高效毛细管电泳法测定钩藤中的钩藤碱

目的分离钩藤碱与异钩藤碱,测定钩藤中的钩藤碱。方法采用高效毛细管电泳法,运行缓冲液为0.05 mol.L-1硼砂溶液(pH6.9)-0.02 mol.L-1羟丙基-β-环糊精和0.05 mo.lL-1胆酸钠的甲醇溶液(85:15),操作电压为12 kV,温度为40℃,检测波长为250 nm。结果钩藤碱0.025~0.496 mg.ml-1与峰面积呈良好的线性关系(r=0.9998),加样回收率为99.8%,RSD=1.91%。结论所用方法可快速有效地分离钩藤碱及异钩藤碱,并测定植物中的钩藤碱。     

钩藤中钩藤碱、异钩藤碱的提取与含量测定在预后中的应用分析

目的:探讨钩藤中钩藤碱、异钩藤碱的提取与含量测定在预后中的应用。方法:采取水煎液以及甲醇提取液的方式对钩藤中的钩藤碱及异钩藤碱进行提取,并使用高效液相色谱系统分别对其提取的含量进行测定。结果:在2.5~80μg/m L的范围内钩藤碱的线性关系良好,此时的回归方程为Y=76.717 0X-0.072 7(r=0.999 8),RSD8.7%;在2.0~80μg/m L的范围内异钩藤碱的线性关系良好,此时的回归方程为Y=87.472 9X-0.366 6(r=0.999 7),RSD7.3%。甲醇提取液的方式所提取钩藤碱和异钩藤碱的含量高于水煎液的方式,并且使用二十倍质量的甲醇量,24 h的冷侵时间以及60 min的超声时间最合适。结论:使用甲醇进行提取在用于钩藤中钩藤碱及异钩藤碱中作用优异,对其质量的控制性好,重复性高,操作方法简单,可提高预后,值得在临床上应用推广。     

Isorhynchophylline ameliorates cognitive impairment via modulating amyloid pathology,tau hyperphosphorylation and neuroinflammation: Studies in a transgenic mouse model of Alzheimer’s disease

Isorhynchophylline (IRN) has been demonstrated to have distinct anti-Alzheimer’s disease (AD) activity in several animal models of AD. In this study, we aimed at evaluating the preventive effect of IRN on the cognitive deficits and amyloid pathology in TgCRND8 mice. Male TgCRND8 mice were administered with IRN (20 or 40 mg/kg) by oral gavage daily for 4 months, followed by assessing the spatial learning and memory functions with the Radial Arm Maze (RAM) test. Brain tissues were determined immunohistochemically or biochemically for changes in amyloid pathology, tau hyperphosphorylation and neuroinflammation. Our results revealed that IRN (40 mg/kg) significantly ameliorated cognitive deficits in TgCRND8 mice. In addition, IRN (40 mg/kg) markedly reduced the levels of Aβ₄₀, Aβ₄₂ and tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and IL-1β, and modulated the amyloid precursor protein (APP) processing and phosphorylation by altering the protein expressions of β-site APP cleaving enzyme-1 (BACE-1), phosphorylated APP (Thr668), presenilin-1 (PS-1) and anterior pharynx-defective-1 (APH-1), as well as insulin degrading enzyme (IDE), a major Aβ-degrading enzyme. IRN was also found to inhibit the phosphorylation of tau at the sites of Thr205 and Ser396. Immunofluorescence showed that IRN reduced the Aβ deposition, and suppressed the activation of microglia (Iba-1) and astrocytes (GFAP) in the cerebral cortex and hippocampus of TgCRND8 mice. Furthermore, IRN was able to attenuate the ratios of p-c-Jun/c-Jun and p-JNK/JNK in the brains of TgCRND8 mice. IRN also showed marked inhibitory effect on JNK signaling pathway in the Aβ-treated rat primary hippocampus neurons. We conclude that IRN improves cognitive impairment in TgCRND8 transgenic mice via reducing Aβ generation and deposition, tau hyperphosphorylation and neuroinflammation through inhibiting the activation of JNK signaling pathway, and has good potential for further development into pharmacological treatment for AD.     

反相HPLC法测定兔血浆异钩藤碱浓度及其药物代谢动力学

用ODS柱分离,甲醇—水(95∶5)为流动相,检测波长UV254nm,建立了兔血浆异钩藤碱浓度的HPLC测定方法。结果显示,血药浓度在0016～16μg·ml^-1范围内呈线性关系,血浆最低检测浓度为0.016μg·ml^-1,绝对回收率为80.5%～85.1%。兔iv IRHY 2及5mg·kg^-1,药代动力学过程符合二室开放模型,T_1/2β分别为1.32h和1.25h。兔经十二指肠给2及5mg·kg^-1后,T_1/2β分别为1.75h和1.26h。生物利用度为42.4%～69.4%。此法简便、快速。IRHY在兔体内吸收迅速,消除也较快。     

Rhynchophylline and isorhynchophylline inhibit NMDA receptors expressed in Xenopus oocytes

Rhynchophylline and isorhynchophylline are major tetracyclic oxindole alkaloid components of Uncaira species, which have been long used as medicinal plants. In this study, the effects of rhynchophylline and isorhynchophylline on the ionotropic and metabotropic glutamate receptor-mediated current responses were examined using Xenopus oocytes injected with total RNA prepared from rat cortices or cerebelli. Rhynchophylline and isorhynchophylline (1-100 microM) per se failed to induce membrane current, but these alkaloids reversibly reduced N-methyl-D-aspartate (NMDA)-induced current in a concentration-dependent but voltage-independent manner. The IC(50) values of rhynchophylline and isorhynchophylline were 43.2 and 48.3 microM, respectively. Substitution of Ba(2+) for Ca(2+) in the recording medium did not alter the extent of rhynchophylline- and isorhynchophylline-induced suppression of NMDA currents. In contrast, neither alkaloid had an effect on the currents mediated by ionotropic kainic acid-type and (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors or by the metabotropic glutamate receptor(1 and 5) (mGlu(1/5)). Rhynchophylline and isorhynchophylline (30 microM) significantly reduced the maximal current responses evoked by NMDA and glycine (a co-agonist of NMDA receptor), but had no effect on the EC(50) values and Hill coefficients of NMDA and glycine for inducing currents. These alkaloids showed no interaction with the polyamine binding site, the Zn(2+) site, proton site or redox modulatory site on the NMDA receptor. These results suggest that rhynchophylline and isorhynchophylline act as noncompetitive antagonists of the NMDA receptor and that this property may contribute to the neuroprotective and anticonvulsant activity of the Uncaira species plant extracts.     

Stereoselective in vitro metabolism of rhynchophylline and isorhynchophylline epimers of Uncaria rhynchophylla in rat liver microsomes

1.?The objective was to investigate the underlying mechanism of the stereoselectivity in the metabolism of rhynchophylline (RIN) and isorhynchophylline (IRN) epimers in rat liver microsomes (RLM).

2.?After incubation, eight metabolites of RIN (M1-5) and IRN (M6-8) reacted at A- and C-ring were identified using LC-Q-TOF/MS. Metabolic pathways included oxidation, hydroxylation, N-oxidation and dehydrogenation. In addition, hydroxylation at A-ring was the major metabolic pathway for RIN whereas the oxidation at C-ring was the major one for IRN.

3.?Enzyme kinetics showed that the intrinsic clearance (CL_int) for IRN elimination was 1.9-fold higher than RIN and the degradation half-life (T_1/2) of RIN was 4.7-fold higher than that of IRN, indicating IRN was more favorable to be metabolized than RIN in RLM.

4.?Data from chemical inhibition study demonstrated CYP3A was the predominant isoform involved in the metabolic elimination of both epimers, as well as the formation of M1-8.

5.?In conclusion, data revealed that due to the spatial configurations at C-7 position, RIN and IRN epimers possessed different hepatic metabolic pathways and elimination rates which were mainly mediated by CYP3A.     

中药钩藤不同药用部位异钩藤碱含量分析

目的：测定钩藤的不同药用部位中异钩藤碱的含量。方法应用高效液相色谱法(HPLC)测定8批不同产地钩藤的不同药用部位中异钩藤碱含量。采用Waters Symmetry C18色谱柱（250 mm×4.6 mm,5μm）,流动相为甲醇-0.01 mol/L醋酸铵缓冲液（pH 8.0）（60∶40）,柱温25℃,进样量20μl,流速1.0 ml/min,检测波长246 nm。结果8批不同产地钩藤药材中均能检测出异钩藤碱,但产地间差异较大;不同药用部位中异钩藤碱的含量：主杆＞带钩茎枝＞无钩茎枝＞无茎枝钩＞叶。结论钩藤不同药用部位大部分含异钩藤碱成分,为扩展钩藤植物的药用部位提供试验依据。