Indole Alkaloids Inhibiting Neural Stem Cell from Uncaria rhynchophylla

Uncaria rhynchophylla is commonly recognized as a traditional treatment for dizziness,cerebrovascular diseases,and nervous disorders in China.Previously,the neuro-protective activities of the alkaloids from U.rhynchophylla were intensively reported.In current work,three new indole alkaloids(1–3),identified as geissoschizic acid(1),geissoschizic acid N4-oxide(2),and 3b-sitsirikine N4-oxide(3),as well as 26 known analogues were isolated from U.rhynchophylla.However,in the neural stem cells(NSCs)proliferation assay for all isolated compounds,geissoschizic acid(1),geissoschizic acid N4-oxide(2),isocorynoxeine(6),isorhynchophylline(7),(4S)-akuammigine N-oxide(8),and(4S)-rhynchophylline N-oxide(10)showed unexpected inhibitory activities at 10 μM.Unlike previous neuro-protective reports,as a warning or caution,our finding showcased a clue for possible NSCs toxicity and the neural lesions risk of U.rhynchophylla,while the structure–activity relationships of the isolated compounds were discussed also.

Study on Germination Rate of Uncaria rhynchophylla (Miq.) Jacks

[Object] The research aimed to find out seed germination conditions and regulations.[Method] Seeds were soaked by clear water and 4 chemical agents of different concentrations(NaHCO3,KHCO3,K2CO3,NaClO)then conducted germination test in illumination box.[Result] Seed germination number and absolute germination rate(above 80%)in water treatment group was obviously higher than other treatments.[Conclusion] No dormancy phenomenon in mature embryo and no inhibition in seed coat,so conventional moist treatment can induct seed germination.

Rapid Analysis of Four Alkaloids in Uncaria rhynchophylla by Core-Shell Column HPLC and Quantitative Analysis of Multi-Components by Single Marker(QAMS)

As a traditional herbal medicine,the major alkaloids in Uncaria rhynchophylla have been proven to have blood pressure-lowering and sedative effects.It is essential to develop an effective method for the determination of the major alkaloids in U.rhynchophylla.In this research,a rapid quantitative analysis involving multi-components analysis by a single marker strategy coupled with core-shell column HPLC was adopted to analyse four alkaloids(corynoxeine,isocorynoxeine,isorhynchophylline,rhynchophylline)in U.rhynchophylla.Isorhynchophylline was selected as the internal reference substance,the content of which was determined by the traditional external standard method.Relative correction factors(RCF)between isorhynchophylline and the other three alkaloids were calculated respectively.The results showed that the QAMS method had good robustness under different HPLC instruments.Nineteen batches of U.rhynchophylla were tested.No significant difference was observed between the results by QAMS and EMS(Correlation coefficient>0.99,p>0.05).The QAMS method could be employed as a rapid,effective technique for the quality control of U.rhynchophylla.

Uncarialines A-E,new alkaloids from Uncaria rhynchophylla and their anticoagulant activity

Uncarialines A-E(1-5),five undescribed monoterpene indole alkaloids,together with five known analogues were obtained from the stems of Uncaria rhynchophylla.Alkaloids 1-3 were unique 3,4-seco-tricyclic alkaloids with a 6/5/10 ring system,while 4 and 5 possessed a rare rearranged scaffold originated from corynantheine-type alkaloids with C-2/C-7 oxidation.Their structures were characterized by a comprehensive analysis of MS,NMR,and ECD.Their effects on blood clotting times of human plasma were evaluated and alkaloid 5 had a slight prolongation effect on both thrombin time and activated partial thromboplastin time(p<0.001).

Pseudotargeted metabolomics for exploring the changes of neurotransmitters profile in aging rat brain and the potential neuroprotective effect of alkaloids from Uncaria rhynchophylla

Background:Aging is an essential risk factor for most neurodegenerative diseases,including Alzheimer’s disease and Parkinson’s disease.However,changes in the levels of neurotransmitters that are associated with aging are not well understood.Methods:Methods such as liquid-liquid extraction,protein precipitation,and solid-phase extraction,using 20 different extraction solvents,were evaluated to optimize the extraction of neurotransmitters.A pseudotargeted metabolomics approach was developed to detect neurotransmitters in brain tissues using ultra-high-performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry.Alkaloids that crossed into the brain were used to evaluate the effect of glutamic acid-induced excitatory neurotoxicity in SH-SY5Y cells.Results:The overall extraction efficiency using protein precipitation was high.The changes in neurotransmitters’levels in the brain exhibited changes during the different growth cycles.The levels of seven neurotransmitters(aspartic acid,tyrosine,isoleucine,leucine,tryptophan,valine,andγ-aminobutyric acid)were significantly different.Meanwhile,alkaloids could reduce the excitatory neurotoxicity of glutamic acid-induced SH-SY5Y cells via suppression of oxidative stress.Conclusion:Significant differences were observed in neurotransmitter profiling between 1-and 8-month-old rats,and the discrepant neurotransmitters were associated with aging.Seven indole alkaloids from Uncaria rhynchophylla,which could cross the blood-brain barrier,were screened and used to explore their protective effects against aging.Uncaria rhynchophylla alkaloids exhibited a neuroprotective effect by inhibiting oxidative stress,indicating that the alkaloid could be a potential therapeutic candidate for neurological disorders caused by glutamic acid toxicity.

Triterpenoids from Uncaria rhynchophylla and Their PTP1B Inhibitory Activity

Uncaria rhynchophylla(Gouteng)is a famous traditional Chinese medicine used for psychiatric and hypotensive purposes in China.In this study,the ethyl acetate(EtOAc)part of U.rhynchophylla was revealed with protein tyrosine phosphatase 1B(PTP1B)inhibitory activity.Subsequent investigation on the EtOAc part yielded one new triterpenoid,3β-formyloxy-6β,19α-dihydroxyurs-12-en-28-oic acid(1)and four known ones,3β,6β,19α-trihydroxyurs-12-en-28-oic acid(2),2-oxopomolic acid(3),3β,19α-dihydroxy-6-oxo-olean-12-en-28-oic acid(4)and sumaresinolic acid(5).The structure of compound 1 was determined by extensive HRESIMS,IR,1D and 2D NMR spectroscopic analyses.Two ursane-type triterpenoids(2 and 3)showed selective inhibition on PTP1B with IC50 values of 48.2 and 178.7μM.The enzyme kinetic study suggested that compounds 2 and 3 were mixtype inhibitors on PTP1B with Ki values of 15.6 and 132.5μM.This investigation manifests the antidiabetic potency of U.rhynchophylla with triterpenoids as the active constituents.

Two new ortho benzoquinones from Uncaria rhynchophylla

The present study was designed to determine the chemical constituents of the stems and hooks of Uncaria rhynchophylla. The chemical constituents were isolated and purified from CH_2Cl_2 fraction by chromatography. Their structures were elucidated by spectroscopic analyses. Their cytotoxicity was tested using MTT method. Two new ortho benzoquinones, 3-diethylamino-5-methoxy-1, 2-benzoquinone(1) and 3-ethylamino-5-methoxy-1, 2-benzoquinone(2), together with a known compound isorhynchophyllic acid(3) were isolated from U. rhynchophylla. These compounds were evaluated for their cytotoxicity against cancer cells A549, Hep G2 and A2780. Compounds 1 and 2 were new ortho benzoquinones and showed weak antiproliferative activities on A549, Hep G2 and A2780 cells. Compound 3 significantly inhibited the proliferation of A549, Hep G2 and A2780 cells with IC_(50) values being 5.8, 12.8 and 11.8 μmol·L^(–1), respectively.

Two folate-derived analogues from an aqueous decoction of Uncaria rhynchophylla

Two new folate-derived analogues,named uncarophyllofolic acids A(1)and B(2),respectively,were isolated from the Uncaria rhynchophylla hook bearing stem(Gouteng in Chinese).The distinct stereochemical structures of 1 and 2 were determined by spectroscopic data analysis in combination with acidic hydrolysis and Marfey’s derivatization,along with comparison of their specific rotation and Cotton effect(CE)data with those of the biogenetically related known derivatives as well as theoretical calculations of electronic circular dichroism(ECD)spectra.A plausible biosynthetic pathway of 1 and 2,associating to folate metabolism and the previously reported orychophragines A-C from Orychophragmus violaceus,is discussed.

钩藤UrTSA基因克隆及组织表达特异性分析

【目的】克隆钩藤碱的合成关键酶即色氨酸合成酶α链基因(UrTSA),并分析其组织表达特异性,为探究该基因的钩藤碱生物合成机制提供理论参考。【方法】从生物项目数据库(BioProject)筛选得到钩藤碱的生物合成相关酶基因UrTSA,以钩藤的根、叶、茎钩和蒴果的cDNA为模板,PCR克隆UrTSA基因的开放阅读框(ORF),并进行生物信息学分析,结合实时荧光定量PCR检测UrTSA基因在钩藤不同组织中的表达模式。【结果】UrTSA基因的ORF为963bp,编码320个氨基酸残基,相对分子量为33924.49Da,理论等电点(pI)为9.11,不稳定指数Ⅰ为33.50,脂溶指数为102.12,总平均亲水性指数为0.117,说明UrTSA蛋白为稳定、脂溶性好的疏水蛋白。UrTSA蛋白亚细胞定位于叶绿体,不含信号肽和跨膜区,为非分泌型蛋白;不具有潜在的糖基化位点,有30个潜在的磷酸化位点,包括16个丝氨酸(Ser)、13个苏氨酸(Thr)和1个酪氨酸(Tyr);二级结构由α-螺旋(43.44%)、无规则卷曲(31.87%)、延伸链(15.94%)和β-折叠(8.75%)组成。UrTSA蛋白与其他11个植物物种的TSA蛋白具有较高的氨基酸序列相似性(69.78%~92.77%),其中与阿拉比卡咖啡(Coffeearabica)和欧基尼奥伊德斯种咖啡(Coffeaeugenioides)TSA蛋白的亲缘关系较近。UrTSA基因在钩藤的根、叶、茎钩和蒴果中均有表达,其中在蒴果、根和叶中相对表达量均极显著高于茎钩中的相对表达量(P<0.01),分别是茎钩的2.17、1.58和1.20倍。【结论】UrTSA基因具有组织表达特异性,主要在蒴果钩藤碱合成中发挥调控作用,为后续研究钩藤碱在钩藤中不同部位的累积及其生物合成途径提供思路和线索。

钩藤内生放线菌Streptomyces sp.IMW-B19化学成分研究

目的 研究钩藤内生放线菌Streptomyces sp.IMW-B19发酵产物的次生代谢产物及部分化合物的生物活性。方法 该菌株的发酵产物采用ODS、Sephadex LH-20、半制备HPLC等色谱技术进行分离纯化,并采用ESI-MS和NMR鉴定化合物的化学结构。使用人肝癌HepG2细胞评价化合物1对11β-羟类固醇脱氢酶1活性的影响。结果 共分离得到11个化合物,经多种波谱数据分析和文献对比,分别鉴定为actiphenol(1)、苯甲酸薄荷酯A(2)、cyclo(L-Pro-L-Phe)(3)、胸苷(4)、11-hydroxy-4-amorphen-15-oic acid(5)、3-吲哚甲酸甲酯(6)、苯乙酸(7)、1-(3-ethylphenyl)-ethane-1,2-diol(8)、亚油酸(9)、亚油酸甲酯(10)和2-氨基-4-甲氧基苯甲酸(11)。化合物1对11β-羟类固醇脱氢酶1具有明显的抑制作用,在20 μmol·L^(-1)时,抑制率达到86.01%。结论 从钩藤内生放线菌Streptomyces sp.IMW-B19发酵产物中共分离得到11个化合物,其中化合物4、5、8为首次从链霉菌属的放线菌中分离得到。化合物1对11β-羟类固醇脱氢酶1具有明显的抑制作用。

钩藤UrLAMT基因及其启动子的克隆与分析

【目的】克隆钩藤(Uncaria rhynchophylla)中马钱苷酸甲基转移酶(loganic acid methyltransferase,LAMT)基因及其启动子,对UrLAMT基因的组织表达及其对生物和非生物胁迫的响应进行分析,并对UrLAMT及其启动子的生物信息学进行分析,为进一步研究UrLAMT转录调控奠定基础。【方法】基于钩藤的转录组数据设计引物,采用PCR从钩藤cDNA中克隆UrLAMT序列,并对其进行生物信息学分析;采用实时荧光定量PCR,分析UrLAMT在钩藤不同组织(根、茎、叶、花、果实、钩)中的表达,以及对外源茉莉酸甲酯(MeJA)、遮光/复光胁迫的响应;利用FPNI-PCR技术克隆UrLAMT上游启动子序列,并验证其活性,结合酵母单杂交试验,分析相应转录因子与UrLAMT启动子的调控关系。【结果】克隆得到钩藤UrLAMT序列,其长度为1137 bp,共编码378个氨基酸。UrLAMT蛋白质相对分子质量为42.64 ku,理论等电点为5.76,为亲水性蛋白,无信号肽,不含跨膜结构,定位在细胞质中;UrLAMT基因在钩藤叶中表达量最高,其次为根;UrLAMT均能响应MeJA和光;其与短小蛇根草和长春花的进化关系较近。UrLAMT启动子序列长度为1141 bp,除核心响应元件外还含有多个光响应元件,UrLAMT启动子在酵母中与光调控转录因子HY5存在相互作用。【结论】获得了UrLAMT基因及其启动子序列,其在钩藤叶中表达量最高,可能受光诱导。

钩藤的药理作用及临床应用研究进展

钩藤(Uncaria rhynchophylla)是广西区域特色药材品种之一,具有息风定惊、清热平肝的功效,药用历史悠久。钩藤生物碱是钩藤的主要药效成分,对缓解药物成瘾、阿尔茨海默症、高血压等疾病具有显著功效,其方药天麻钩藤饮、复方钩藤降压片等在临床上广泛应用。随着现代医学研究技术的进步,钩藤生物碱及其方药的治病机理正在不断被发现。本文收集近十年钩藤相关研究文献,以钩藤的药效物质为基础,总结钩藤生物碱及其方药的药理研究成果,分析钩藤的临床应用情况,以期为钩藤的新药研发或新应用提供理论借鉴。

HPLC法测定新乐康片相关药材中8种活性成分含量

建立HPLC法同时测定新乐康片中三味原料药材钩藤、酸枣仁和萝芙木中钩藤碱、异钩藤碱、斯皮诺素、酸枣仁皂苷A、酸枣仁皂苷B、育亨宾、阿义吗啉及利血平等8种活性成分的含量。采用Titank C18(250 mm×4.6mm,5μm)色谱柱,以乙腈-0.4%磷酸水溶液(含0.05%三乙胺)为流动相,梯度洗脱,检测波长204 nm,流速为1.0 mL/min,柱温为30℃。上述三味药材相关成分之间分离度良好,在λ=204 nm条件下,其在分别的线性范围内与响应值之间的线性关系良好(R^(2)≥0.9990);8种活性成分的平均加样回收率98.37%~101.21%,RSD<2%,仪器精密度、供试品溶液的稳定性与方法重复性均良好。建立的HPLC法可一法对钩藤、酸枣仁和萝芙木三味中药材中8种活性成分含量进行测定,为药业在生产新乐康片前期选材上节约了测定时间,提高了生产效率,同时为新乐康片中活性成分HPLC含量测定方法的建立及其质量标准升级提供参考。

钩藤不同加工方法对DPPH自由基清除能力的研究

目的研究钩藤37种加工方法的醇提物对DPPH自由基清除能力的影响。方法采用DPPH(1,1-二苯基-2-苦味基肼)自由基清除法,以抗坏血酸为阳性对照,采用紫外-可见分光光度仪测定吸光度,根据公式计算不同加工方法样品的IC_(50)值(清除率为50%时样品浓度),进一步评价钩藤不同加工方法的抗氧化活性。结果37种加工方法的醇提物清除自由基能力具有显著差异。作用较强的加工方法为蒸制法,IC_(50)值最低的依次为蒸制60 min,蒸1次(0.2438 mg/mL)<蒸制45 min,蒸1次(0.2591 mg/mL)<蒸制15 min,蒸1次(0.2613 mg/mL);而发汗法作用较弱,其中以直接发汗3天的抗氧化活性最差,IC_(50)值为0.4823 mg/mL。结论钩藤不同加工方法对DPPH自由基的清除能力不同,高温加热处理可提高其抗氧化活性,研究结果为该药材产地加工方法的综合质量评价及抗氧化活性研究提供重要参考。

钩藤中不同成分降压作用的差异

目的 :比较钩藤中提取的异钩藤碱、钩藤碱、钩藤总碱及非生物碱部分的降压作用。方法 :大鼠麻醉后经颈总动脉插管记录外周血压和经股静脉微量输注实验用药。结果 :实验表明钩藤中 4种成分的降压强度为异钩藤碱(4 2 .0 % ) >钩藤碱 (32 .1% ) >钩藤总碱 (2 1.3% ) >钩藤非生物碱 (12 .4% )。结论 :提示钩藤中主要降压成分为异钩藤碱和钩藤碱。

不同生物有机肥对钩藤产量、品质及土壤生物性状的影响

针对贵州"剑河钩藤"野生资源减少,人工栽培产量低、品质差的问题,从改善钩藤产地土壤入手,筛选适宜的生物有机肥,培肥地力,进而提高钩藤产量及品质。结果表明:配施生物肥对钩藤的产量、品质和土壤生物性状都有不同程度地提高和改良作用,其中gz-Ⅳ号生物肥对钩藤农艺性状、产量以及土壤养分的提高效果最好;gz-Ⅲ号生物肥对钩藤品质以及土壤生物性状的改良效果最好。

钩藤不同溶剂提取物及总生物碱抗氧化活性研究

采用流动注射化学发光法,对钩藤石油醚提取物(PE)、氯仿提取物(CE)、乙酸乙酯提取物(AE)和乙醇提取物(EE)及总生物碱(Alk)的抗氧化活性进行测定,并与Vc的抗氧化活性进行比较。结果表明,钩藤4种溶剂提取物及总生物碱都具有较强的抗氧化活性,抗氧化活性强弱顺序为:EE>Vc>AE>CE>Alk>PE。

钩藤叶化学成分的研究

目的研究茜草科植物钩藤Uncaria rhynchophylla(Miq.)Jacks.叶的化学成分。方法用多种色谱方法和重结晶法进行分离和纯化,经理化、光谱和色谱分析等方法鉴定结构。结果从钩藤叶中分离得到5个化合物,鉴定为喜果苷(vincoside lactam,Ⅰ)、异去氢钩藤碱(isocorynoxeine,Ⅱ)、异钩藤碱(isorhynchophylline,Ⅲ)、去氢钩藤碱(corynoxeine,Ⅵ)、钩藤碱(rhynchophylline,Ⅴ)。结论化合物Ⅲ为首次从钩藤叶中分离得到,通过2D-NMR技术完善了化合物Ⅲ的核磁数据。

蒙药材珍珠杆及其伪品的紫外/荧光/红外光谱法鉴别研究

文章用紫外、荧光及红外三种光谱法对四种蒙药材进行了鉴别研究。分别用甲醇、石油醚、氯仿对蒙药材珍珠杆及其代用品钩藤、接骨木和混伪品桂枝进行了超声提取,对提取物分别进行了紫外光谱和荧光光谱测试,并对上述四种药材粉末的红外光谱进行了比较。结果表明,四种药材的紫外光谱特征峰相异。用荧光光谱鉴别时,对药材的提取溶剂有选择性,以甲醇作提取溶剂时四种药材的荧光光谱能有明显的差异。四种药材粉末的红外光谱有明显区别。

钩藤总碱预处理对海马神经元急性缺氧的保护作用

目的:观察钩藤总碱预处理对急性缺氧后海马神经元电压门控性Na+电流的影响。方法:原代培养大鼠海马神经元,分为常氧、急性缺氧和钩藤总碱预处理进行实验。采用膜片钳全细胞记录技术,检测海马神经元钠电流(INa)的变化。结果:急性缺氧后,细胞INa幅度明显降低,INa阈值右移;而经钩藤总碱预处理的海马神经元INa的降低幅度明显减轻。结论:钩藤总碱预处理提高了海马神经元对急性缺氧的耐受性,对神经元有显著保护作用。