2，3-二氢-1-氨基-2-羟基-4-硝基茚的合成研究

最近几年,国外开发了不少以茚为母体的系列药物和兽药。如：以系列抗HIV药物L-689,502;L-685,927;L-735,524等、系列平滑肌迟缓药SARs及系列抗精神分裂药物。此外,作为农药应用,有日本开发的除草剂一茚草酮及美国杜邦公司于1992年开发,并于2001年登记上市的氨基甲酸酯类杀虫剂——茚虫威等。     

4-氯-2-氧代-1,2-二氢喹啉-3-甲醛的合成工艺研究

目的：对4-氯-2-氧代-1,2-二氢喹啉-3-甲醛的合成工艺进行研究。方法以苯胺为原料通过酯的氨解、环合、胺亚甲基化、水解反应得到目标化合物。结果合成了目标化合物,并利用MS和1H-NMR确证了结构,此路线所得产品收率为62.1%。结论此路线操作简单,成本低廉,设计合理,收率高,适合该化合物的工业化生产。     

1-[3-（3-苄基-4-乙氧基苄基）-4-氯苯基]-1,6-二脱氧-β-D-吡喃葡萄糖的合成工艺研究

目的：设计并合成1-[3-（3-苄基-4-乙氧基苄基）-4-氯苯基]-1,6-二脱氧-β-D-吡喃葡萄糖。方法以5-溴-2-氯-4＆#39;-乙氧基二苯甲烷为原料,通过Friedel-Crafts酰基化、还原、亲核加成、还原乙酰化、脱乙酰化反应的得到目标化合物。结果根据理化性质和波谱数据鉴定了目标化合物的结构,总收率为32%,质量分数为98.48%。结论1-[3-（3-苄基-4-乙氧基苄基）-4-氯苯基]-1,6-二脱氧-β-D-吡喃葡萄糖的合成为tianagliflozin中杂质的研究提供了方便。     

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??OBJECTIVE To prepare inclusion complex of SBE₇-??-CD with trimethoprim(TMP) and optimize the preparation process, to evaluate the products by structural characterization and molecular simulation. METHODS The TMP/SBE₇-??-CD inclusion complex was prepared by the ultrasound-freeze-dry method and the preparation process was optimized by Box-Behnken Design-response surface method(BBD-RSM). Inclusion complex was characterized by FT-IR, DSC, XRPD and ¹H-NMR. Molecular docking method was used to simulate 3-dimensional conformations of the inclusion complex and the binding energy was calculated. The dissolution and stability were tested. RESULTS The optimum conditions of TMP/SBE₇-??-CD inclusion complex were: temperature(52 ??), time(45 min), and the ratio of SBE₇-??-CD and TMP(mol/mol, 1.7??1). All characterizations(FT-IR, DSC, XRPD and ¹H-NMR) indicated the formation of TMP/SBE₇-??-CD inclusion complex. The best 3-dimensional docking conformation was consistent with the characterizations, and the binding energy was -9.015 kcal??mol^-1. The TMP dissolution rate of the inclusion complex increased significantly, the hygroscopicity is strong. CONCLUSION The preparation process of TMP/SBE₇-??-CD inclusion complex optimized by BBD-RSM is reasonable and feasible. The characterizations of inclusion complex are reliable. The molecular simulation is corresponded to the characterized results and provided reliable theoretical basis for inclusion experiments.     

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??OBJECTIVE To prepare small interfering RNA(siRNA) lipid complexes (TPOS-L/siRNA) modified by D-α-tocopheryl poly (2-ethyl-2-oxazoline) succinate (TPOS).METHODS The conventional siRNA lipid complexes (CLs/siRNA) and PEGylated CLs/siRNA (PEG-L/siRNA) were used as controls. CLs/siRNA was prepared by mixing blank CLs and siRNA directly of equal volume according to the electrostatic interaction of positive and negative charges. The encapsulation efficiency, morphology, stability, in vitro release and cell uptake of TPOS-L/siRNA were investigated.RESULTS The CLs/siRNA had obvious lipid bilayer structure, the encapsulation efficiency (EE) was (86.68±1.41)%, and the particle size of CLs/siRNA was less than 200 nm. The modification with TPOS or PEG-DSPE had no significant effect on the EE and particle size of CLs/siRNA, which could endow the lipid complexes with good stability. In addtion, TPOS-L/siRNA had good pH-sensitive property, and could respond to slightly acidic environment, which significantly enhanced the cell uptake.CONCLUSION TPOS can construct good siRNA carrier and increase the stability and pH sensitivity of the nanocarrier.     

铁皮石斛1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶基因的克隆与表达分析

目的从铁皮石斛Dendrobium officinale中克隆1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶[1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphatereductase,HDR]基因,并分析其在铁皮石斛不同组织中的表达差异以及不同信号分子诱导下的表达模式。方法采用RT-PCR和RACE等方法获得铁皮石斛HDR基因(Do HDR)全长,利用DNAMAN和MEGA6.0对其他物种的HDR基因编码的氨基酸序列进行同源性分析和进化关系分析,使用实时荧光定量分析HDR基因的表达模式。结果成功获得Do HDR基因,Gen Bank登录号为KC344827,全长1 658 bp,编码460个氨基酸,与其他科属植物的同源性达到80%以上。Do HDR基因在铁皮石斛叶片中表达量最高,从高到低依次是根、茎、原球茎;且受到脱落酸(abscisic acid,ABA)、水杨酸(salicylic acid,SA)信号分子的诱导。结论从铁皮石斛中获得Do HDR基因,为进一步阐明铁皮石斛萜类化合物合成途径中该基因的重要作用奠定了理论基础。     

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??Endogenous neurotoxin 1-methyl-6,7-dihydroxy-1,2,3,4 -tetrahydroisoquinoline (salsolinol,Sals), an endogenous dopamine metabolite, were shown to be toxic to dopaminergic neurons in vitro as well as in vivo, and was known to be involved in the pathogenesis of Parkinson??s disease (PD). Sals is a more realistic model for selective toxicity to nigral dopaminergic neurons, and mimic the natural course of PD that develops slowly, allowing the brain to adapt to progressive damage. Sals lead to neurotoxicity in dopaminergic cells through induction of oxidative stress and apoptotic dopaminergic cell death,which made it as an important tool drugs to study the pathogenesis of PD.     

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??OBJECTIVE To prepare the inclusion complex of Lignum dalbergia odorifera oil with hydroxyl-??-cyclodextrin(HP-??-CD), and to optimize the preparation process of it. METHODS The inclusion complex was prepared by the stirring-freeze-dry method. The preparation process was optimized by central composite design-response surface method (CCD-RSM),with the colligation score which was calculated by the yield of inclusion, the utilization rate of volatile oil and the content of trans-nerolidol as index. The inclusion complex was verified by phase-solubility method, DSC,UV and microscopical identification. RESULTS The optimum inclusion technology was: inclusion solvent 5% ethanol, stirring rate 500 r??min^-1, HP-??-CD to volatile oil 33??1, inclusion temperature 42 ??,inclusion time 2.5 h. The formation of inclusion complex can change the solubility, optical and thermodynamic properties of volatile oil. CONCLUSION The preparation process of inclusion complex of Lignum dalbergia odorifera oil with HP-??-CD optimized by CCD-RSM is reasonable and feasible, and provide a reliable experiment basis for its application.     

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??OBJECTIVE To isolate and purify 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid from the roots of Actinidia eriantha Benth, establish its determination method, and compare the contents in different extracted parts and samples from different sources. METHODS An HPLC-PDA method was established for the content determination. The contents of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid in the roots of Actinidia eriantha Benth from five different sources and different extracted parts were determined by standard curve method. RESULTS The content of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid was higher in the samples from Shouning county of Fuzhou city and Yunhe county of Lishui city while lower in those from Yongjia county of Wenzhou city and Lishui city. And in different extracted parts, the content was the highest in dichloromethane part, lower in ethanol part, and the lowest in methanol part. CONCLUSION The method is stable and accurate with good reproducibility and can be used for the determination of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid in the roots of Actinidia eriantha Benth. The content of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid is different in Actinidia eriantha Benth samples from different sources, which is the highest in dichloromethane part.     

高效液相色谱法测定2-(2,6-二氯苯基氨基)苯乙酸-4-(4-苯基-1,2,5-噁二唑-2-氧化物-3-)甲氧基苯甲酯原料药的研究

 目的采用反相高效液相色谱法建立2-(2,6-二氯苯基氨基)苯乙酸-4-(4-苯基-1,2,5-二唑-2-氧化物-3-)甲氧基苯甲酯(ZLR-8)原料药质量控制的方法。方法色谱柱为Lichrospher ODS柱(4.6 mm×250 mm),流动相为甲醇-乙腈-水(55∶20∶10),检测波长为226 nm,流速为1.0 mL·min^-1。结果中间体及各降解产物对样品测定不产生干扰,ZLR-8的浓度为11.18～223.6mg·L^-1时与峰面积呈良好线性关系(r=0.999 8,n=5),重复性良好(RSD%=0.47,n=6),检测限为54μg·L^-1。结论该方法可以用于ZLR-8原料药的含量测定和有关物质检查。     

红参的组分调配研究

目的建立通过批次红参药材中人参皂苷Rg1、Re、Rb1的组分调配达到组分最优化效果的方法。方法用高效液相色谱法测定各批次红参药材,用统计学方法计算特定条件下的预测含量,再测定经过组分调配的样品含量。结果组分调配的样品含量与经计算预测含量符合程度中,人参皂苷Rg1、Re、Rb1含量相对偏差分别为-1．88％、-1．03％、0．72％。结论建立通过不同批次红参药材中人参皂苷Rg1、Re、Rb1的组分调配达到组分最优化效果的方法可行。     

西洋参、红参X射线衍射傅里叶指纹图谱鉴定

目的:建立X射线衍射傅里叶(Fourier)指纹图谱鉴定西洋参与红参。方法:对28个西洋参样品,14个红参样品粉碎,建立指纹图并寻找指纹图共同的几何拓扑。结果:西洋参有34个(进口)和39个(国产)特征标记峰,红参有52个(朝鲜)和48个(国产)特征标记峰,它们几何拓扑无差异。结论:微观衍射物理图象鉴定人参类药材是可行的。     

人参药材等级标准

目的:通过对人参药材的性状与主要化学成分指标进行分析,探索人参药材性状与成分指标间的关联性,建立新的人参等级评价标准,为人参药材的质量评价提供更加全面而科学的依据。方法:对48批人参样品的外观性状特征进行量化测量,测定9种人参皂苷指标性成分的含量,将内外指标测定结果进行相关性分析、主成分分析及聚类分析,依据分析结果合理划分人参药材等级并构建等级评价标准。结果:一等人参药材:主根直径1.72 cm,芦头长度2.61 cm,单支参重14.15 g,人参皂苷Rb_1质量分数0.612 1%,人参皂苷Re质量分数0.385 8%,人参皂苷Rg_1质量分数0.320 8%,无破疤、杂质、虫蛀、霉变。二等人参药材:主根直径为1.55～1.72 cm,芦头长度1.74～2.61 cm,单支参重10.24～14.15 g,人参皂苷Rb_1质量分数0.496 8%～0.612 1%,人参皂苷Re质量分数0.323 3%～0.385 8%,人参皂苷Rg_1质量分数0.263 6%～0.320 8%,无破疤、杂质、虫蛀、霉变。三等人参药材:主根直径为1.29～1.55 cm,芦头长度1.34～1.74 cm,单支参重6.90～10.24 g,人参皂苷Rb_1质量分数0.389 5%～0.496 8%,人参皂苷Re质量分数0.235 2%～0.323 3%,人参皂苷Rg_1质量分数0.217 1%～0.263 6%,无杂质、虫蛀、霉变。四等人参药材:主根直径为1.29 cm,芦头长度1.34 cm,单支参重6.90 g,人参皂苷Rb_1质量分数0.389 5%,人参皂苷Re质量分数0.235 2%,人参皂苷Rg_1质量分数0.217 1%,无杂质、虫蛀、霉变。结论:以主根直径、芦头长度、单支参重为人参药材等级标准划分的外观指标,以人参皂苷Rg,Re,Rb_1的含量作为药材内在质量评价指标。制定人参商品规格分为4级,综合了外观和内在指标,具有科学性、全面性的特点,可作为人参药材等级标准的划分依据。     

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??OBJECTIVE To establish an HPLC-UV method for the quantitative determination of ginsenosides Rg₁, Re, Rf, Rb₁, Rc, Rb₂ and the qualitative determination of ginsenosides Rb₃ and Rd in Red Ginseng. This method is used to make different between the imported Red Ginseng and China Red Ginseng.METHODS The analysis were performed on a YMC-Pack ODS-A column(4.6 mm??100 mm,3 ??m), the mobile phase was acetonitrile -0.1% phosphoric acid at the flow rate of 0.6 mL??min^-1, the detection wavelength was set at 203 nm,the column temperature was maintained at 30 ??.RESULTS The method herein is effective. The data of samples was subjected to t test and principal component analysis(PCA)in order to find the marker constituents. According to the information of t test and PCA,ginsenoside Rg₁ and ginsenoside Rd were the main factor to classify Korean Red Ginseng and Chinese Red Ginseng.CONCLUSION The peak area ratio of ginsenoside Rg₁ to ginsenoside Rd is used as the quality control parameters. This method is suitable to classify Korean Red Ginseng and Chinese Red Ginseng.     

UFLC/Q-TOF-MS结合主成分分析法考察人参与黄连共煎前后人参皂苷类成分的变化

目的:利用超快速液相-四级杆-飞行时间串联质谱(UFLC/Q-TOF-MS)结合主成分分析法考察人参-黄连共煎前后人参皂苷类成分的变化。方法:采用UPLC T3 C18色谱柱(2.1 mm×100 mm,1.8μm),流动相0.3%甲酸-0.3%甲酸乙腈梯度洗脱,负离子模式下采集质谱数据,应用Markview1.2.1等软件进行主成分分析(PCA),以黄连水煎液为空白,比较人参水煎液与人参-黄连共煎中人参皂苷类成分的变化。结果:3种溶液中发现10个差异性人参皂苷类化合物,其中齐墩果酸和2个未知化合物含量显著上升,20-葡萄糖Rf和人参皂苷Rf,Ra3,Rb1,Ra2,Rb3,Rs2含量显著下降。结论:负离子模式下方法能很好区分共煎前后人参皂苷类成分的变化,提示人参皂苷类成分水解生成的齐墩果酸型苷元可能是人参与黄连共煎后的物质基础。     

正交设计优选丹参-人参组分抗肝癌配伍研究

目的:优选丹参-人参活性组分抗肝癌优化配伍并对其作用机制作初步研究。方法:以人肝癌细胞SMMC-7721为研究对象,人正常肝细胞L-O2为对照,采用CCK-8法以对肝癌细胞增殖抑制和正常肝细胞保护作用为筛选指标,正交设计优选丹参-人参活性组分抗肝癌有效组合,确定优化配伍;应用实时细胞分析技术(RTCA DP)检测丹参-人参组分优化配伍对肝癌细胞SMMC-7721和正常肝细胞L-O2增殖的影响,并进行时效关系考察;采用Annexin V/PI双染法经高内涵细胞成像系统(HCS)检测丹参-人参组分优化配伍对肝癌细胞SMMC-7721凋亡的影响。结果:丹参-人参组分配伍抗肝癌SMMC-7721细胞的优化组合为丹参总酚酸、人参总皂苷和人参多糖,其配伍剂量分别为10,10,5 mg·L-1;优选的丹参-人参组分配伍能显著抑制肝癌细胞的增殖,呈现时间依赖性,而对正常肝细胞的增殖抑制作用不明显;丹参-人参组分优化配伍能诱导肝癌细胞凋亡,对正常肝细胞的凋亡无显著影响。结论:丹参-人参组分优化配伍具有潜在的特异选择性抗肝癌作用,其机制与抑制肝癌细胞增殖和诱导细胞凋亡相关。     

丹参-人参组分配伍对肺癌A549增殖、凋亡和骨架的影响

中药组分配伍抗肿瘤研究是近年来治疗恶性肿瘤的新方向。该研究通过正交设计优选丹参-人参活性组分抗肺癌A549的有效配伍,并探讨其对肺癌A549细胞增殖、凋亡和骨架的影响。研究发现,正交设计优选出丹参-人参组分配伍抗肺癌的最佳组合为丹参总酚酸、人参总皂苷和人参多糖的最佳配伍剂量为5,10,5 mg·L-1。并利用CCK-8法结合实时细胞分析技术检测丹参-人参组分配伍对肺癌细胞增殖的量效和时效关系,结果显示丹参-人参组分配伍能以时间和剂量依赖性的方式显著抑制肺癌A549细胞的增殖。进一步采用流式细胞术分析丹参-人参组分配伍对肺癌细胞凋亡的影响和高内涵细胞成像系统分析丹参-人参组分配伍对肺癌细胞骨架蛋白F-actin表达的影响,结果显示丹参-人参组分配伍能够显著诱导肺癌A549细胞凋亡和显著降低肺癌A549细胞骨架的面积,且呈一定的剂量依赖性。综上表明丹参-人参组分配伍能抑制肺癌A549细胞的增殖和生长,具有治疗肺癌的应用前景,其机制与诱导肿瘤细胞凋亡及减少微丝形成相关。     

黄连与人参配伍后生物碱的含量变化

目的:探讨黄连与人参不同配伍剂量对水煎液中盐酸小檗碱、盐酸药根碱含量的影响。方法:人参黄连分别按1∶0,2∶1,1.5∶1,0.5∶1比例配伍,煎煮制备水煎液,采用HPLC测定不同配伍剂量水煎液中黄连生物碱的含量。结果:人参-黄连不同配伍样品中盐酸小檗碱质量分数分别为36.68,39.41,44.33 mg·g-1,盐酸药根碱分别为8.607,8.349,10.790 mg·g-1,二者含量随人参用量的减少而增加。结论:黄连-人参配伍后不利于盐酸小檗碱和盐酸药根碱的溶出,按0.5∶1配伍时黄连中2种生物碱含量均最高。     

人参-三七-川芎提取物延缓高糖诱导的小鼠血管衰老的机制探讨

目的:从单磷酸腺苷活化蛋白激酶/哺乳动物雷帕霉素靶蛋白(AMPK/mTOR)通路探讨人参-三七-川芎提取物对高糖诱导的小鼠血管衰老的保护作用。方法:130只雄性C57BL/6小鼠先随机分为空白组和高糖组,高糖组腹腔注射链脲佐菌素(STZ),并用高脂饮食连续性喂养7个月后再次随机分组,分为模型组、人参-三七-川芎提取物低剂量组(0.819 g·kg~(-1))、人参-三七-川芎提取物高剂量组(1.638 g·kg~(-1))及二甲双胍组(150 mg·kg~(-1)),灌胃给药,每天1次,连续9周,期间检测小鼠体质量和血糖变化。给药结束后,苏木素-伊红(HE)染色检测小鼠胸主动脉形态,蛋白免疫印迹法(Western blot)检测小鼠胸主动脉周期蛋白依赖激酶抑制因子2A(p16),周期依赖性蛋白激酶抑制因子1A(p21),AMPK,p-AMPK,mTOR,pmTOR,肝激酶B1(LKB1),p-LKB1,核糖体蛋白s6激酶(p70s6k),p-p70s6k蛋白表达情况。结果:与空白组相比,模型组小鼠体质量显著降低、血糖显著升高(P0.01),在药物干预9周后,各给药组小鼠体质量无明显差异,血糖值较模型组明显下降(P0.05,P0.01)。与空白组相比,模型组内膜损伤严重且有增生,中膜明显增生且排列不整齐,给药各组内膜损伤不明显,中膜少量增生,p16,p21,mTOR,p-mTOR,p70s6k,p-p70s6k蛋白表达明显升高(P0.05,P0.01),AMPK,p-AMPK,LKB1,p-LKB1蛋白表达明显下降(P0.05,P0.01)。药物干预后,各给药组p16,p21,mTOR,p-mTOR,p70s6k,p-p70s6k蛋白表达明显下降(P0.05,P0.01),AMPK,p-AMPK,LKB1,p-LKB1蛋白表达明显升高(P0.05,P0.01)。结论:高糖能够诱导小鼠主动脉衰老,人参-三七-川芎提取物可能通过AMPK/mTOR通路改善由高糖诱导的小鼠主动脉衰老。     

人参-知母-赤芍提取物对血管性痴呆大鼠海马神经元的保护作用及机制

目的:观察人参-知母-赤芍提取物对血管性痴呆大鼠海马N-甲基-D-天冬氨酸受体1(NMDAR1)的影响,探讨其保护海马神经元的作用机制。方法:60只SPF级雄性SD大鼠,随机分为正常组,假手术组,模型组,人参-知母-赤芍提取物组(0. 20 g·kg-1)和美金刚组(2. 1 mg·kg-1),每组12只。采用双侧颈总动脉反复夹闭合并腹腔注射硝普钠的方法建立血管性痴呆大鼠模型,造模后,正常组,假手术组,模型组给予同等体积生理盐水,每日1次,连续14 d。采用Morris水迷宫评价各组大鼠学习记忆能力;苏木素-伊红(HE)染色观察海马CA1区病理改变;蛋白免疫印迹法(Western blot)检测海马神经元胞膜NMDAR1的蛋白表达水平;免疫组化法(IHC)检测海马NMDAR1的表达情况;实时荧光定量PCR(Real-time PCR)检测海马组织中NMDAR1 mRNA的表达水平。结果:与正常组和假手术组比较,模型组大鼠逃避潜伏期显著延长(P 0. 01),在平台所在象限停留时间及穿越平台的次数显著减少(P 0. 01),海马CA1区神经细胞层次减少,排列紊乱、出现核固缩、神经元丢失,NMDAR1蛋白及mRNA表达显著升高(P 0. 01);与模型组比较,人参-知母-赤芍提取物组,美金刚组逃避潜伏期明显缩短(P 0. 05,P 0. 01),在平台所在象限停留时间及穿越平台的次数明显增加(P 0. 05,P 0. 01),海马CA1区神经元数目与形态有明显改善,海马神经元NMDAR1蛋白及NMDAR1 mRNA表达明显降低(P 0. 05)。结论:人参-知母-赤芍提取物能够改善血管性痴呆大鼠的学习记忆能力,减轻海马CA1区神经元的损伤,其机制可能与下调海马神经元NMDAR1的表达有关。