(-)，(+)黄皮酰胺对乙酰胆碱酯酶的抑制作用

用Ellman比色法测定乙酰胆碱酯酶(AChE)的活性， 观察(-)，(+)黄皮酰胺对小鼠脑组织海马，前脑皮层和红细胞膜AChE活性的影响， 以探索药物作用机制并比较左右旋体黄皮酰胺作用的异同.结果表明:(-), (+)黄皮酰胺对海马和皮层AChE均有抑制作用, (-)黄皮酰胺对小鼠脑皮层和海马AChE抑制的IC₅₀(95%可信限)值分别为0.31(0.27-0.36)和0.33(0.28-0.39)mmol·L^-１; (+)黄皮酰胺对小鼠脑皮层和海马AChE抑制的IC₅₀(95%可信限)值分别为0.71(0.53-0.94)和0.77(0.55-1.07)mmol·L^-１. (-), (+)黄皮酰胺对红细胞膜AChE的抑制作用是可逆性的，抑制特点属于竞争与非竞争混合型抑制， (-)黄皮酰胺的K_i值为0.26 mmol·L^-１, K_i′值为1.205 mmol·L^-１；（+）黄皮酰胺K_i值 为0.72 mmol·L^-1, K_i′值为1.856 mmol·L^-１. 提示：(-)黄皮酰胺作用强于(+)黄皮酰胺. 黄皮酰胺的抑制AChE作用存在手性选择性.     

黄皮酰胺对映体在大鼠肝微粒体中的酶促反应动力学黄皮酰胺对映体在大鼠肝微粒体中的酶促反应动力学

目的研究黄皮酰胺（clausenamide,Clau）对映体在大鼠肝微粒体中的酶促反应动力学并比较其立体选择性差异。方法应用反相HPLC法测定Clau对映体在体外代谢系统中的产物，并用Eadie-Hofstee作图法分析数据、求算酶促反应动力学参数K_m和V_max以及肝代谢速率V_max/K_m。结果在体外代谢系统中，左旋黄皮酰胺主要生成7-羟-Clau、5-羟-Clau和4-羟-Clau，其优势代谢途径为7位羟化；7位羟化代谢的V_max/K_m值高于5位和4位。右旋黄皮酰胺的4位羟化反应K_m最小、V_max最大, 因此代谢速率最高，是左旋体4位羟化的8倍；而其7-羟-Clau和5-羟-Clau 的产生量很小。结论黄皮酰胺对映体在大鼠肝微粒体中的羟化代谢存在明显的底物立体选择性差异。     

(-),(+)黄皮酰胺对鼠脑内 NMDA- 受体的影响

用[³H]MK-801放射配体竟争结合法测定了(-),(+)黄皮酰胺对大鼠前脑,海马,皮层等部位突触膜的NMDA-R的作用,以探讨其促智机制。同时用饱和实验分析po给药10d后,小鼠脑内该受体密度的变化。结果表明:(-),(+)黄皮酰胺对脑内各部位的NMDA受体均无特异亲和力。但(-)黄皮酰胺在体给药10d后能使小鼠脑内NMDA受体密度显著增高,并呈一定的量效关系。提示黄皮酰胺的药理作用有光学选择性;(-)黄皮酰胺增加脑内NMDA受体密度为其促智作用提供了重要理论依据。     

右旋和左旋黄皮酰胺在大鼠体内代谢转化的研究

目的　研究黄皮酰胺在大鼠体内的代谢转化途径。方法　收集ip给药后大鼠的尿液、粪便及血液进行分析,寻找已知的代谢产物并通过HPLC-DAD和HPLC-MS分析寻找未知的代谢产物,确定大鼠体内的主要代谢途径。并通过比较(+),(-)-黄皮酰胺代谢的差异,初步研究其代谢转化的立体选择性。结果　HPLC分析发现,大鼠肝微粒体中所分离得到的6个主要代谢产物均在体内存在,(+),(-)-黄皮酰胺的代谢有明显的差异,根据MS碎片信息确定了一个新的代谢产物的结构,即N-去甲黄皮酰胺。结论　手性黄皮酰胺主要在肝脏中发生羟基化代谢反应,并有明显的立体选择性。     

左旋黄皮酰胺在大鼠肝微粒体中的代谢转化研究

用大鼠肝微粒体体外温孵法进行了左旋黄皮酰胺[(-)-clausenamide]代谢转化研究,优化了温孵体系,建立了反相HPLC-DAD同时分离检测左旋黄皮酰胺及其体外代谢产物的分析方法。用硅胶低压柱色谱、制备TLC及制备HPLC分离纯化了两个代谢产物并进行了光谱鉴定。结果表明,两个代谢物分别确定为6-和5-位羟基取代的黄皮酰胺。     

左旋黄皮酰胺在大鼠体内的排泄

左旋黄皮酰胺[（-）-clausenamide]是从芸香科黄皮属植物黄皮[Clausena lansium（lour） sheels]叶的水浸膏中分离得到的有效成分，经不对称合成和拆分制备而得。药效学研究表明，左旋黄皮酰胺促进突触体谷氨酸释放，增加大鼠脑皮层厚度和海马CAL区突触数及NMDA受体密度，提高小鼠脑皮层和海马的胆碱乙酰转移酶活性，对抗樟柳碱引起的乙酰胆碱含量降低。这些结果表明左旋黄皮酰胺具有较好的促智和神经保护作用以及潜在的抗老年痴呆作用；右旋体作用不明显，且有较强的毒性。     

Inhibitory effect of(-),( )clausenamide on acetylcholinesterase in vitro

用Ｅｌｍａｎ比色法测定乙酰胆碱酯酶（ＡＣｈＥ）的活性，观察（－），（＋）黄皮酰胺对小鼠脑组织海马，前脑皮层和红细胞膜ＡＣｈＥ活性的影响，以探索药物作用机制并比较左右旋体黄皮酰胺作用的异同．结果表明：（－），（＋）黄皮酰胺对海马和皮层ＡＣｈＥ均有抑制作用，（－）黄皮酰胺对小鼠脑皮层和海马ＡＣｈＥ抑制的ＩＣ５０（９５％可信限）值分别为０．３１（０．２７－０．３６）和０．３３（０．２８－０．３９）ｍｍｏｌ·Ｌ－１；（＋）黄皮酰胺对小鼠脑皮层和海马ＡＣｈＥ抑制的ＩＣ５０（９５％可信限）值分别为０．７１（０．５３－０．９４）和０．７７（０．５５－１．０７）ｍｍｏｌ·Ｌ－１．（－），（＋）黄皮酰胺对红细胞膜ＡＣｈＥ的抑制作用是可逆性的，抑制特点属于竞争与非竞争混合型抑制，（－）黄皮酰胺的Ｋｉ值为０．２６ｍｍｏｌ·Ｌ－１，Ｋｉ′值为１．２０５ｍｍｏｌ·Ｌ－１；（＋）黄皮酰胺Ｋｉ值为０．７２ｍｍｏｌ·Ｌ－１，Ｋｉ′值为１．８５６ｍｍｏｌ·Ｌ－１．提示：（－）黄皮酰胺作用强于（＋）黄皮酰胺．黄皮酰胺的抑制ＡＣｈＥ作用存在手性选择性．     

(-),(+)黄皮酰胺对樟柳碱引起的小鼠脑内乙酰胆碱含量降低及记忆障碍的影响

为证实黄皮酰胺体内抗记忆缺失作用是否也存在手性选择性,并且进一步探讨其作用的机制,用高效液相色谱 电化学检测器法测定乙酰胆碱含量并结合行为学实验,比较了(-),(+)黄皮酰胺对小鼠脑内乙酰胆碱含量及对小鼠记忆障碍的影响。结果表明:(-)黄皮酰胺剂量依赖性地对抗樟柳碱引起的脑皮层、海马、纹状体内乙酰胆碱含量降低,(+)黄皮酰胺在同样剂量却无此作用。行为学实验也得到相似结果。提示:黄皮酰胺的药理作用存在手性选择性;(-)黄皮酰胺能改善樟柳碱所致的记忆障碍,这与其逆转樟柳碱引起的脑内乙酰胆碱含量降低作用相关。     

(-),(+)黄皮酰胺对大鼠海马突触传递功能的不同影响

采用细胞外电生理记录法记录了低频刺激所诱发的麻醉大鼠海马齿状回颗粒细胞层群峰电位(PS)并比较了侧脑室注射(-),(+)黄皮酰胺对PS及强直刺激所诱导的长时程增强效应(LTP)作用。结果表明:低剂量(1nmol)时,(-)或(+)黄皮酰胺都对基础状态下的PS没有影响。对LTP(+)黄皮酰胺也无作用,(-)黄皮酰胺表现为增强作用;将剂量提高至4nmol时,(-)黄皮酰胺对基础PS和LTP都有增强作用且对LTP的作用表现出一定的剂量依赖性,(+)黄皮酰胺对PS仍无影响,对LTP有一定的抑制作用。提示黄皮酰胺对海马齿状回突触传递活动的作用具有光学选择性,这一结果有力地支持了(-)黄皮酰胺促智作用的行为学和神经生化学研究结果。     

黄皮化学成分的研究进展

黄皮是芸香科黄皮属植物,原产于我国热带和亚热带地区的一种特种果树,具有很高的药用价值,果、叶、根、种等均可入药。黄皮果实有化痰、消食的功效,民间用其叶煮水洗浴治疗疥癫,消风肿等。研究黄皮化学成分主要在黄皮的叶和根茎等。从黄皮叶中分离得到3个酰胺类化合物,黄皮酰胺、新黄皮酰胺、桥环黄皮酰胺;从黄皮果挥发油中分离得到43个化合物,主要有萜品烯-4-醇,桧萜等;黄皮果肉中具有丰富的氨基酸,且含有大量黄酮。现代药理学研究证明黄皮的粗提物及有效化学成分具有广泛的药理活性。黄酮是一类天然的抗氧化剂;黄皮酰胺能明显抑制由酒精中毒诱发的肝脂质过氧化反应,提高谷胱甘肽过氧化物酶活性,清除自由基,抵抗氧化损伤,并对β-淀粉样多肽25-35片段诱导以及糖尿病诱导的大鼠学习记忆功能障碍具有明显的改善作用。本文对黄皮的化学成分研究做一综述,为其药学、药理学作用的深入研究提供思路。     

synapsinⅠ参与(-)黄皮酰胺增强齿状回突触传递功能

目的观察synapsinⅠ在(-)黄皮酰胺促进大鼠海马齿状回突触传递功能中的作用。方法用电生理方法观察(-)黄皮酰胺对基础突触传递功能的影响;采用Westernblot方法及共聚焦显微镜检测了(-)黄皮酰胺对synapsinⅠ磷酸化的时间、浓度依赖关系,以及确定促进synapsinⅠ激活的上游蛋白激酶。结果在整体动物中,(-)黄皮酰胺可明显增加海马齿状回群峰电位,并且脑室给药5 min即可促进海马synapsinⅠ磷酸化增强,15 min时皮层synapsinⅠ磷酸化增强最明显。0.1、1、10μmol.L-1(-)黄皮酰胺可浓度依赖性促进PC12细胞中synapsinⅠ激活,且10μmol.L-1(-)黄皮酰胺在1~2 min均可激活突触体和PC12细胞中synapsinⅠ。PKA抑制剂H89可抑制(-)黄皮酰胺对synapsinⅠ的磷酸化。结论(-)黄皮酰胺通过PKA促进synapsinⅠ磷酸化而增强基础突触传递活动。     

左旋黄皮酰胺对冈田酸和β淀粉样肽25-35神经毒性的保护作用

探讨左旋黄皮酰胺对冈田酸(okadaic acid，OA)诱导的人神经瘤细胞(SH-SY5Y)和去卵巢(ovariectomy，OVX)及单侧侧脑室注射Aβ_25-35所致神经元损伤的保护作用。通过MTT试验、LDH释放测定试验、Hoechst 33258荧光染色试验以及SH-SY5Y细胞检测，考察左旋黄皮酰胺拮抗冈田酸诱导的细胞毒作用。通过避暗试验、电镜检测、Nissl体染色及HE染色，考察左旋黄皮酰胺对去卵巢及侧脑室注射Aβ_25-35大鼠神经元的保护作用。左旋黄皮酰胺可明显拮抗冈田酸诱导的细胞毒作用，提高去卵巢及侧脑室注射Aβ_25-35大鼠的学习记忆能力，保护海马及皮层神经元。左旋黄皮酰胺可拮抗冈田酸及Aβ_25-35诱导的神经毒性，具有神经保护作用。     

(-),( )黄皮酰胺对鼠脑内 NMDA- 受体的影响

用［３Ｈ］ＭＫ８０１放射配体竟争结合法测定了（－），（＋）黄皮酰胺对大鼠前脑，海马，皮层等部位突触膜的ＮＭＤＡＲ的作用，以探讨其促智机制。同时用饱和实验分析ｐｏ给药１０ｄ后，小鼠脑内该受体密度的变化。结果表明：（－），（＋）黄皮酰胺对脑内各部位的ＮＭＤＡ受体均无特异亲和力。但（－）黄皮酰胺在体给药１０ｄ后能使小鼠脑内ＮＭＤＡ受体密度显著增高，并呈一定的量效关系。提示黄皮酰胺的药理作用有光学选择性；（－）黄皮酰胺增加脑内ＮＭＤＡ受体密度为其促智作用提供了重要理论依据。     

（一）黄皮酰胺体外对胆碱能神经元生长的促进作用

AIM: To study the neurotrophic effects of (-) and (+)clausenamide on frontal cortex neurons in culture. METHODS: The activity of the choline acetyltransferase (ChAT) was determined by spectrophotometric method; protein content was assayed by Folin phenol method. RESULTS: (-)Clausenamide increased the activity of ChAT and protein content in cultured neurons, as well as stimulated proliferation of neuronal cells, support survival and neurite outgrowth of neurons. The neurotrophic action of (-)clausenamide (0.001-10 mumol.L-1) was similar to that of nerve growth factor. The (+)clausenamide had no neurotrophic action, even at high concentrations (0.1-10 mumol.L-1), but neurons were damaged. CONCLUSION: (-)Clausenamide stimulated central cholinergic neuron development.     

Different effects of nine clausenamide ennatiomers on liver glutathione biosynthesis and glutathione S-transferase activity in mice

AIM: To study the effects of nine synthetic clausenamide with different stereo structures on liver glutathione (GSH) biosynthesis and glutathione S-transferase (GST) activity in mice. METHODS: The nine test compounds were racemic mixtures and their ennatiomers of clausenamide, neoclausenamide and epineoclausenamide. Mice were administered clausenamide 250 mg/kg once daily for 3 consecutive days, ig, and were killed 24 h after the last dosing. The mouse liver cytosol GSH and GST were determined with related biochemical methods. RESULTS: Nine clausenamides exhibited different effects on liver GSH and GST. Of nine clausenamides, only (+) and (+/-)clausenamide markedly increased liver cytosol GSH content. The mechanism of increasing liver GSH content of (+)clausenamide is mainly due to stimulating the key limiting enzyme gamma-glutamylcysteine synthetase (gamma-GCS) activity for GSH biosynthesis. The other test clausenamides had no such effect on liver GSH. All of the nine clausenamides induced a significant increase of GST activity. CONCLUSION: The effects of clausenamide ennatiomers on liver GST and GSH varied with the alterations of their spatial structures. (+)Clausenamide stimulated liver GSH biosynthesis through enhancing gamma-GCS activity.     

黄皮酰胺的抑制脂质过氧化和脑保护作用

黄皮酰胺是类似脑复康结构的一种化合物。实验表明po能明显延长小鼠断头后的张口呼吸时间和sc亚硝酸钠225 mg/kg后的动物存活时间。po黄皮酰胺100 mg/kg·bid×3能抑制由p0 50%乙醇(15 ml/kg)诱发小鼠肝脂质过氧化反应,使TBA反应值下降,明显激活肝和脑组织胞浆液中谷胱甘肽过氧化酶的活力。大鼠脑基底动脉条收缩实验结果表明,黄皮酰胺10^-5mol/L可明显抑制由5-HT,PGF_2x和花生四烯酸引起的血管收缩,提示黄皮酰胺有缓解血管痉挛,增加脑血流量的作用。     

(-)Clausenamide facilitates synaptic transmission at hippocampal Schaffer collateral-CA1 synapses

Clausenamide is a chiral compound isolated from leaves of the traditional Chinese herb Clausena lansium (lour) Skeels. It has been shown that (-)clausenamide, but not (+)clausenamide, improved learning and memory in amnesia animal models. However, the precise mechanism of clausenamide's actions remains unknown. Here we used an electrophysiological approach to observe the effect of (-)clausenamide on facilitating field excitatory postsynaptic potential (f-EPSP) in the CA1 area of hippocampal slices from rats. The results showed that (-)clausenamide enhanced synaptic transmission at doses 0.1, 1 and 10 μM. The increase of f-EPSP induced by (-)clausenamide was completely inhibited by preincubation with nimodipine (L-voltage-dependent calcium channel blocker, 10 μM), but there was no change when nimodipine was added after (-)clausenamide application. However, ryanodine (ryanodine receptors blocker, 100 μM) attenuated the slope of f-EPSP before or after (-)clausenamide incubation. The data suggested that (-)clausenamide promoted calcium influx to trigger intracellular calcium release which was responsible for potentiating synaptic transmission. Intracellular calcium release induced by (-)clausenamide promoted the activation of CaMKIIα at concentrations of 0.1, 1 and 10 μM, and pretreatment with KN93 (CaMKIIα inhibitor, 10 μM) completely blocked the enhancement of synaptic transmission induced by (-)clausenamide. cAMP response element-binding protein (CREB) was activated by (-)clausenamide and inhibited by KN93 preincubation, but H89 (PKA inhibitor, 10 μM) had no effect, indicating that (-)clausenamide facilitated synaptic transmission by a PKA-independent pathway. Collectively, (-)clausenamide facilitated synaptic transmission by promoting calcium influx to trigger intracellular calcium release, subsequently activating CaMKIIα-CREB signal pathway.     

Activation of ERK1/2-CREB pathway during potentiating synaptic transmission of (-)clausenamide in rat dentate gyrus

To investigate the signal mechanism of (-)clausenamide ((-)-3-hydroxy-5-(hydroxy-phenyl-methyl)-1-methyl-4-phenyl-pyrrolidin-2-one, 1) and for understanding its effect on synaptic transmission, electrophysiological recording was done for basal synaptic transmission determination. Western blot analysis was employed to examine the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cAMP responsive element-binding protein (CREB). Immunohistochemistry and tissue in situ hybridization were applied to detect the expression of Zif268. The results showed that (-)clausenamide (1) increased the population spike of hippocampal dentate gyrus. The phosphorylation of ERK1/2 in hippocampus and cortex was increased and reached the maximum at 5 min and 30 min, respectively. (-)Clausenamide (1) promoted the phosphorylation of CREB, the downstream protein of ERK1/2. The expression of Zif268 protein and mRNA increased in both hippocampal dentate gyrus and cortex. Therefore, (-)clausenamide (1) activated the ERK1/2-CREB pathway, which may provide an explanation for its effect on potentiating synaptic transmission and improving learning and memory.     

中药特征:诱导多靶点效应（英文）

In the past decades,single target drugs showed less therapeutic action and more side effects in treatment of complicated diseases such as tumor,AIDS,inflammation,diabetes,stroke and neurodegenerative disorders.The reason for this is that complicated diseases have multiple pathogenesis and multiple genes or pathological changes occur in many organs or in different kinds of cells.Therefore,scientists of worldwide hope to develop multi-target drugs recently traditional Chinese medicine(TCM)and their many effective components are characterized by inducing multi-target effects,the following 2 components isolated from TCM were proved in our laboratory to have multi-target effects which benefit complicated diseases.Anti-stroke drug-salvianolic acid B(Sal B):In MCAO rats,Sal B was shown to have many biological activities.Firstly,Sal B could past through blood-brain barrier(BBB)and could repair damage of BBB induced by cerebral ischemia.Secondly,Sal B improved blood flow in ischemic hemisphere with no steal blood and without hypotention,inhibited platelet aggregation a thrombosis but no hemorrhagic risk.More importantly,Sal B could inhibit three risk factors-intracellular Ca2+ overload,excessive regeneration of free radicals,excitotoxicity which aggravate ischemic brain injuries.Thirdly,Sal B activated organism protective mechanism such as increasing neurogenesis,angiogenesis,and many anti-oxidative substances and improving energy supply.Taking all these results we believe that sal B is a good anti-stroke agent.Anti-dementia drug-(-)clausenamide:(-)Clausenamide is a novel compound isolated from clausena lansium(Lour)which is the first chiral compound having anti-dementia effect in recent years.As the content in the plant is very low,after long term of effort this compound now been chemically synthesized by our institute and the production are has reached semi-industry scale that satisfies the demand for clinical trial and hereafter therapeutic use.For pharmacology,(-)clausenamide improved cognition and inhibited Aβpathogenesis including inhibition of Aβtoxicity and tau hyperphosphorylation.According to the new theory″Synaptic loss=AD″,agood anti-dementia drug must be able to improve synaptic plasticity and promote synaptogenesis.Fortunately,(-)clausenamide happened to be such compound.As proved in our study that(-)clausenamide increased synaptic plasticity both in efficacy and structure.For latter,(-)clausenamide increased synaptic density and expression of growth associate protein(GAP-43)in the brain significantly.Now(-)clausenamide has been developed to phaseⅡ of clinical trial.