天麻成分对羟基苯甲醛抗血小板聚集作用及急性毒性研究

为探讨天麻成分对羟基苯甲醛的抗血小板聚集作用,本实验以二磷酸腺苷(ADP)、花生四烯酸(AA)、血小板活化因子(PAF)为诱导剂,探讨了对羟基苯甲醛的体外抗血小板聚集活性;采用ADP静脉注射致小鼠肺栓塞方法以及下腔静脉结扎致大鼠静脉血栓方法,考察了对羟基苯甲醛的体内抗血小板聚集活性;并对经口给药的对羟基苯甲醛的急性毒性进行了检测。结果表明,天麻成分对羟基苯甲醛对ADP诱导的家兔体外血小板聚集有明显的对抗作用,其半数抑制率(50%inhibitory concentration,IC50)为2mmol/L,对PAF诱导的家兔体外血小板聚集无明显影响;天麻成分对羟基苯甲醛能明显降低小鼠肺栓塞死亡率,并显著对抗大鼠下腔静脉血栓的形成。说明天麻成分对羟基苯甲醛在体外、体内均具有显著的抗血小板聚集活性。急性毒性研究结果表明天麻成分对羟基苯甲醛经口给药的小鼠半数致死量(lethal dose 50,LD50)为1.23 g/kg。     

华北绣线菊二萜生物碱抗血小板聚集活性研究

采用Born氏比浊法观察华北绣线菊小叶变种中分离得到的总碱和 9个hetisine型C2 0 二萜生物碱及其衍生物体外对血小板活化因子 (PAF)、花生四烯酸 (AA)和二磷酸腺苷 (ADP)三种诱导剂引起的血小板聚集活性的影响 ,并初步探讨了构效关系。结果表明 ,华北绣线菊小叶变种中总碱对PAF和ADP诱导的血小板聚集均有明显的抑制作用 ;9个hetisine型C2 0 二萜生物碱中 ,有 8个显著抑制PAF诱导的血小板聚集 ,其活性与分子结构明显相关 ;此外 ,hetisine型生物碱及其衍生物对ADP诱导的血小板聚集亦有一定的抑制作用 ,但总碱及生物碱对AA诱导的聚集影响不明显。提示hetisine型C2 0 二萜生物碱具有抗血小板聚集活性。     

滇丹参注射液对兔血小板功能的影响

观察云南产滇丹参对血小板聚集功能的影响.方法采用Bom氏比浊法,测定滇丹参体内、体外对抗ADP、PAF、AA诱导的兔血小板聚集的作用.体外每种药物浓度分别为40 g/L、20 g/L、10 g/L、5 g/L、2.5 g/L,体内实验分为7组,即生理盐水组、两种丹参低中高剂量组分别为5 g/kg、10 g/kg、20 g/kg,每组6只.结果与对照组相比,滇丹参体外显著抑制ADP、AA诱导的血小板聚集,抑制效应呈浓度-效应关系(P＜0.05,0.01),IC50为33.7 g/L(ADP)、18.1 g/L(AA).滇丹参也显著抑制PAF诱导的血小板聚集,其最大抑制率为36.8%;体内实验结果显示,滇丹参在高剂量时可显著抑制ADP、PAF和AA诱导的血小板聚集(P＜0.05,0.01),且均具有剂量依赖性.滇丹参在药后20 min开始显效,40 min达到最大抑制作用,抑制率分别为95.6%(ADP)、91.5%(PAF)和88.5%(AA).结论以上结果表明,滇丹参体外、体内显著抑制血小板聚集,且其抗血小板聚集作用优于丹参,为进一步开发和利用滇丹参提供了依据.     

尖吻蝮蛇毒活性肽K组分对血小板聚集和超微结构的影响

目的应用电镜研究尖吻蝮蛇毒活性肽(K组分)对血小板聚集和超微结构的影响,以探讨其抗血小板聚集的机制。方法取2周内未服任何药物健康志愿者静脉血,观察K组分对二磷酸腺苷(ADP)和胶原(collagen)诱导的血小板聚集作用的影响。将已调好的PRP分为5组,A组为空白对照组,B、D组为加入等体积的生理盐水组,C、E组为加K组分组。B、C组分别加血小板聚集诱导剂ADP,D、E组分别加血小板聚集诱导剂胶原,各组分别制成超薄切片样品进行电镜观察、摄片,测出其聚集抑制率。结果K组分能抑制由ADP和胶原诱导的血小板聚集,剂量与抑制率成量效关系,IC50经直线回归分别为0.067和0.088μmol/L。ADP和胶原组血小板形态不规则,突起明显增多。K组分组血小板形态基本规则,膜表面清晰光滑,颗粒较ADP与胶原组明显增加,胞浆空泡化现象减轻。结论K组分明显抑制ADP和胶原诱导的血小板聚集反应及其超微结构的改变。     

滇产胡椒属植物醇提物抗血小板活性研究

采用Born氏比浊法观察了12种滇产胡椒属植物的乙醇提取物对血小板活化因子(PAF)、花生四烯酸(AA)和二磷酸腺苷(ADP)引起血小板活化聚集的影响.结果表明,这12种醇提物均明显抑制PAF诱导的血小板聚集,其中部分醇提物对AA和ADP引起的聚集亦有显著拮抗作用.结果提示,该12种滇产胡椒属植物醇提物具有较高的抗血小板活性     

紫锥菊愈伤组织诱导及生物活性成分分析

以紫锥菊(Echinacea purpurea)无菌苗为材料,研究了不同激素配比条件下不同外值体愈伤组织的诱导及所诱导的愈伤组织中的主要成分多糖和总酚,并应用高效液相色谱法对根愈伤组织提取物与紫锥菊根提取物中的酚酸类物质进行了比较分析.结果表明,紫锥菊不同部位愈伤组织诱导的最适宜激素配比不同,其中根愈伤组织诱导的最佳激素配比为0.5 mg/L 2,4-D 0.5 mg/L 6-BA.叶、茎、根诱导愈伤组织的多糖含量分别为10.15、11.84、14.49 mg/g干重;总酚含量分别为52.16、28.144、7.99 mg/g干重.根愈伤组织总酚和多糖含量均较高,其提取物的酚酸类物质的高效液相色谱与紫锥菊根提取物图谱主峰一致,且生长速度快、质地疏松,是细胞培养获取紫锥菊主要生物活性成分的适宜材料.     

Spiramine N-6,一种新的抗血小板和抗血小板-中性粒细胞相互作用的物质

Spiramine N-6属粉花秀线菊植物中提取分离的二十碳二萜生物碱。本实验采用Born,Shen和Hamburger等方法分别观察了spiramine N-6在体外和体内对兔血小板聚集功能的影响。应用荧光分光光度法测定其对血小板5-羟色胺释放反应的作用,同时评价spiramine N-6对激活的血小板与中性粒细胞之间粘附反应的影响。结果表明：spiramine N-6在体外选择性抑制血小板活化因子(PAF)诱导的血小板聚集,并呈量效关系,其IC50=26μmol／L,对花生四烯酸(AA)或腺苷二磷酸(ADP)引起的血小板聚集无明显作用;spiramine N-6静注后明显抑制PAF、AA和ADP诱导的血小板聚集。Spiramine N-6呈浓度依赖性减少AA和PAF引起血小板5-羟色胺的释放,其IC50分别为64．7和33．5μmol／L。Spiramine N-6明显阻抑激活的血小板与中性粒细胞间的粘附率,其IC50为78．6μmol／L。结果提示spiramine N-6作为二十碳二萜生物碱具有较强的抗血小板和阻抑血小板一中性粒细胞相互作用的生物活性。     

天麻醒脑胶囊对家兔血小板聚集和花生四烯酸诱导大鼠脑血栓形成的影响

采用比浊方法测定天麻醒脑胶囊在体内和体外对腺苷二磷酸（ADP）、花生四烯酸（arachidonic acid,AA）和血小板活化因子（platelet activating factor,PAF）诱导家兔血小板活化聚集的影响。采用从经大鼠颈内动脉注射诱发同侧大脑半球脑血栓形成方法评价天麻醒脑胶囊的抗脑血栓形成作用。天麻醒脑胶囊在体外呈浓度依赖性明显抑制从和ADP引起的血小板聚集,其半数抑制浓度（50％of inhibitory concentration,IC50）分别为1．83和3．25g/L。0．5和1g/kg的天麻醒脑胶囊于灌胃后明显抑制从诱导的家兔血小板聚集,本品1g/kg时显著阻抑ADP引起的血小板聚集。天麻醒脑胶囊在体内外对PAF诱导的血小板聚集均无明显影响。1、2g/kg天麻醒脑胶囊组的右侧与左侧脑重差值均显著减小,显著降低右脑伊文思蓝吸光度与右脑重的比值。结果提示,天麻醒脑胶囊具有较强的抗血小板和减轻脑血栓形成作用,有利于血小板聚集性增高的血栓栓塞性疾病的防治。     

基于斑马鱼模型和动态分子对接技术探讨西洋参抗缺氧作用及潜在靶点CSCD

本研究利用斑马鱼模型和动态分子对接技术研究西洋参抗缺氧(hypoxia)的作用及潜在靶点。以AB系斑马鱼为实验动物,无水硫酸钠为造模剂诱导形成斑马鱼幼鱼缺氧模型,综合评价西洋参的抗缺氧作用。借助网络药理学技术筛选西洋参活性成分以及缺氧相关共有靶点;并使用STRING平台和Cytoscape 3.8.2软件构建蛋白-蛋白作用网络图,寻找西洋参抗缺氧可能的潜在靶点;利用动态分子对接技术验证活性成分与关键靶点的结合能力和稳定性。斑马鱼体内实验显示,西洋参提取物可明显增加斑马鱼在缺氧条件下的存活率,减轻因缺氧导致的神经行为状态(P<0.05),且呈剂量依赖性相关。共筛选获得西洋参7个潜在活性成分和5个抗缺氧潜在靶点;动态分子对接结果显示,西洋参关键活性成分与靶点之间有良好的结合能力,其中TNF、HSP90AA1与活性成分稳定结合,可能为西洋参抗缺氧的关键潜在靶点。综上,本实验建立的斑马鱼幼鱼抗缺氧模型可以快速简便地评价西洋参样品的抗缺氧活性,动态分子对接的结果显示该作用可能通过TNF以及HSP90AA1等靶点发挥作用,为后续西洋参抗缺氧机制研究提供了参考依据。     

大孔吸附树脂对紫锥菊提取物中菊苣酸分离纯化的研究

采用大孔吸附树脂分离纯化紫锥菊提取物中免疫活性成分菊苣酸,7％(v／v)的甲醇-水溶液洗脱,HPLC法对产品中菊苣酸含量进行检测分析。该法能把紫锥菊提取物中菊苣酸含量(4％)提高9倍左右,回收率达到95％以上,且操作简单,可用于菊苣酸的分离纯化。     

The evaluation and structure-activity relationships of 2-benzoylaminobenzoic esters and their analogues as anti-inflammatory and anti-platelet aggregation agents

Forty-seven 2-benzoylaminobenzoic esters were synthesized and evaluated in anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Most 2-benzoylamino-4-chlorobenzoic acid derivatives showed selective inhibitory effects on arachidonic acid (AA)-induced platelet aggregation. Among them, compounds 6b and 7b exhibited more potent inhibitory effects (ca. 200-fold) than aspirin. Additionally, compounds 1a and 5a showed strong inhibitory effects on neutrophil superoxide generation with IC(50) values of 0.65 and 0.17 microM, respectively. However, compounds 6d and 6e exhibited dual inhibitory effects on platelet aggregation and neutrophil elastase (NE) release; therefore, these two compounds may be new leads for development as anti-inflammatory and anti-platelet aggregatory agents.     

The evaluation of 2,8-disubstituted benzoxazinone derivatives as anti-inflammatory and anti-platelet aggregation agents

A series of 2,8-disubstituted benzoxazinones were synthesized and subjected to anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Among them, 2-(2'-substituted-phenyl)-benzoxazinones exhibited significant inhibitory effect to target assays. Additionally, all of them were more potent than aspirin on AA-induced platelet aggregation, and these suggested that 2-(2'-substituted-phenyl)-benzoxazinones also possess aspirin-like activity. On the other hand, the compounds 6 and 16 showed inhibitory effects on neutrophil elastase release and superoxide generation.     

Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets

The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.     

Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats

We have previously reported that Chinese traditional medicine rutaecarpine (Rut) produced a sustained hypotensive effect in phenol-induced and two-kidney, one-clip hypertensive rats. The aims of this study are to determine whether Rut could exert antihypertensive and anti-platelet effects in spontaneously hypertensive rats (SHR) and the underlying mechanisms. In vivo, SHR were given Rut and the blood pressure was monitored. Blood was collected for the measurements of calcitonin gene-related peptide (CGRP), tissue factor (TF) concentration and activity, and platelet aggregation, and the dorsal root ganglia were saved for examining CGRP expression. In vitro, the effects of Rut and CGRP on platelet aggregation were measured, and the effect of CGRP on platelet-derived TF release was also determined. Rut exerted a sustained hypotensive effect in SHR concomitantly with the increased synthesis and release of CGRP. The treatment of Rut also showed an inhibitory effect on platelet aggregation concomitantly with the decreased TF activity and TF antigen level in plasma. Study in vitro showed an inhibitory effect of Rut on platelet aggregation in the presence of thoracic aorta, which was abolished by capsazepine or CGRP(8-37), an antagonist of vanilloid receptor or CGRP receptor. Exogenous CGRP was able to inhibit both platelet aggregation and the release of platelet-derived TF, which were abolished by CGRP(8-37). The results suggest that Rut exerts both antihypertensive and anti-platelet effects through stimulating the synthesis and release of CGRP in SHR, and CGRP-mediated anti-platelet effect is related to inhibiting the release of platelet-derived TF.     

Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation.

The effects of aqueous extracts of raw and boiled garlic and onions were studied in vitro on the collagen-induced platelet aggregation using rabbit and human platelet-rich plasma. A dose dependant inhibition of rabbit platelet aggregation was observed with garlic. Onion also showed dose-dependent inhibitory effects on the collagen-induced platelet aggregation but this inhibition was of a lesser magnitude compared to garlic when related to dose. The concentration required for 50% inhibition of the platelet aggregation for garlic was calculated to be approximately 6.6 mg ml(-1) plasma, whereas the concentration for onion was 90 mg ml(-1) plasma. Boiled garlic and onion extracts showed a reduced inhibitory effect on platelet aggregation. Garlic but not onion significantly inhibits human platelet aggregation in a dose-dependent fashion. The potency of garlic in inhibiting the collagen-induced platelet aggregation is approximately similar to that of rabbit platelets (8.8 mg ml(-1) produced 50% inhibition of platelet aggregation). The results of this study show that garlic is about 13 times more potent than onion in inhibiting platelet aggregation and suggest that garlic and onion could be more potent inhibitors of blood platelet aggregation if consumed in raw than in cooked or boiled form.     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.     

ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationship between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation

Four spin-labeled probes (5-doxylstearic acid (5-NS), its methyl ester (5-NMS), 16-doxylmethylstearate (16-NMS) and 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl (CAT-15)) were used to monitor membrane fluidity change in bovine platelets induced by three alkyl alcohols, benzyl alcohol and two phenolic compounds. The relationship between the increase in membrane fluidity induced by these compounds and their inhibitory effects on platelet aggregation was observed. Experiments with the four probes showed that n-hexyl alcohol induced decreases in the order parameter of 5-NS and apparent rotational correlation times of the other probes at the same minimal alcohol concentration. The decreases were observed in the concentration range that inhibited aggregation. n-Amyl alcohol and n-butyl alcohol decreased the values of the parameters of the above mentioned only at higher concentrations that were dependent on their hydrophobicities. Like alkyl alcohols, benzyl alcohol and phenolic compounds decreased the values of the parameters in the concentration ranges in which these compounds inhibited platelet aggregation. The concentration of these compounds causing 50% inhibition of platelet aggregation, the IC50 values, and data on 5-NS-labeled platelets indicated that they inhibited aggregation and decreased the value of the order parameter at lower concentrations relative to their Poct values in comparison to the effective concentrations of alcohols. Phenolic compounds also decreased the values of the apparent rotational correlation times of 5-NMS and 16-NMS. These results indicate that the inhibition of platelet aggregation by alcohols and phenolic compounds is due to membrane perturbation in wide range in depths within the lipid bilayer.     

Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC

A fast and reliable HPLC method for the determination of caffeic acid derivatives (caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid) in various species of the genus Echinacea has been developed. Extraction of root samples by magnetic stirring with 80% methanol aqueous solution at room temperature allowed the complete recovery of all compounds of interest. Root extracts were analysed on a reversed-phase column with gradient elution and photodiode array detection. Caffeic acid derivatives showed differential qualitative and quantitative distributions in Echinacea species. The total amount of phenolic compounds ranged from 33.95 to 0.32 mg/g. The highest contents of caffeic acid derivatives were found in E. paradoxa var. paradoxa, E. paradoxa var. neglecta and E. purpurea, followed by E. angustifolia var. angustifolia, E. simulata, E. pallida and E. laevigata, whilst E. tennesseensis, E. sanguinea and E. atrorubens had low amounts of phenolic compounds. The radical scavenging activities of methanolic extracts of roots of Echinacea species was evaluated in vitro using the DPPH* radical scavenging method. The EC50 values of the samples ranged from 122 to 1223 microg/mL. The radical scavenging activities of the root extracts were correlated with the content of phenolic compounds, with a correlation coefficient (r2) of 0.923.     

Evaluation of the bioactivities of extracts of endophytes isolated from Taiwanese herbal plants

The endophytic extracts from 19 endophytes, isolated from 13 species of Taiwanese plants, were evaluated for biological activity, including cytotoxicity, anti-platelet aggregation, and anti-inflammatory activity. The extracts of 12 endophytes exhibited inhibitory effects on collagen-induced platelet aggregation with IC₅₀ values of 19.85–87.64 μg/ml. Four strains, Rahnella aquatilis, Pantoea agglomerans, Rhodotorula sp., and Penicillium paxilli, also showed inhibitory effects on thrombin-induced platelet aggregation with IC₅₀ values of 42.80–61.54 μg/ml. Additionally 12 extracts of endophytes exhibited cytotoxicities with IC₅₀ values of 0.12–19.83 μg/ml. However, eight extracts revealed inhibitory effects on superoxide anion generation induced by fMLP (N-formyl-l-methionyl-l-leucyl-l-phenylalanine) in human neutrophils. The extract of Rahnella aquatilis showed anti-platelet aggregation activity, and bioassay-directed fractionation led to the isolation of six compounds, including one isoalloxazine: lumichrome (1); two isoflavones: genistein (2) and daidzein (3); two cyclic peptides: cyclo-Pro-Val (4) and cyclo-Pro-Phe (5); and one benzenoid: methyl 2,4,5-trimethoxybenzoate (6). These results indicated that endophytes from Taiwanese herbal plants could be useful sources for research and development of bioactive lead compounds from nature.     

Inhibition of fibrinogen binding to human platelets by S-nitroso-N-acetylcysteine

Because of the central role of fibrinogen binding in platelet aggregation and recent evidence implicating S-nitrosothiol compounds in the platelet inhibitory effects of endogenous and exogenous organic nitrate compounds, we examined the effect of the S-nitrosothiol S-nitroso-N-acetylcysteine (SNOAC) on fibrinogen binding to gel-filtered human platelets. We found that SNOAC markedly inhibited the binding of fibrinogen to normal human platelets in a dose-dependent fashion and that this inhibitory effect was the result of both an increase in the apparent Kd of the platelet receptor for the fibrinogen molecule (from 6.8 x 10(-7) to 1.8 x 10(-6) M, a 2.7-fold increase) and a decrease in the total number of fibrinogen molecules bound to the platelet (from 76,200 to 38,250, a 50% decrease). In addition, we noted a rapid, dose-dependent rise in platelet cyclic GMP levels following exposure of platelets to SNOAC which was significantly inversely correlated with fibrinogen binding and was accompanied by inhibition of intracellular calcium flux in response to a variety of platelet agonists. Similar dose-dependent inhibition of fibrinogen binding was found in the presence of cyclic GMP analogues and was significantly enhanced by inhibition of platelet cyclic GMP phosphodiesterase. These results describe the inhibition of platelet fibrinogen binding by an S-nitrosothiol compound, help define the biochemical mechanism by which S-nitrosothiols inhibit platelet aggregation, and lend support to the view that cyclic GMP is an important inhibitory intracellular mediator in human platelets.