黄藤素衍生物的合成及其抑制乙酰胆碱酯酶活性研究

对黄藤素进行结构修饰,采用160~180℃高温热解使黄藤素选择性在9-位脱甲基,再分别与一系列酰氯发生酯化反应,最终获得12种黄藤素衍生物,经~1H-NMR、~(13)C-NMR分析确定了各衍生物的化学结构,分别为:9-O-苯甲酰基-黄藤素(9-O-Benzoyl-fibrauretin)、9-O-(2-甲基苯甲酰基)-黄藤素(9-O-(2-Methylbenzoyl)-fibrauretin)、9-O-(4-甲基苯甲酰基)-黄藤素(9-O-(4-Methylbenzoyl)-fibrauretin)、9-O-(3,5-二甲基苯甲酰基)-黄藤素(9-O-(3,5-Dimethylbenzoyl)-fibrauretin)、9-O-(4-(氯甲基)苯甲酰基)-黄藤素(9-O-(4-(Chloromethyl)benzoyl)-fibrauretin)等共12种化合物,均为新化合物。采用以碘化硫代乙酰胆碱为底物、来源于苍蝇头部的乙酰胆碱酯酶(ACh E)为酶源的体外活性测定方法,测定了黄藤素及其衍生物的ACh E抑制活性。结果表明,大部分黄藤素酰氯衍生物体外ACh E抑制活性均强于黄藤素,其中化合物9-O-(4-甲基苯甲酰基)-黄藤素、9-O-(3,5-二甲基苯甲酰基)-黄藤素、9-O-(4-(氯甲基)苯甲酰基)-黄藤素对ACh E的抑制作用显著,活性强度强于阳性药盐酸多奈哌齐,具有开发成抗阿尔茨海默症药物的潜力。     

毛细管电泳结合高效液相色谱-质谱用于天然产物活性成分的筛选

发展了毛细管电泳(CE)和高效液相色谱-质谱(HPLC-MS)相结合的用于天然产物中活性成分筛选和鉴定的方法。该方法中,用HPLC半制备柱对天然产物粗提物进行分离纯化,再用CE对HPLC纯化后的组分进行活性测试。根据HPLC-MS/MS提供的二级质谱数据,即可确定活性成分的化学结构。以乙酰胆碱酯酶为实验模型,对我们发展的筛选方法进行了验证。从黄连粗提物中确定了药根碱、巴马汀等7种活性成分,并通过CE测定了它们的半抑制率(IC₅₀)值。与传统的天然产物分离纯化和活性筛选方法相比,该方法具有简单、微量、快速、准确的优点。本文建立的方法为天然产物粗提物中活性成分的筛选提供了新技术。     

6-羟基石松碱与乙酰胆碱酯酶的相互作用

目前治疗阿尔茨海默症(Alzheimer’s disease,AD)的主要药物为乙酰胆碱酯酶抑制剂(AChEI).为探讨石松碱类化合物对AChE的抑制作用,以6-羟基石松碱(6-hydroxylycopodine,HLD)为对象,应用核磁共振、分子对接、酶活性测定、分子动力学模拟及自由能分析,研究了HLD与AChE的相互作用.研究发现6-羟基石松碱对乙酰胆碱酯酶具有混合型抑制作用,其结合作用主要来自氢键和范德华作用.     

高效液相色谱-质谱联用快速筛选并鉴定黄芩甲醇提取物中抗氧化活性成分

建立了高效液相色谱-质谱联用技术快速筛选和鉴定黄芩甲醇提取物中抗氧化活性成分的方法.在黄芩甲醇提物中加入适量的二苯基三硝基苯肼( DPPH)作为实验组,避光室温反应30 min后直接经液相色谱分析,并与不加入DPPH的空白组进行比较.有抗氧化活性的成分因会与DPPH反应而使峰面积减少,而不具有抗氧化活性的成分峰面积则不变.基于色谱数据可以获得有抗氧化活性组分的相对活性强度,基于质谱数据可获得活性组分的结构信息用于结构鉴定.本方法成功地从黄芩甲醇提取物中筛选并鉴定出了黄芩苷、千层纸素A-7-O-B-D-葡萄糖醛酸苷、汉黄芩苷和千层纸素A4种较强的抗氧化活性成分.本方法利用HPLC-MS技术对复杂体系(如中药提取物)中小分子的分离和鉴定优势,可以直接筛选出黄芩甲醇提取物中的抗氧化活性成分,而不需要繁琐的前期分离和纯化,并且不需要仪器改装工作,有利于实现复杂体系中抗氧化活性成分的高通量筛选.     

S-烃基-1-烃基-3-[4-(苯并噻唑-2-巯基)苯基]异硫脲的合成及生物活性

以2-巯基苯并噻唑(1)为原料,经缩合和还原得到2-(4-氨基苯硫基)苯并噻唑(3),再与异硫氰酸苯甲酰酯或异硫氰酸烃基酯反应得到取代硫脲(5和7),最后与卤代烃反应得到20个新的S-烃基-1-烃基-3-[4-(苯并噻唑-2-巯基)苯基]异硫脲化合物(6和8),其结构经IR,1H NMR,MS及元素分析确证.初步的药理试验表明,20个目标化合物均有不同程度的iNOS抑制活性,化合物8a,8b,8c,8e,8f,8i,8j和8k的iNOS抑制活性强于阳性对照药氨基胍,其中化合物8j的iNOS抑制活性比氨基胍强26倍多.     

高效液相色谱-质谱-二苯基三硝基苯肼在线筛选与鉴别茶叶中抗氧化活性成分

将基于在线高效液相色谱-二苯基三硝基苯肼(HPLC-DPPH)快速筛选自由基清除剂的方法与电喷雾飞行时间质谱(ESI-TOF/MS)结合,建立了茶叶粗提物中抗氧化活性成分在线筛选与鉴别的方法.本方法是在HPLC色谱柱分离后进行分流,一路进入ESI-TOF/MS用于各化合物的快速鉴别,另一路流出液与稳定的自由基DPPH混合,实现在线筛选自由基清除剂的作用.本方法用于茶水中抗氧化成分的快速筛选与鉴别,筛选出11个具有明显DPPH自由基清除作用的化合物,通过ESI-TOF/MS在线分析获得的质谱信息,结合相关文献和数据库,实现了各化合物的快速鉴别.11个化合物分别为茶氨酸、Theogallin、没食子儿茶素、茶碱、色氨酸、表没食子儿茶素、表没食子儿茶素没食子酸酯、表儿茶酚、没食子酸酯、没食子儿茶素没食子酸酯及儿茶素没食子酸酯.本方法效率高、稳定性好,是复杂天然产物中抗氧化剂快速筛选与鉴别的有力工具.     

8-烷基小檗碱的合成

对比研究了乙醚、二氧六环和四氢呋喃(THF)三种溶剂对8位长链烷基取代小檗碱衍生物中间体合成产率的影响. 以四氢呋喃(THF)为反应溶剂, 8-癸基和8-月桂基盐酸小檗碱的产率是以乙醚为溶剂的4倍以上.     

在线亲和固相萃取-高效液相色谱-二极管阵列检测-四极杆飞行时间质谱快速筛选中药中α-葡萄糖苷酶结合成分

中药活性成分的快速分离分析一直是中药研究的重点和热点问题。该文建立了在线亲和固相萃取-高效液相色谱-二极管阵列检测-四极杆飞行时间质谱技术快速筛选中药中与α-葡萄糖苷酶有结合作用的活性成分的方法。四通阀和六通进样阀组成接口界面,收集亲和固相萃取柱中与α-葡萄糖苷酶有结合作用的活性成分,接着进入液相色谱系统进行分析鉴定。阳性对照品((+)-儿茶素)和阴性对照品(水杨酸)混合物筛选确定了方法的特异性和准确性。接着,以玉竹为研究对象,筛选出9种主要的α-葡萄糖苷酶抑制活性化合物,包括5种苯乙基肉桂酰胺类化合物,4种双氢高异黄酮类化合物。结果表明建立的方法简便、快速、特异性强,可用于任何复杂体系中α-葡萄糖苷酶结合活性成分的筛选。     

S-烃基-1-烃基-3-[4-(苯并咪唑-2-巯基)苯基]异硫脲的合成及其iNOS抑制活性

以2-巯基苯并咪唑(1)为原料,经缩合和还原得到2-(4-氨基苯硫基)苯并咪唑(3),再与异硫氰酸苯甲酰酯或异硫氰酸烃基酯反应得到取代硫脲(5和7),最后与卤代烃反应得到20个新的S-烃基-1-烃基-3-[4-(苯并咪唑-2-巯基)苯基]异硫脲化合物(6和8),其结构经IR,¹HNMR,MS及元素分析确证.初步的药理试验表明,20个目标化合物均有不同程度的iNOS抑制活性,其中化合物6b,8d和8f的iNOS抑制活性与阳性对照药氨基胍相当.     

虎杖和大黄中α-葡葡萄糖苷酶抑制剂的超滤质谱研究

通过体外酶活性实验结合离心超滤LC-ESI-MSn技术从富含蒽醌类成分的中药虎杖和大黄提取物中筛选α-葡萄糖苷酶抑制剂。首先,以4-硝基苯-α-D-吡喃葡萄糖苷(PNPG)为底物,对虎杖和大黄提取物的体外酶抑制活性进行了初步评价,结果表明,虎杖和大黄提取物对α-葡葡萄糖苷酶的半抑制浓度分别为0.027和0.050 g/L。其次,利用离心超滤技术对虎杖和大黄提取物种的潜在的α-葡葡萄糖苷酶抑制剂进行了筛选,并通过LC-ESI-MSn对筛选得到的潜在抑制剂结构进行了鉴定,结果显示,虎杖和大黄中各鉴定得到12和7种,共计16种活性小分子成分。其中大黄素等7种化合物属于蒽醌类;虎杖苷等5种化合物属于多元酚类;莲花掌甘等4种化合物都具有没食子酰基取代基。结果表明,蒽醌类化合物具有较好的α-葡萄糖苷酶抑制活性,可以为开发新的安全有效的富含蒽醌类成分的中药降糖药物奠定基础。     

基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立

以可重复使用的固定化酶代替游离态酶, 建立一种基于比色分析的乙酰胆碱酯酶(AChE)抑制剂体外筛选新模型. 采用以氨基化硅胶为载体固定的AChE优化了实验条件, 用AChE抑制剂阳性对照物他克林和毒扁豆碱对该模型进行验证, 还对模型技术参数进行评价, 并将新模型用于单体化合物及天然产物粗提物AChE抑制活性评价. 结果表明, 最佳实验条件为: 固定化酶用量55 μL, 底物浓度5 mmol/L, 甲醇、 乙醇及体积分数不高于6%的二甲基亚砜水溶液均可作为样品溶剂; 模型验证及模型技术参数评价结果良好, 该模型对AChE抑制剂筛选有较好的特异性和灵敏度, 可用于筛选AChE抑制剂. 该模型具有适用性强、 固定化酶可重复使用及结果可靠等优点, 是单体化合物及天然产物粗提物AChE抑制剂活性评价的有效方法.     

Synthesis,Biological Evaluation,and In Silico Studies of New Acetylcholinesterase Inhibitors Based on Quinoxaline Scaffold

A quinoxaline scaffold exhibits various bioactivities in pharmacotherapeutic interests. In this research, twelve quinoxaline derivatives were synthesized and evaluated as new acetylcholinesterase inhibitors. We found all compounds showed potent inhibitory activity against acetylcholinesterase (AChE) with IC₅₀ values of 0.077 to 50.080 µM, along with promising predicted drug-likeness and blood–brain barrier (BBB) permeation. In addition, potent butyrylcholinesterase (BChE) inhibitory activity with IC₅₀ values of 14.91 to 60.95 µM was observed in some compounds. Enzyme kinetic study revealed the most potent compound (6c) as a mixed-type AChE inhibitor. No cytotoxicity from the quinoxaline derivatives was noticed in the human neuroblastoma cell line (SHSY5Y). In silico study suggested the compounds preferred the peripheral anionic site (PAS) to the catalytic anionic site (CAS), which was different from AChE inhibitors (tacrine and galanthamine). We had proposed the molecular design guided for quinoxaline derivatives targeting the PAS site. Therefore, the quinoxaline derivatives could offer the lead for the newly developed candidate as potential acetylcholinesterase inhibitors.     

Tyrosinase inhibitor screening in traditional Chinese medicines by electrophoretically mediated microanalysis

A capillary‐electrophoresis‐based method for the screening of tyrosinase inhibitors in traditional Chinese medicines was developed. The method integrated electrophoretically mediated microanalysis with sandwich mode injection, partial filling, and rapid polarity switching techniques, and carried out on‐column enzyme reaction and the separation of substrate and product. The conditions were optimized including the background electrolyte, mixing voltage, and the incubation time. Finally, the screening of nine standard natural compounds of traditional Chinese medicines was carried out. The inhibitors can be directly identified from the reduced peak area of the product compared to that obtained without any inhibitor. Chlorogenic acid (100 μM) showed inhibitory activity with the inhibitory percentage of 19.8%, while the other compounds showed no inhibitory activity. This method has great application potential in drug discovery from traditional Chinese medicines.     

Chemical Constituents from the Wild Atractylodes macrocephala Koidz and Acetylcholinesterase Inhibitory Activity Evaluation as Well as Molecular Docking Study

Screening the lead compounds which could interact both with PAS and CAS of acetylcholinesterase (AChE) is an important trend in finding innovative drugs for Alzheimer’s disease (AD). In this paper, four sesquiterpenes, i.e., atractylenolide III (1), atractylenolide IV (2), 3-acetyl-atractylon (3) and β-eudesmol (4), were obtained from the wild Atractylode macrocephala grown in Qimen for the first time. Their structures were elucidated mainly by NMR spectroscopy. To screen the potential dual site inhibitors of AChE, the compounds 1, 2, 3, as well as a novel and rare bisesquiterpenoid lactone, biatractylenolide II (5), which was also obtained from the tilted plant in our previous investigation, were evaluated their AChE inhibitory activities by using Ellman’s colorimetric method. The results showed that biatractylenolide II displayed moderate inhibitory activity (IC₅₀ = 19.61 ± 1.11 μg/mL) on AChE. A further molecular docking study revealed that biatractylenolide II can interact with both the peripheral anionic site (PAS) and the catalytic active site (CAS) of AChE. These data suggest that biatractylenolide II can be considered a new lead compound to research and develop more potential dual site inhibitors of AChE.     

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay

Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI‐FTMS) for screening irreversible AChE inhibitors. Due to its good salt‐tolerance and low sample consumption, MALDI‐FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE‐inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC‐MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non‐AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI‐FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.     

Acetylcholinesterase-Inhibiting Activity of Salicylanilide <em>N</em>-Alkylcarbamates and Their Molecular Docking

A series of twenty-five novel salicylanilide N-alkylcarbamates were investigated as potential acetylcholinesterase inhibitors. The compounds were tested for their ability to inhibit acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.). Experimental lipophilicity was determined, and the structure-activity relationships are discussed. The mode of binding in the active site of AChE was investigated by molecular docking. All the discussed compounds expressed significantly higher AChE inhibitory activity than rivastigmine and slightly lower than galanthamine. Disubstitution by chlorine in C'_(3,4) of the aniline ring and the optimal length of hexyl-undecyl alkyl chains in the carbamate moiety provided the most active AChE inhibitors. Monochlorination in C'₍₄₎ exhibited slightly more effective AChE inhibitors than in C'₍₃₎. Generally it can be stated that compounds with higher lipophilicity showed higher inhibition, and the activity of the compounds is strongly dependent on the length of the N-alkyl chain.     

Screening of acetylcholinesterase inhibitors in natural extracts by CE with electrophoretically mediated microanalysis technique

An electrophoretically mediated microanalysis (EMMA) method for screening acetylcholinesterase (AChE) inhibitors in natural extracts is described. In this method, solutions of AChE and the mixture of the substrate and the natural extract were successively injected into the capillary, and mixed electrophoretically by applying a voltage for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at UV 230 nm represents the enzyme activity. Since the extract is mixed with the substrate, there is no need to separate the components before testing the inhibition. The inhibitory activity of the natural extract as a whole can be easily found if the peak area of the product is reduced. This makes the present method suitable for screening inhibitors in complex mixtures, such as natural extracts. Compared to the commonly used spectrometric method for screening of AChE inhibitors, the major advantage of the present method is the elimination of Ellman reagent, which is essential for the spectrometric method. This not only simplifies the experimental procedure but also minimizes false-positive results. Moreover, it is an obvious advantage of combining the separation power with the on-column enzyme assay for further investigating which compound(s) is/are responsible for the inhibition. The method was validated using a commercially available AChE inhibitor tacrine and a small chemical library containing four AChE inhibitors and 32 natural extracts. Inhibitors in natural extracts were identified with the present method.     

Automated docking of 82 N-benzylpiperidine derivatives to mouse acetylcholinesterase and comparative molecular field analysis with 'natural' alignment

Automated docking and three-dimensional Quantitative Structure-Activity Relationship studies (3D QSAR) were performed for a series of 82 reversible, competitive and selective acetylcholinesterase (AChE) inhibitors. The suggested automated docking technique, making use of constraints taken from experimental crystallographic data, allowed to dock all the 82 substituted N-benzylpiperidines to the crystal structure of mouse AChE, because of short computational times. A 3D QSAR model was then established using the CoMFA method. In contrast to conventional CoMFA studies, the compounds were not fitted to a reference molecule but taken in their 'natural' alignment obtained by the docking study. The established and validated CoMFA model was then applied to another series of 29 N-benzylpiperidine derivatives whose AChE inhibitory activity data were measured under different experimental conditions. A good correlation between predicted and experimental activity data shows that the model can be extended to AChE inhibitory activity data measured on another acetylcholinesterase and/or at different incubation times and pH level.     

Bioassay-Guided Isolation of Anti-Alzheimer Active Components from the Aerial Parts of Hedyotis diffusa and Simultaneous Analysis for Marker Compounds

Previous studies have reported that Hedyotis diffusa Willdenow extract shows various biological activities on cerebropathia, such as neuroprotection and short-term memory enhancement. However, there has been a lack of studies on the inhibitory activity on neurodegenerative diseases such as Alzheimer’s disease (AD) through enzyme assays of H. diffusa. Therefore, H. diffusa extract and fractions were evaluated for their inhibitory effects through assays of enzymes related to AD, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and on the formation of advanced glycation end-product (AGE). In this study, ten bioactive compounds, including nine iridoid glycosides 1–9 and one flavonol glycoside 10, were isolated from the ethyl acetate and n-butanol fractions of H. diffusa using a bioassay-guided approach. Compound 10 was the strongest inhibitor of cholinesterase, BACE1, and the formation of AGEs of all isolated compounds, while compound 5 had the lowest inhibitory activity. Compounds 3, 6, and 9 exhibited better inhibitory activity than other compounds on AChE, and two pairs of diastereomeric iridoid glycoside structures (compounds 4, 8, and 6, 7) showed higher inhibitory activity than others on BChE. In the BACE1 inhibitory assay, compounds 1–3 were good inhibitors, and compound 10 showed higher inhibitory activity than quercetin, the positive control. Moreover, compounds 1 and 3 were stronger inhibitors of the formation of AGE than aminoguanidine (AMG), the positive control. In conclusion, this study is significant since it demonstrated that the potential inhibitory activity of H. diffusa on enzymes related to AD and showed the potential use for further study as a natural medicine for AD treatment on the basis of the bioactive components isolated from H. diffusa.     

磁珠富集与液相色谱-质谱联用结合筛选枳壳中脂肪酶抑制剂

提出了一种磁珠收集与液相色谱-质谱联用(LC-MS)结合用于快速筛选中药提取物中的脂肪酶抑制剂的方法. 通过制备键合脂肪酶的磁珠, 将其与枳壳总黄酮孵育后, 筛选出枳壳中脂肪酶的配体. 通过LC-MS分析发现, 4个化合物均是潜在的脂肪酶抑制剂, 鉴定其分别为柚皮苷、 新橙皮苷、 橙皮苷和枸橘苷. 对4个化合物进行了体外脂肪酶抑制活性验证. 研究结果表明, 新橙皮苷、 橙皮苷和枸橘苷具有显著的脂肪酶抑制活性, IC₅₀分别为48.04, 52.45和46.18 μg/mL, 其中枸橘苷具有脂肪酶抑制活性为首次报道. 结果表明, 磁珠富集与LC-MS集成技术能够用于快速发现中药活性成分.