；用容量法测定钩藤碱的含量。结果：方法简便，重现性好，质量可控。结论：可作为潜阳降压胶囊制订质量标准的参考依据。     

高效液相色谱法测定萝藤安神片中钩藤碱的含量

目的:建立藤安神片中钩藤碱的HPLC测定方法。方法:Diamonsil ODS C18(200mm×4．6 mm,5μm)柱,甲醇-水(48:52)含10 mmol·L-1三乙胺溶液,用醋酸调pH=5．0为流动相,流速1．0 ml·min-1检测波长为254 nm。结果:钧藤碱的线性范围是0．20-0．99μg(r=0．999 8),平均回收率为97．9％,RSD=1．0％。结论:方法简单准确,可作为该制剂的质量控制。     

采用HPLC-MS-AIS法测定钩藤中四种生物碱含量

目的 采用高效液相-质谱联用-自体内标法（HPLC-MS-AIS）测定钩藤中四种主要的吲哚类生物碱（钩藤碱、异钩藤碱、去氢钩藤碱、异去氢钩藤碱）含量。方法 采用C₁₈色谱柱（3.0 mm×50 mm，3.3 μm）；0.1%甲酸水溶液∶乙腈=82∶18为流动相；流速0.5 mL/min；柱温30℃。质谱条件：电喷雾离子源；正离子多反应监测模式；毛细管电压为4 000 v；选择质子数/电荷数（m/z）：385.25/160.10（钩藤碱）、385.30/160.10（异钩藤碱）、383.25/160.15（去氢钩藤碱）、383.25/160.15（异去氢钩藤碱）作为检测离子对；进样量2 μL。结果 钩藤碱、异钩藤碱、去氢钩藤碱、异去氢钩藤碱线性范围分别为2.30~600.00 ng/mL（r=0.999 3）、2.30~600.00 ng/mL（r=0.999 2）、2.47~650.00 ng/mL（r=0.999 4）、2.47~650.00 ng/mL（r=0.999 2），精密度和稳定性的相对标准偏差（RSD）均<5.00%，准确度为92.40%~104.10%，加样回收率为95.90%~104.60%。结论 采用HPLC-MS-AIS法测定钩藤中四种生物碱含量简便准确，可作为钩藤药材质量控制的新方法。