【作者】 丁阳平；

【导师】 李学刚；

【作者基本信息】 西南大学 ， 药用资源化学， 2011， 博士

【摘要】 小檗碱又叫黄连素(Berberine, BBR),是从黄连和黄柏等中药材中提取出来的异喹啉生物碱。在传统中医中被长期用于治疗由细菌感染引起的胃肠道疾病。大量研究表明,小檗碱还有抗心律失常、抗传染性原虫、抗氧化、降糖、调脂、降压和强心等作用。本研究主要以小檗碱抑菌及抗氧化为研究目标,设计合成了8-烷基小檗碱、13-烷基(苄基)小檗碱、8-烷基-13-溴-小檗碱、13-卤代小檗碱、8-13-二烷基小檗碱、8-烷基-13-苄基小檗碱、系列酚类小檗碱及系列8-烷基酚类小檗碱,并进行了抑菌、降糖、抗氧化和对a-淀粉酶等活性作用研究。实验方法与结果如下：1.小檗碱衍生物的合成1.1 C8烷基及C13卤代小檗碱衍生物的合成采用格氏试剂和卤素分别在C8和C13进行取代,生成8-烷基小檗碱、8-烷基-13-溴-小檗碱及13-卤代小檗碱。并对13-卤代小檗碱及8-烷基-13-溴-小檗碱的合成工艺进行优化。其中小檗碱及烷基小檗碱的最佳溴代反应体系为：反应温度为30℃、反应物比例为6：1、冰醋酸为60 mL时最佳添加量为0.5000g,反应顺序是先加溴,后滴加小檗碱及烷基小檗碱。13-氯代小檗碱的最佳反应条件为：反应时间6-10min、反应温度为30℃、氯气制备的温度(用温度来控制氯气流速)60℃、冰醋酸在30 mL时最佳加样量为0.2000g。研究发现反应完成后加入少许三氯化铁,能使产物析出沉淀,这样既简化了产物的回收,又提高产物产率。碘代反应因反应氧化体系不同,碘取代的位置也不同,在HNO3/I2/AcOH体系中取代的是13位,而在KI/KIO4/AcOH/H2O体系中取代的可能是11位。1.213-及8,13-二取代小檗碱衍生物的合成小檗碱经还原、烷基化和氧化三步生成13烷基小檗碱。具体步骤是先利用硼氢化钠将小檗碱还原成二氢小檗碱,再利用烯胺烷基化生成13烷基或苄基二氢小檗碱取代衍生物,最后用NCS进行氧化。8,13-二取代小檗碱是以8-辛基二氢小檗碱作为先导物,其后的合成方法与13烷基小檗碱的合成相同。1.3酚类小檗碱及酚类烷基小檗碱衍生物的合成实验采用N,N-二甲基甲酰胺、间苯三酚/硫酸体系、无水三氯化铝/吡啶及三溴化硼/二氯甲烷,分别生成9-羟基-小檗碱衍生物、2,3-二羟基-小檗碱衍生物、2,3,9-三羟基-小檗碱衍生物和2,3,9,10-四羟基-小檗碱衍生物及其相应烷基化酚类小檗碱。2.小檗碱衍生物抗菌活性研究实验考查了8-烷基小檗碱、13-烷基(或苄基)小檗碱、8-辛基-13-烷基小檗碱、8-辛基-13-苄基小檗碱、8-烷基-13-溴代小檗碱和卤代小檗碱对9种常见微生物的抑菌能力。结果发现8-烷基小檗碱的抗菌规律跟前人报道一致,8-辛基小檗碱活性最强；当C13引入一系列烷基后也能增强其抗菌活性,并且也有与8-烷基小檗碱相同的抗菌规律,即,在八个碳原子内随着碳链的延长抗菌活性增加,只是抗菌活性要小于相应8-烷基小檗碱；当小檗碱C13位引入卤素后也能增强小檗碱的抗菌能力,增加强度与它们吸电子能力有关,只是抗菌活性远不及烷基小檗碱；在8-烷基小檗碱的C13引入溴后,其抗菌活性强于相应的8-烷基小檗碱,说明C8的烷基与C13的溴具有协同增效作用；当C8和C13同时引入烷基时,并不是8,13-二辛基-小檗碱抗菌活性最强,而是13-丁基-8-辛基-小檗碱抗菌活性最强。3.小檗碱衍生物对部分酶类作用研究用碘-淀粉比色法测定了小檗碱及其衍生物对a-淀粉酶的作用,结果发现小檗碱及其衍生物对a-淀粉酶具有很强的抑制能力,其中氯代小檗碱对a-淀粉酶的IC50值为0.042 mg/mL比小檗碱提高了2倍,2,3,9-三羟基小檗碱和2,3,9,10-四羟基小檗碱的IC50值为0.02 mg/mL,比小檗碱提高了5倍。但所有小檗碱衍生物对ACE酶的作用不明显。4.小檗碱衍生物的降糖作用研究本文考查了8-烷基小檗碱、8-烷基-13-溴代小檗碱及13-卤代小檗碱对HepG2细胞降糖的影响,结果发现13-氯代小檗碱的降糖作用最强。M’IT细胞毒性实验表明,小檗碱引入烷基后细胞毒性增强,而13-氯代小檗碱的毒性最弱,因此认为氯代小檗碱的降糖可能是通过降低对HepG2细胞的毒性来实现的。5.小檗碱及酚类小檗碱抗氧化作用研究实验选用DPPH和羟自由基作为研究对象考查多酚类小檗碱对自由基的清除效果,其中2,3,9,10-四羟基小檗碱作用最强,对DPPH的IC50值为0.13 mg/mL,但仍不及BHA,对羟自由基的IC50值为0.27 mg/mL,作用要强于Vc。多酚类小檗碱对已存在的亚硝酸盐并没有太大作用,但能阻断强致癌物亚硝胺的生成,其IC50值为0.46 mg/mL。实验选用8-烷基-2,3,9,10-四羟基小檗碱来考查油脂的抗氧化能力,结果发现,8-烷基-2,3,9,10-四羟基小檗碱均能降低油脂的过氧化值和酸价,起到良好抗油脂氧化效果,其中8-十二烷基-2,3,9,10-四羟基小檗碱衍作用最强。更多还原

【Abstract】 Berberine is an isoquinoline alkaloids isolated from Chinese herbs Coptidis Rhizoma or Cortex Phellodendri,was initially used as anti-microbial agent, anti-diarrhea, anti-inflammation, and anti-cardiovascular ipid-modulating, antihyperglycemic, antihypertension and antitumor diseases. To increase the pharmaceutica and antioxidant activity, some berberine derivative were synthesized, such as 8-alkyl-berberine,8-alkyl-13-bromo-berberine,13-halogenated-berberine,13-alkyl(or benzyl)-berberine,8,13-dialkyl-berberine,8-alkyl-13-benzyl-berberine, phenol- berberine and 8-alkyl- phenol-berberine. Some pharmacological effects of the berberine derivatives were studied including the antimicrobial activity, antihyperglycemic and antioxidant. The methods and results are as follows:1. Synthesis of berberine derivatives1.1 Synthesis of 8-alkyl-berberine,8-alkyl-13-bromo-berberine and 13-halogenated- berberine8-alkyl on the ring C were synthesized according to the pervious methods. All bromination reaction were performed in the similar way. The optimum conditions of synthesis of 8-alkyl-13-bromo-berberine are that the temperature is 30℃, the ratio of Br2 to 8-alkyl-berberine is 6:1,0.5000 g 8-alkyl-berberine is added in 60 mL glacial acetic acid.The order of reactants addition and the reaction temperature were the tow key factors to synthesize these bromo-substituted compounds. The yield of adding Br2 before adding berberine or alkyl-berberine was far better than reverse order. Adding Br2 before adding berberine or alkyl-berberine was far better than reverse order in terms of the yield.. Chlorination of berberine was comparably easy via chlorinating reagent chlorine. The high yield of chloro-berberine can be accomplished in shorter reaction time and at room temperature. However, in order to improve the yield, the following points must be obeyed. Firstly, all the reagent and solvent must be dried due to the stronger oxidizability of chlorine than the other two. Secondly, berberine must be quickly added after chlorine saturation. Lastly, small amount of FeCl3 must be added at the time when the experiment finished. FeCl3 is prone to cause production precipitation, so high-purity production would be easily obtained through recrystallization instead of column chromatography.Iodinations were more difficult to substitution compared to other halogen due to the lower electrophilicity of iodine.So the oxidant must be added for iodination.The substitute of iodination is different according to oxidant.C13 is iodinated in HNO3/I2/AcOH and C11 is iodinated in KI/KIO4/AcOH/H2O, maybe.1.2 Synthesis of 13-alkyl (or benzyl)-berberine,8-octyl-13-alkyl-berberine,8-octyl-13-benzyl-berberineA number of 13-substitued berberine derivatives were thus from berberine in 3steps via dihydroberberine or in 2 steps via 8-acetonyldiydroberberine derivatives.In the first synthetic approach, berberine was reduced with sodium borohydride in pyridine to afford dihydroberberine, followed by enamine alkylation with electrophiles, and subsequent oxidation with NCS(N-chlorosuccinimide) to give the 13-substitued berberine salt derivatives.The target compounds were also prepared with acetone and aqueous sodium hydroxide solution to afford 8-acetonydihydroberberine and subsequent enamine alkylation with electrophiles followed by elimination of acetone to afford the salt derivatives. The synthesis of 8-octyl-13-alkyl-berberine and 8-octyl -13-benzyl-berberine start from 8-octyldiydroberberine.The following steps are according to the first synthetic approach.1.3 Synthesis of phenol-berberine and alky- phenol-berberine derivativesBerberrubine derivatives,2,3-dihydroxyl-berberine derivatives,2,3,9-trihydroxyl-berberine derivatives and 2,3,9,10-tetrahydroxyl-berberine derivatives were prepared with dimethylform amide, phloroglucol/H2SO4, AlCl3/pyridine, BBr3/dichloromethane, respectively.2. Anti-microbial of berberine derivativesMICs of 8-alkyl-berberine,8-alkyl-13-bromo-berberine,13-halogenated-berberine,13-alkyl(or benzyl)-berberine,8,13-dialkyl-berberine,8-alkyl-13-benzyl-berberine homologues against 9 microorganisms were determined by turbidimetric method respectively.8-alkyl-berberine and 13-alkyl-berberine share the same result that the antimicrobial activity increased as the length of aliphatic chain elongated, but decreased gradually when the alkyl chain exceeded 8 carbon atoms. 8-octyl-berberine and 13-octyl-berberine showed the highest antimicrobial activity. But the antimicrobial activity of 8-alkyl-berberine was stronger than conrresponding 13-alkyl-berberine. Halogenation could further increase subtlely the antimicrobial activity of 8-alkyl-berberine and berberine,so the the antimicrobial activity of 8-alkyl-13-bromo-berberine and 13-halogenated-berberine were better than 8-alkyl-berberine and berberine. An interesting finding is that the 8,13-dioctyl-berberine did not showed the highest antimicrobial activity among all compounds, but 13-butyl-8-octyl-berberine.3. Effect of berberine derivatives on a-Amylase and ACEThe effect of 13-halogenated-berberine, berberine and phenol-berberine on a-Amylase were determined by iodine-starch colorimetry method. The result showed that berberine and its derivatives strongly inhibited a-Amylase.2,3,9-trihydroxyl-berberine and 2,3,9,10-tetrahydroxyl-berberine showed the best inhibiting a-Amylase activity among all the synthesized compounds, which was over 5-fold higher than its precursor (berberine). But the derivatives had little influence on ACE.4. Antihyperglycemic of berberine derivativesIn our study the glucose-lowering effect of berberine and its derivative was not due to an increment of cell number. On the contrary, they depressed the growth of HepG2 cells. Chloro-berberine and bromo-berberine were better than berberine in promoting glucose consumption. That did not indicate chloro-berberine and bromo-berberine carried out the mechanism different from berberine, but they cause less toxicity in HepG2 cells. The glucose-lowering effect of 8-octyl-berberine would abolished when its concentration increased from 0.5 to 4μg/ml, just because of its remarkably cytotoxicity. So the inhibition of cell proliferation increased by 8-octyl -berberine might has potentially beneficial anticancer effects because HepG2 cells are immortalized cell lines. So chloro-berberine which is less cytotoxicity might have more glucose-lowering effect in vitro than BBR. Therefore we infered that it might also be a good option in treating hyperglycemic patients with liver impairment.5. Antioxidant of berberine and its phenol-berberine derivativesAntioxidant of berberine and its phenol-berberine derivatives were tested by scavenging free radical, such as DPPH and·OH.2,3,9,10-tetrahydroxyl-berberine showed the best scavenging free radical capacity among all the other phenol-berberine derivatives.The IC50 of 2,3,9,10-tetrahydroxyl-berberine on DPPH was 0.13 mg/mL, but higher than that of BHA. Its IC50 was 0.27 mg/mL on·OH free radical, lower than Vc. These berberine derivatives had little influence on scavenging nitricol, but could block the formation of nitrosamines which was strong carcinogens. Restraining the oxidation of grease of 8-alkyl-2,3,9,10-tetrahydroxyl-berberine derivatives was investigated in the paper. The result showed that 8-alkyl-2,3,9,10-tetrahydroxyl-berberine derivatives could decrease POV and acid value of grease comparing with the placebo group. The activity increased as the length of aliphatic chain elongated and 8-dodecyl-2,3,9,10-tetrahydroxyl- berberine showed the best antioxidative activities on grease.更多还原