瑞香狼毒抗癫痫活性部位筛选及活性部位中总黄酮含量测定

目的探索瑞香狼毒抗癫痫活性部位、活性部位中总黄酮含量测定方法及总黄酮含量与抗癫痫活性之间的关系。方法采用乙醇提取和不同溶剂萃取等手段,通过惊厥实验模型对瑞香狼毒各提取部位的活性筛选,确定其抗癫痫活性部位;采用紫外分光光度法对活性部位中总黄酮进行了含量测定。结果初步确定乙醚提取部位与丙酮提取部位为其抗癫痫活性部位,乙醚提取部位表现出较强的抗癫痫活性和较高的死亡率,丙酮提取部位显示了更强的综合治疗效果;乙醚活性部位中总黄酮含量为8.51%,丙酮活性部位中总黄酮含量为5.07%。结论紫外分光光度法测定其活性部位中总黄酮含量,准确性高、重现性好、简便易行;初步推断其活性部位中总黄酮含量越高,其抗癫痫活性作用也可能越强。     

黄芩抗乙酰胆碱酯酶活性部位的RRLC-DAD-ESI-MS/MS分析

目的：筛选黄芩体外抑制乙酰胆碱酯酶（AChE）的活性部位，并对其化学成分进行分析。方法：黄芩提取物经反相C₁₈填料进行固相萃取（SPE）制备不同浓度甲醇醇梯度洗脱部位，以体外抑制AChE能力评价其抗阿尔茨海默症（AD）作用，采用高分离快速液相色谱-质谱联用技术（RRLC-MS/MS）对黄芩抑制AChE活性部位化学成分进行分析。结果：黄芩提取物的50%甲醇洗脱部位的AChE抑制活性明显强于其他洗脱部位并存在明显的量效关系，为其体外抑制的活性部位，经液质联用分析共鉴定出15种化合物。结论：黄芩具有体外抑制AChE活性，其主要有效部位为50%甲醇洗脱部位，其主要有效成分为黄芩苷为代表的黄酮类成分。     

分子蒸馏技术筛选退热止痛散镇痛活性部位研究

目的:筛选退热止痛散的镇痛活性部位。方法:采用超临界CO2流体萃取技术从退热止痛散中提取镇痛活性部位,采用分子蒸馏技术对其进行分离纯化,以镇痛药效为指标,筛选更纯化的镇痛活性部位。结果:分离得到的镇痛活性部位具有很好的镇痛效果。结论:该工艺为基于该活性部位的新药开发提供理论依据。     

白及不同提取部位对小鼠止血活性实验

目的确定白及的止血活性部位,为其止血活性成分的筛选提供线索和依据。方法用70%乙醇(EtOH)对白及原药材进行渗漉提取,然后,依次用石油醚(Pet)、乙酸乙酯(EtOAc)、正丁醇(n-BuOH)对醇浸膏进行萃取,分别制得Pet部分、EtOAc部分、n-BuOH部分和H2O部分,通过测定小鼠凝血时间和出血时间,考察其活性部位。结果 n-BuOH部分和H2O部分具有止血作用,而EtOAc部分具有延长凝血、出血时间作用。结论白及中具有止血作用的活性成分主要存在于n-BuOH部分和H2O部分,而EtOAc部分具有活血作用。     

抗有机磷酸酯药物的合成及其构效关系的研究

本文报道一类双吡啶季铵盐化合物,对抗小鼠有机磷酸酯化合物中毒有效,其中一些化合物的抗毒效价超过了文献报道的HI-6,HGG-42的水平。为探讨其构效关系,用EHMO方法计算了23个化合物的分子轨道指数,从中发现吡啶环Ⅱ和羰基可能是化合物的活性部位,影响抗毒作用的因素有二个,并再次设计合成了二个新化合物,量化计算预测的抗毒效价与药理结果一致。化合物与胆碱N受体结合有较强的选择性,根据计算,提出了其可能的作用模式,推测N受体活性部位除有文献报道的阴离子部位、亲酯中心、疏水区域外,还可能有轨道作用部位。     

顺-3-甲基芬太尼的4-N-丙酰基结构类似物的合成与镇痛活性及构效关系的研究

以不同电性的基团取代顺-3-甲基芬太尼中4-N-丙酰基上的乙基,合成某些顺-3-甲基芬太尼的结构类似物。药理试验结果表明,所合成的化合物均有典型的吗啡样作用。化合物3的镇痛活性略强于顺-3-甲基芬太尼。应用半经验的INDO方法对4个代表化合物进行了量子化学计算,讨论了电子结构与镇痛活性间的关系,化合物3由于氯乙烯基的引入具有与顺-3-甲基芬太尼不同的电子结构特征,氯乙烯基可能作为电子接受体参与了与受体的作用。     

4种海星的α-葡萄糖苷酶抑制活性研究

目的寻找具有降糖活性作用的海星并确定其活性部位。方法首先采用正常和糖尿病小鼠口服糖耐量试验,比较4种海星粗提物改善口服糖耐量的作用,然后利用体外α-葡萄糖苷酶抑制剂模型进一步确定具有改善口服糖耐量作用的海星中抑制α-葡萄糖苷酶的活性部位。结果罗氏海盘车粗提物对正常小鼠和糖尿病小鼠餐后0.5、1 h血糖均有显著降低作用,其中的正丁醇部位具有很强的α-葡萄糖苷酶抑制活性。结论罗氏海盘车的正丁醇部位具有α-葡萄糖苷酶抑制作用。     

顺-3-甲基芬太尼的4-N-丙酰基结构类似物的合成与镇痛活性及构效关系的研究

以不同电性的基团取代顺-3-甲基芬太尼中4-N-丙酰基上的乙基,合成某些顺-3-甲基芬太尼的结构类似物。药理试验结果表明,所合成的化合物均有典型的吗啡样作用。化合物3的镇痛活性略强于顺-3-甲基芬太尼。应用半经验的INDO方法对4个代表化合物进行了量子化学计算,讨论了电子结构与镇痛活性间的关系,化合物3由于氯乙烯基的引入具有与顺-3-甲基芬太尼不同的电子结构特征,氯乙烯基可能作为电子接受体参与了与受体的作用。     

基于半体内法研究利胆排毒方抗内毒素活性部位

摘 要 目的：观察利胆排毒方活性部位半体内抗内毒素作用强度,进一步确认抗内毒素作用的活性部位。方法: 利胆排毒方提取液采用不同极性溶剂进行萃取,获取活性部位。采用偶氮化显色基质法测定该部位抗内毒素活性,比较不同剂量组不同提取部位的内毒素灭活量。结果: 各给药组大鼠血浆对内毒素灭活量均高于空白对照组,差异有统计学意义（P<0.05）;全方提取液各剂量组给药大鼠血浆对内毒素有灭活作用,但组间差异无统计学意义（P＞0.05）;水溶性部位给药大鼠血浆各组也表现出对内毒素的灭活作用,且高剂量组与低剂量组差异有统计学意义（P<0.05）。结论:半体内抗内毒素作用实验结果可见水溶性部位抗内毒素作用与全方提取液相当,且初步呈现出一定的量效关系,则水溶性部位很大程度上保留了全方提取液的有效成分,可进一步确认为利胆排毒方抗内毒素活性部位。     

T肽研究进展

ＨＩＶ包膜中ｇｐ１２０糖蛋白与ＣＤ４抗原结合是ＨＩＶ感染Ｔ淋巴细胞的基础。Ｔ肽是ｇｐ１２０糖蛋白中的一多肽序列。人工合成的Ｔ肽是ｇｐ１２０糖蛋白与ＣＤ４抗原结合的竞争性抑制剂，它具有单核细胞趋向性激活作用，对ｇｐ１２０糖蛋白所致的脑细胞死亡具有保护作用。在临床上Ｔ肽对ＡＩＤＳ病人的躯体状况改善及精神恢复具有一定作用，对治疗牛皮癣也有一定效果。对Ｔ肽构效关系研究表明，其序列中活性部位是－Ｔｈｒ＾５－     

Binding of naloxone to human T lymphocytes

Purified T lymphocytes have a specific binding site for naloxone, the opiate antagonist. The KD for the site was 50.6 +/- 2.4 nM, while the Hill coefficient (n) was 1.67 +/- 0.16, indicating a degree of positive cooperativity of ligand binding. The bound naloxone was partially displaceable by various opiate agonists including morphine (56%), beta-endorphin (61%), met5- and leu5-enkephalin (40% each), [D-ala2, D-leu5]-enkephalin (78%) and [D-ala2, D-leu5]-enkephalinamide (66%). Virtually all of the binding capacity was recovered in the particulate membrane fraction after sonic lysis of the cells. There was great interindividual variability in Bmax between samples, suggesting a possible mechanistic basis for the variability in drug action seen between different individuals.     

High affinity [3H]ethylketazocine binding: evidence for specific kappa receptors

A study has been made of the inactivation of mu(mu) ([3H]-dihydromorphine), delta (delta) ([3H](D-ala2-D-leu5)enkephalin) and kappa (kappa) ([3H]ethylketazocine) opiate receptor binding sites by N-ethylmaleimide (NEM) and it was observed that in contrast to mu and delta sites, the kappa sites of rat brain membrane preparations were resistant to low concentrations of N-ethylmaleimide. Furthermore, this kappa site was selectively protected, from inactivation with high concentrations of N-ethylmaleimide, by the kappa agonists ethylketazocine and (-)-alpha-(1R,5R,9R)-5,9-dimethyl-2-(L-tetrahydrofurfuryl)-2'-hydroxy-6,7-benzo morphan (MR-2034) but not by morphine or (D-ala2-D-leu5)-enkephalin. These studies suggest that a unique kappa receptor is present in the rat CNS.     

Pharmacological differentiation of presynaptic inhibitory alpha-adrenoceptors and opiate receptors in the cat nictitating membrane.

1 The action of morphine, naturally occurring and synthetic opiate peptides on [3H]-noradrenaline release induced by nerve stimulation was studied in the isolated nerve muscle preparation of the cat nictitating membrane under experimental conditions in which the alpha-presynaptic receptors were blocked by phentolamine 1 microM. 2 Morphine and the naturally occurring peptides: [Met5]-enkephalin, [Leu5]-enkephalin and beta-endorphin reduced 3H-transmitter overflow and responses to nerve stimulation from the cat nictitating membrane, effects which were completely antagonized by naloxone 0.3 microM. The relative order of potency for the inhibition of the stimulation-induced 3H-transmitter overflow at the level of the IC50 (microM) was as follows: [Met5]-enkephalin (0.020 microM) greater than or equal to [Leu5]-enkephalin (0.036 microM) > morphine (0.3 microM) > beta-endorphin (1 microM). 3 The synthetic opiate pentapeptides: BW 180 C (Tyr-D-Ala-Gly-Phe-D-Leu), and BW834 C (Tyr-D-Ala-Gly-pClPhe-DLeu), which are resistant to enzymatic degradation were more potent than the enkephalins in reducing the stimulation-evoked transmitter overflow from the cat nictitating membrane. On the other hand, the tetrapeptide BW832 C, which lacks the D-leucine terminal of BW180 C l was less potent than the enkephalins in inhibiting neurotransmission. 4 In the presence of phenoxybenzamine 1 microM, 3H-transmitter overflow was increased 8 fold and the inhibition of neurotransmission by methionine-enkephalin was not affected. Exposure to phenoxybenzamine 10 microM increased [3H]-noradrenaline overflow 15 fold and antagonized the effects of methionine enkephalin on transmitter release. 5 In the cat nictitating membrane the inhibitory presynaptic opiate receptors are different from the presynaptic alpha-autoreceptors which regulate the release of noradrenaline elicited by nerve depolarization through a negative feed-back mechanism.     

The regulation of delta-opiate receptor density on 108CC15 neuroblastoma X glioma hybrid cells.

The effect of exogenous substances on the expression of opiate receptors on 108CC15 neuroblastoma X glioma hybrid cells has been studied. Cell differentiation by culture in the presence of N6-O2-dibutyryl adenosine 3',5'-cyclic monophosphate induced a three fold increase in opiate receptor density. When the cells were grown in the presence of 10(-5) M morphine hydrochloride for up to 23 days, opiate receptor densities were reduced by only 30% when compared with matched controls. Culture in the presence of 10(-7) M D-Ala2-D-Leu5-enkephalin produced opiate receptor down regulation of 73% compared to controls after only 4 h of treatment. The down regulation process could be inhibited by continued exposure to D-Ala2 D-Leu5-enkephalin at concentrations greater than 4 nM; below this concentration down regulation was rapid and irreversible. A model to explain these observations is described.     

Discrimination of three opiate receptor binding sites with the use of a computerized curve-fitting technique

The presence of different types of opiate binding sites was investigated with the use of a computerized, weighted, nonlinear least-squares regression program. The experimental data were obtained from four groups. Each of three labeled opiate ligands was displaced using each of the same unlabeled ligands. The resulting nine different ligand combinations of each group were evaluated by use of a curve-fitting program. The four groups consisted of the kappa ligand ethylketocyclazocine, the sigma ligand SKF 10047, and the oripavine derivatives etorphine and diprenorphine, each in conjunction with the delta opiate receptor ligand (D-Ala2,D-Leu5)-enkephalin and the mu opiate receptor ligand dihydromorphine. The binding model which best fitted each of the four groups suggested the existence of three different binding sites in the rat brain homogenate. Two of these sites conform to the previously described mu and delta sites. A third site (R3) displayed high affinity for ethylketocyclazocine, SKF 10047, etorphine, and diprenorphine but very low affinity for dihydromorphine and [D-Ala2,D-Leu5]enkephalin. Naloxone, cyclazocine, and dynorphin-(1--13) had high affinity for R3. Behavioral data support the interpretation that the R3 site may represent a kappa site at which SKF 10047 acts antagonistically.     

Morphine tolerance increases mu-noncompetitive delta binding sites

In light of more recent knowledge concerning endogenous opioid peptides and their multiple opiate receptors, we reevaluated the effects of morphine tolerance on opiate receptor binding parameters. Rats were implanted with morphine or placebo pellets, and [3H][D-Ala2,D-Leu5]enkephalin ([3H]DADL) was used to label brain membranes. Utilizing the technique of binding surface analysis, we observed a selective 47% up-regulation of lower affinity [3H]DADL binding sites (mu-noncompetitive delta binding sites) in morphine pelleted rats. To corroborate these results, we treated brain membranes with the site directed alkylating agent FIT (N-phenyl-N-[1-(2-p-isothiocyanato)phenyl-ethyl)-4-piperidinyl] propanamide), which results in membranes highly enriched with the lower affinity [3H]DADL binding site. Scatchard plots of [3H]DADL binding to FIT-treated membranes also revealed that chronic morphine treatment produced a 60-65% up-regulation of the mu-noncompetitive delta binding site. These data indicate that chronic morphine alters a selective subpopulation of opiate receptors that may play a role in the mechanisms of opiate tolerance and physical dependence.     

Morphiceptin: biphasic inhibition of mu-opiate receptor ligands

The interaction between morphiceptin and the morphine (mu) opiate receptor present in rat brain membranes has been examined. Detailed competitive displacement curves of morphiceptin against the mu receptor ligands [3H]fentanyl and [3H]naloxone were biphasic, with Hill coefficients of 0.78 and 0.60 respectively. Hoftsee plots of these displacement curves suggested that 30-35% of the morphiceptin binding was to a high affinity site and the residual binding was to a site with lower affinity. These results indicate that morphiceptin binding to the mu opiate receptor does not obey the law of mass action, and raises the possibility that morphiceptin distinguishes subclasses of mu binding site.     

Design, synthesis, and binding characteristics of an opiate receptor mimetic peptide

An artificial tetracontapeptide that mimicked the recognition/binding properties of naturally occurring opioid receptors was designed, synthesized, and purified to homogeneity. The design of the primary structure of the receptor mimetic peptide (RMP) accounted for secondary structure prediction rules and for the stereochemical anatomy of various enkephalin and morphine derivatives. The affinity of a series of opioid and nonopioid peptides to RMP was determined from their potency in displacing the binding of enzymatically prepared (14C)-[Leu]-enkephalin. The competition studies revealed that the binding is specific for endogenous opiate peptides, stereoselective for the naturally occurring L isomer of [Leu]-enkephalin, and discriminative for closely related opioid peptides. The thermodynamic parameters associated with the binding of [Leu]-enkephalin to RMP were evaluated from equilibrium studies at different temperatures. The van't Hoff plot of the resulting data was curvilinear. The formation of the ligand--RMP complex was characterized by a decrease both in entropy and in enthalpy with temperature. The thermodynamic behavior provided some evidence that hydrophobic interactions played a prominent role in stabilizing the [Leu]-enkephalin--RMP complex.     

Action of enkephalin analogues and morphine on brain acetylcholine release: differential reversal by naloxone and an opiate pentapeptide.

1 Methionine (Met)-enkephalin, leucine (Leu)-enkephalin and their synthetic analogues were tested for effects on the spontaneous release of cortical acetylcholine (ACh) in vivo. The ability of naloxone to reverse the action of enkephalins on ACh release was compared with its action against morphine. An enkephalin analogue, structurally related to Met-enkephalin, was tested for opiate antagonistic activity in ACh release experiments. 2 Intraventricular administration of Met-enkephalin, Leu-enkephalin, D-Ala2-Met5-enkephalinamide (DALA) and D-Ala2-D-Leu5-enkephalin (DALEU) produced a dose-related inhibition of cortical ACh release. Met- and Leu-enkephalin were very similar both in their potency and the time course of their action on ACh release. Both DALA and DALEU were more potent and had a longer duration of action than Leu-enkephalin. Systemic injections of two pentapeptides, D-Met2-Pro5-enkephalinamide and D-Ala2-MePhe4-Met5(O)-ol-enkephalin (33,824), produced a sustained inhibition of cortical ACh release. 3 Naloxone, administered systemically following the depression of ACh release induced by either intraventricular injections of enkephalins (DALA or DALEU), or systemic injections of enkephalins (D-Met2-Pro5-enkephalinamide or 33,824), reversed this depression and restored the release to baseline levels. The effect of D-Met2-Pro5-enkephalinamide on the release of ACh was reversed by naloxone with difficulty. Naloxone also reversed the inhibitory effect of systemic morphine and this reversal was associated with a large overshoot of ACh release. The latter was never observed in the enkephalin experiments. 4 Intraventricular injection of the pentapeptide, D-Ala2-D-Ala3-Met5-enkephalinamide (TAAPM), at doses that did not influence the basal ACh release, blocked or reversed the inhibitory effect of morphine on this release. This peptide did not block the effect of the non-opiate, chlorpromazine, under similar conditions. In two experiments TAAPM failed to reverse the inhibition of ACh release produced by systemically injected enkephalin, D-Met2-Pro5-enkephalinamide. 5 Effects of morphine and enkephalin on ACh release are discussed in terms of their action on difference opiate receptors.     

Peripheral analgesic actions of opioid peptides and morphine analogues

Opiates and opioid peptides were administered in the order of 10(-9)-10(-6) mol peripherally, and their action on pain sensitivity was investigated by the modified formalin test which has two characteristic pain responses (the first and the second phase) in the mouse hindpaw. Opioid peptides (20-500 pmol) had dose-dependent analgesia against both first and second phases, and their action ranked dynorphin greater than [D-Ala2, Met5]-enkephalinamide greater than [Met5]-enkephalin. EKC and morphine (0.4-2.5 nmol) inhibited pain response of the first phase, but produced hyperalgesia in the second phase dose-dependently. Lidocaine hydrochloride had peripheral analgesic action, but was about 500-10000 times weaker than these substances. So, these peripheral analgesic actions have a different mechanism from that of local anesthetic action. N-methyl levallorphan which is thought to be a peripherally selective narcotic antagonist reversed these peripheral analgesic actions at the first and second phases and also prevented the hyperalgesic effects of EKC and morphine at the second phase. Naloxone reversed analgesia at only the first phase. These results suggest that an analgesic mechanism by opioids may exist at the peripheral site as well. Furthermore, it is estimated that a receptor exists which is antagonized by N-methyl levallorphan but not by naloxone and that there is a system of hyperalgesia by EKC and morphine in pain modulation.