Simultaneous Determination of Six Quaternary Ammonium Alkaloids in Coptidis Rhizoma by UPLC

Objective To establish a new, rapid, and reliable reversed-phase ultra performance liquid chromatography (RP-UPLC) method for the simultaneous determination of six quaternary ammonium alkaloids (QAAs) in Coptidis Rhizoma. Methods The effect of different experimental parameters on the analysis of QAAs by RP-UPLC was evaluated. Results Optimal resolution was achieved with an Acquity UPLC BEH C18 column using a gradient elution profile and a mobile phase consisting of water spiked with 10 mmol/L ammonium bicarbonate (A, pH adjusted to 10.0 by ammonia water) and acetonitrile (B), at a flow rate of 0.30 mL/min and wavelength of 345 nm. The column temperature was set at 30 ℃. The proposed method was found to be reproducible, precise, and rapid according to the method validation. Conclusion The proposed method, which is compatible with MS analysis and the preparation of QAA, provides some helpful insights into the quality control of Coptidis Rhizoma.     

莲子心总生物碱的毒性试验研究

摘要 目的： 探讨莲子心总生物碱的毒副作用,以对莲子心总生物碱作出安全性评价。方法 ：小鼠急性毒性试验中一次性给予最大灌胃量5000mg/kg莲子心总生物碱,观察小鼠中毒症状。长期口服毒性实验,Sprague-Dawley(SD)大鼠随机分为四组,分别按照400mg/kg?d、200 mg/kg?d、100 mg/kg?d的剂量给予SD大鼠灌胃实验室自制莲子心总生物碱,对照组给予等量体积0.3%羧甲基纤维素钠,连续服用30天。结果 ：在急性毒性试验中给予5000mg/kg莲子心总生物碱对小鼠不产生毒性作用。在长期毒性试验中,除不同剂量给药组SD大鼠血清尿素氮与对照组具有显著性差异(P<0.05),高、中剂量组SD大鼠血糖含量与对照组有明显差异(P<0.05)外余下指标与对照组相比均无明显差异。高、中、低剂量组大鼠肝组织病理切片与对照组相比均明显异常,表现为肝小叶结构正常。结论 ： 莲子心总生物碱对小鼠最大耐受剂量为5000mg/kg,同时灌胃给药400mg/kg/天莲子心总生物碱对大鼠无毒副作用。     

Real-time toxicity prediction of Aconitum stewing system using extractive electrospray ionization mass spectrometry

Due to numerous obstacles such as complex matrices, real-time monitoring of complex reaction systems (e.g., medicinal herb stewing system) has always been a challenge though great values for safe and rational use of drugs. Herein, facilitated by the potential ability on the tolerance of complex matrices of extractive electrospray ionization mass spectrometry, a device was established to realize continuous sampling and real-time quantitative analysis of herb stewing system for the first time. A complete analytical strategy, including data acquisition, data mining, and data evaluation was proposed and implemented with overcoming the usual difficulties in real-time mass spectrometry quantification. The complex Fuzi (the lateral root of Aconitum)–meat stewing systems were real-timely monitored in 150 min by qualitative and quantitative analysis of the nine key alkaloids accurately. The results showed that the strategy worked perfectly and the toxicity of the systems were evaluated and predicated accordingly. Stewing with trotters effectively accelerated the detoxification of Fuzi soup and reduced the overall toxicity to 68%, which was recommended to be used practically for treating rheumatic arthritis and enhancing immunity. The established strategy was versatile, simple, and accurate, which would have a wide application prospect in real-time analysis and evaluation of various complex reaction systems.     

Composition of Ophiopogon Polysaccharide, Notoginseng Total Saponins and Rhizoma Coptidis Alkaloids Inhibits Myocardial Apoptosis in Diabetic Atherosclerosis Rabbits

Objective: To investigate the effects of Composition of Ophiopogon polysaccharide, Notoginseng total saponins and Rhizoma Coptidis alkaloids (CONR) on myocardial apoptosis of diabetic atherosclerosis (DA) rabbits Methods: Sixty male New Zealand white rabbits were randomly divided into 6 groups [control group, model group, CONR high-dose group (450 mg/kg), CONR medium-dose group (150 mg/kg), CONR low-dose group (50 mg/kg), and simvastatin group] by using a completely random method, 10 in each group. DA model was established by intravenously injected alloxan combined with high-fat diet and abdominal aortic balloon injury. After mediation for 10 weeks, fasting blood glucose (FBG), glycosylated hemoglobin (GHB), glycosylated serum protein (GSP), fructoseamine (FRA), aldose reductase (AR), advanced glycation end products (AGEs) in serum were measured by enzyme linked immunosorbent assay (ELISA) method; the expression of receptor of AGEs (RAGE) in myocardial tissue were observed by immunohistochemical method; and p-Jun N-terminal kinase (p-JNK), caspase-3, B-cell lymphoma-2 (bcl-2) protein expression in myocardial tissue were measured by Western blotting. The myocardial apoptosis was detected by TdT-mediated dUTPnick-end labeling (TUNEL)method, and apoptosis index (AI) was calculated. Results: Compared with the control group, serum FBG, GHB, GSP, FRA, AR, AGEs and the expression of myocardium RAGE, p-JNK, caspase-3 proteins, as well as apoptosis index (AI) were significantly increased and bcl-2 protein was significantly decreased in the model group (P<0.01). Compared with the model group, the levels of serum FBG, GHB, GSP, FRA and AR showed a significant decline in CONR high- and medium-dose groups (P<0.01). FBG and GHB showed a significant decline in CONR low-dose group (P<0.01). Compared with the model group, the expression of serum AGEs and myocardium RAGE, p-JNK and caspase-3 protein as well as AI were significantly decreased and bcl-2 protein was significantly up-regulated in all treatment groups (P<0.01); high-dose CONR had the most significant effect on abovementioned indices compared with other treatment groups (P<0.01). Middle-dose CONR had better effect on serum AGEs compared with the low-dose group (P<0.01); middle-dose CONR and simvastatin groups had better effect on the expression of caspase-3, bcl-2 protein, myocardium apoptosis compared with the CONR low-dose group (P<0.01). Conclusion: CONR may effectively inhibit myocardial apoptosis on DA rabbits by intervening AGEs-RAGE and JNK, caspase-3, and bcl-2 protein expressions.     

Pinellia Total Alkaloids Modulate the GABAergic System in Hippocampal Formation on Pilocarpine-Induced Epileptic Rats

Objective: To investigate the neuromodulatory effect of pinellia total alkaloids(PTA) on the gamma-aminobutyric acidergic(GABAergic) system in epileptic rats, and preliminarily evaluate the anti-epileptic effect of PTA. Methods: Ninety-one male Sprague-Dawley rats were randomized to a control group(n=17) or an epileptic group(n=74) using computer-generated random numbers. Status epilepticus(SE) was induced with pilocarpine in the epileptic group. Epileptic rats that survived SE were randomly divided into 4 groups, namely an epilepsy group(n=13), a topiramate(TPM, 60 mg/kg) group(n=12), a high-dose PTA(800 mg/kg) group(n=12), and a low-dose PTA(400 mg/kg) group(n=10). Treatments were given intragastrically once daily for 14 days. The control group and epilepsy group received normal saline. Spontaneous recurrent seizures(SRSs) were monitored 8-h daily for 7 days after treatment. Then, the hippocampal formation tissues were collected. GABA level was measured using enzyme-linked immunosorbent assay. Protein and mRNA expression levels of glutamate decarboxylase 65(GAD65), GABA transporter-1(GAT-1), GABA transaminase(GABA-T), and GABAA receptor(GABAAR) α4, α5, γ2 and δ subunits were measured using Western-blotting analysis and quantitative polymerase chain reaction. Results: PTA lowered the incidence and frequency of SRS(both doses vs. the TPM group, P0.05). Compared with the epilepsy group, PTA increased the levels of GABA(both doses P0.01) and GAD65(mRNA, 800 mg/kg, P0.01), and suppressed the levels of GAT-1(mRNA, 800 mg/kg, P0.01; 400 mg/kg, P0.05), GABA-T(mRNA, both doses P0.01), and GABAAR δ subunit(protein, 800 mg/kg, P0.05) and γ2 subunit(protein, both doses P0.01). PTA upregulated the low-expressed mRNA levels of GABAAR α5 subunit(400 mg/kg, P0.01), δ subunit(800 mg/kg, P0.05), and γ2 subunit(400 mg/kg, P0.05). Conclusions: PTA regulated the GABAergic system through modulating GABA levels and the expression levels of GAD65, GAT-1, GABA-T, and GABAAR α4, α5, γ2 and δ subunits. PTA may exert antiepileptic effects on the pilocarpine-induced epilepsy model.     

海南粗榧总碱中化学成分研究

目的研究海南粗榧Cephalotaxus hainanensis的生物碱类成分。方法采用硅胶、Sephadex LH-20和ODS等多种柱色谱技术进行分离纯化,根据化合物理化性质及波谱数据鉴定化合物结构。结果从海南粗榧总碱中分离得到13个化合物,分别鉴定为三尖杉碱(1)、异三尖杉酮碱(2)、三尖杉酮碱(3)、三尖杉胺G(4)、(2R,3S,4S,5S)-2,3-二羟基三尖杉碱(5)、乙酰三尖杉碱(6)、脱氧三尖杉酯碱(7)、(R)-丽江三尖杉碱(8)、(S)-丽江三尖杉碱(9)、3-表里哈灭里辛碱异构体(10)、1,4-N-二异丙胺蒽醌(11)、苯甲酸(2-乙基)-正己酯(12)和β-谷甾醇(13)。结论化合物3~6和10~13为首次从海南粗榧中分离得到。     

雷公藤研究进展及其质量标志物的预测分析

雷公藤是一味传统中药,所含化学成分丰富,具有多方面的药理活性。在刘昌孝院士提出中药质量标志物(Q-marker)新概念的基础上,系统综述了雷公藤化学成分及药理活性,并根据质量标志物的定义结合雷公藤的传统药效、传统药性、临床用药新用途、可测成分、入血成分和配伍研究进行分析,对雷公藤质量标志物进行预测,以期为雷公藤药材质量的深入研究提供参考。     

Cytotoxic monoterpenoid-type alkaloids from the aerial parts of Melodinus morsei

Three new monoterpenoid alkaloids, melomorines A–C (1–3), along with melohemsine B (4), were isolated from the 70% ethanol extract of the aerial parts of Melodinus morsei. Structural elucidation of all the compounds was performed on the basis of spectral data. All the compounds were tested in vitro for cytotoxic activities. As a result, alkaloids 1, 2, and 4 exhibited cytotoxicities against all the five tested tumor cell lines with IC₅₀ value of less than 20 μM.     

苦参碱类生物碱的镇痛作用研究进展

苦参碱类生物碱（包括有苦参碱、氧化苦参碱、氧化槐定碱、槐果碱、氧化槐果碱等）具有镇痛作用,镇痛特征是以中枢性为主、无成瘾性和耐药性,综述其镇痛作用及作用机制研究进展。苦参碱类生物碱的中枢性镇痛作用机制可能是：激活大麻素受体-2和上调电压门控钙离子N-型通道表达,促进抑制性神经递质（γ-氨基丁酸、甘氨酸）合成和释放,下调GAT-1表达,使突触间隙γ-氨基丁酸浓度升高以及上调γ-氨基丁酸-A受体表达,从而增强γ-氨基丁酸能神经功能。γ-氨基丁酸能神经功能的增强可抑制兴奋性神经递质谷氨酸过度表达并下调NMDA受体和蛋白激酶Cγ表达,降低NMDA受体对谷氨酸的兴奋性,从而下调电压门控钙离子L-型通道的表达,抑制钙离子内流,阻滞CaMKⅡ/CREB通路,减轻炎症反应和产生镇痛作用。     

巴豆生物碱对Lewis小鼠肺腺癌细胞生长和凋亡的影响及机制研究

目的 通过研究巴豆生物碱（CA）对Lewis小鼠肺腺癌细胞生长及对凋亡相关蛋白（Survivin、Caspase-3、Bax）、YAP蛋白的调控作用,探讨CA对肺腺癌细胞生长和凋亡的影响及其具体的作用机制。方法 培养Lewis鼠源性肺腺癌细胞（LLC）,复制小鼠皮下成瘤模型,分为5组,每组10只,分别为对照组、低剂量CA组、中剂量CA组、高剂量CA组及顺铂组。皮下成瘤1周后给予药物干预,第22天对5组小鼠取瘤称重,计算抑瘤率并通过光学显微镜观察各组病理形态学的变化,判断CA对瘤体生长的抑制作用;Western blotting检测Survivin、Caspase-3、Bax及YAP蛋白相对表达量;qRT-PCR法检测Survivin、Caspase-3、Bax及YAP mRNA相对表达量。结果 中剂量CA组和高剂量CA组小鼠的一般生存状态较对照组、低剂量CA组及顺铂组好。低剂量CA组、中剂量CA组、高剂量CA组及顺铂组肿瘤细胞出现不同程度细胞凋亡现象,对照组肿瘤细胞多为坏死细胞。低剂量CA组、中剂量CA组、高剂量CA组及顺铂组的抑瘤率分别是13.91％、14.83％、27.84％和68.45％,4组抑瘤率比较,差异有统计学意义（P <0.05）,各组抑瘤率依次升高,顺铂组肿瘤生长最慢。不同剂量CA组中Survivin mRNA相对表达量低于对照组（P <0.05）,CA剂量越高,Survivin mRNA相对表达量越低;而不同剂量CA组中Caspase-3和Bax mRNA相对表达量高于对照组（P <0.05）,CA剂量越高,Caspase-3和Bax mRNA的相对表达量越高;各组YAP mRNA的相对表达量比较,差异无统计学意义（P >0.05）。结论 CA对Lewis肺腺癌小鼠瘤体生长有抑制作用,并引起细胞凋亡。CA通过促进Bax和Caspase-3表达及抑制Survivin表达来调控Lewis小鼠肺腺癌细胞凋亡;其调控机制可能是通过下调Survivin的表达,从而使得Survivin对Caspase-3的抑制作用减弱,而Bax上调可进一步激活Caspase-3,从而诱导Caspase级联效应促进细胞凋亡,为肺腺癌治疗提供了新思路。