千层塔中产石杉碱甲内生真菌的研究

从千层塔中分离产石杉碱甲内生真菌。采用平板分离法分离千层塔内生真菌,通过抑制乙酰胆碱酯酶活性和抗氧化活性筛选,结合薄层层析和HPLC测定活性菌株中的石杉碱甲含量,通过形态学ITS-rDNA序列分析鉴定内生真菌。共分离得到94株内生真菌,筛选到S29、L44、S94共3株乙酰胆碱酯酶抑制活性和清除DPPH·自由基活性都在50%以上,筛选到一株S29能产石杉碱甲的菌株。摇瓶发酵产量为每克干菌体的石杉碱甲含量为50.64μg。形态学与ITS-rDNA序列分析法鉴定该内生真菌为Podospora sp.属。药用植物千层塔体内Podospora sp.属的内生真菌可产石杉碱甲。     

武夷山脉石杉科植物石杉碱甲含量的研究

石杉植物因所含Hup A为高效、可逆、高选择性的中枢性乙酰胆碱酯酶抑制剂而受关注。实地采集标本并用改进的HPLC法测定了武夷山脉4种石杉科植物及不同居群间的石杉碱甲(Hup A)含量。结果显示石杉碱甲在长柄石杉、四川石杉、有柄马尾杉和华南马尾杉中均有分布,其中以有柄马尾杉中含量最高,达0.1510%;含量最低的是四川石杉,为0.016%。该地区优势种长柄石杉（千层塔）11个居群的石杉碱甲含量测定显示武平县梁野山和三元区中村乡两个居群含量较高,分别为0.0636%和0.0519%。对石松科部分种进行石杉碱甲测定,认为藤石松等不含石杉碱甲。结果表明武夷山脉地区拥有优良的石杉碱甲植物资源。     

石杉碱甲的研究进展

本文综述了1984年以来对石杉碱甲的药理作用、构效关系、合成及其天然来源等方面的研究进展;详细罗列了巳确认的石杉碱甲天然来源,以及从中分离出的其它生物碱;探讨了石碱杉甲的结构特征与其抑制乙酰胆碱酯酶活性能力之间的关系。     

石杉碱甲对氧自由基的影响

目的:研究石杉碱甲对氧自由基的影响,探讨石杉碱甲抗老年性痴呆的机制.方法:按50 mg/kg·d给大鼠皮下注射D-半乳糖6周后,按石杉碱甲0.1mg/kg·d、0.2mg/kg·d给药2周,检测血清和脑组织中SOD活力和MDA含量.结果:石杉碱甲能明显提高大鼠血清和脑组织中SOD的活力,同时降低MDA的含量.结论:加快氧自由基的清除、减少自由基损伤是石杉碱甲抗老年性痴呆的机制之一.     

HPLC法检测蛇足石杉内生真菌胶胞炭疽发酵液中石杉碱甲和石杉碱乙的含量

建立了高效液相色谱(HPLC)测定蛇足石杉(Huperzia serrate)内生真菌胶胞炭疽发酵液中石杉碱甲(Huperzina A)和石杉碱乙(Huperzine B)含量的方法,并以此方法检测胶胞炭疽发酵液中石杉碱甲和石杉碱乙含量的积累。内生真菌发酵液经氯仿萃取、甲醇溶解、过滤后进行高效液相检测分析,选用Agilent Eclipse plus-C18色谱柱(250 mm×4.6 mm,5μm),以0.015 mol/L乙酸铵(p H 6.8)和甲醇溶液(70∶30)为流动相进行等度洗脱,流速1 mL/min,检测波长为308 nm,连续检测内生真菌胶胞炭疽发酵液中第6–15天石杉碱甲和石杉碱乙的含量积累。结果表明,发酵提取液中的石杉碱甲和石杉碱乙可在25 min内进行很好的分离和分析,石杉碱甲在1.50-48.00μg/mL范围内线性关系良好(相关系数r为0.999 5),石杉碱乙在0.25-7.50μg/mL范围内线性关系良好(相关系数r为0.999 7),石杉碱甲和石杉碱乙的平均加标回收率分别为106.83%、108.06%,相对标准偏差(RSD)分别为3.34%、3.60%。该方法简便、快速、精密度高、结果准确,适用于内生真菌发酵液中石杉碱甲和石杉碱乙含量检测。在发酵过程中,内生真菌发酵液中石杉碱甲和石杉碱乙的含量呈现先增后减,随后有所增加继而又减少的趋势。石杉碱甲和石杉碱乙的含量分别在内生真菌发酵第14天、第8天达到最高,分别为12.417 0μg/mL、4.660 3μg/mL。该方法学的建立为内生真菌胶胞炭疽合成石杉碱甲与石杉碱乙的机制研究提供了检测手段,从而有利于药物新资源的开发。     

湘西蛇足石杉中石杉碱甲、乙和丙含量的测定

采用超声萃取法提取湘西蛇足石杉总生物碱,用高效液相色谱法同时测定其石杉碱甲、乙和丙含量,并分析其在植株不同部位的分布。结果表明:湘西4个样地蛇足石杉中石杉碱甲、乙和丙含量基本一致,分别达到0.5‰、0.3‰和0.04‰;但植株不同部位三种石杉碱含量差异显著,其中石杉碱甲和乙的分布均为叶>茎>根,石杉碱丙则是根大于叶和茎。由此可知,湘西蛇足石杉具有资源优势,石杉碱在植株的分布具有明显的部位选择性;采用HPLC可同时检测石杉碱甲、乙和丙,方法简单快速、准确可靠。     

蛇足石杉产石杉碱甲内生真菌SNZ-12的分离及鉴定北大核心CSCD

为了获取具有应用价值的产石杉碱甲内生菌,本文从蛇足石杉分离得到60株内生真菌,采用高效液相色谱法和质谱法分别检测各个真菌的提取物,发现内生真菌SNZ-12的提取物具有与石杉碱甲标品相近的色谱特征峰和保留时间(8. 994 min)以及相同的质谱特征峰((M+H)+=243. 1),说明内生真菌SNZ-12可以产石杉碱甲,其石杉碱甲产量约为1. 01 mg/L;并通过结合内生真菌SNZ-12的形态特征和18S r DNA序列分析,将其鉴定为尖孢镰刀菌(Fusarium oxysporum)。本文结果为利用微生物发酵生产石杉碱甲研究提供了潜在的菌种资源。     

蛇足石杉的药学研究进展

蛇足石杉是蕨类植物门,石松纲石松目,石杉科石杉属的多年生草本植物,主要生长在山顶岩石缝隙中、山地密林下或沟谷阴湿环境中,分布特点散而广,全草可入药,民间用于治疗跌打损伤、瘀血肿痛、内伤出血、肿痈疔毒、毒虫叮咬、烧伤烫伤等症。该植物中主要成分为生物碱、三萜和黄酮等,其中以活性成分石杉碱甲为代表性成分,石杉碱甲是一种高选择性乙酰胆碱酯酶抑制剂,对阿尔茨海默病和重症肌无力疗效显著,还可以有效改善记忆力和恢复意识障碍。为了更好的开发和利用蛇足石杉的药用资源,本文综述了国内外有关该植物的药化、药理、药分、药剂和组培等方面的研究进展。     

蛇足石杉中产石杉碱甲内生真菌的分离和鉴定

【目的】从野生蛇足石杉(Huperzia serrata)中分离筛选产石杉碱甲的内生真菌。【方法】采用薄层层析及高效液相色谱法对内生真菌代谢产物进行测定和分析以期分离获得产石杉碱甲菌株,运用形态及ITS序列分析方法对产石杉碱甲菌株进行鉴定,并利用连续传代方法考察菌株遗传稳定性。【结果】经筛选获得一株产石杉碱甲内生真菌NSH-5,经形态学鉴定及ITS序列分析鉴定为轮枝镰孢菌(Fusarium verticillioides),其石杉碱甲产量为11.76 mg/100 m L,菌株经20次连续传代后遗传稳定。【结论】NSH-5菌株为一株具有产石杉碱甲能力的轮枝镰孢菌,该菌株的发现为生物合成石杉碱甲提供了新的菌种资源。     

蛇足石杉产石杉碱甲内生真菌SNZ-12的分离及鉴定

为了获取具有应用价值的产石杉碱甲内生菌,本文从蛇足石杉分离得到60株内生真菌,采用高效液相色谱法和质谱法分别检测各个真菌的提取物,发现内生真菌SNZ-12的提取物具有与石杉碱甲标品相近的色谱特征峰和保留时间(8. 994 min)以及相同的质谱特征峰((M+H)+=243. 1),说明内生真菌SNZ-12可以产石杉碱甲,其石杉碱甲产量约为1. 01 mg/L;并通过结合内生真菌SNZ-12的形态特征和18S r DNA序列分析,将其鉴定为尖孢镰刀菌(Fusarium oxysporum)。本文结果为利用微生物发酵生产石杉碱甲研究提供了潜在的菌种资源。     

Synthesis of more potent analogues of the acetylcholinesterase inhibitor,huperzine B

The synthesis and acetylcholinesterase inhibition activity of analogues of huperzine B are reported. These new racemic analogues show a better AChE inhibitory activity than the natural product huperzine B.     

Rational design and synthesis of potent aminoglycoside antibiotics against resistant bacterial strains

Based on the structural information of biomacromolecule-aminoglycoside complexes, a series of kanamycin B analogues were rationally designed and synthesized. A convenient approach to the construction of kanamycin derivatives, in which the C4′-position on ring I of neamine moiety was modified, was developed. Most synthetic analogues exhibited good to excellent antibiotic activity against some typical drug-resistant bacteria. The disclosed results suggested that the C4′-position of aminoglycosides such as kanamycin may be an ideal site for modification to gain new modifying enzyme-resistant aminoglycoside antibiotics.     

Huperzine A--human serum albumin association: chromatographic and thermodynamic approach

The synthesis of six new huperzine analogues was reported. Each product presents an amidification of the free amine on huperzine A. The synthesis strategy of these new huperzine A derivatives is based on a condensation with an acyl anhydride. The binding on HSA of two molecule series (huperzine and benzodiazepine, respectively) was investigated with high performance liquid affinity chromatography (HPLAC) using an HSA column. A thermodynamic approach showed that binding huperzine A on HSA involved hydrophobic and Van der Waals interactions. A comparative thermodynamic study with benzodiazepine molecules was carried out to determine the potential binding site of huperzine derivatives on HSA.     

Novel 4-azasteroidal N-glycoside analogues bearing sugar-like D ring: synthesis and anticancer activities

A series of novel N-glycoside analogues with 4-azasteroid moiety bearing sugar-like D ring were conveniently synthesized by constructing the core dihydropyran ring embedded in 4-azasteroidal skeleton which was prepared from 4-aza-5α-androst-3,17-dione 1 in four steps. The structure of 6b were unambiguously proved by the appropriate X-ray structural analysis. Anticancer activity was found for all of the analogues with purinyl moiety against breast cancer (MCF-7), human neuroblastoma (SK-N-SH), cervical cancer cell (HeLa) and prostatic cancer (PC-3), while the analogue 7 containing 1,2,4-triazole heterocycle as the nucleobase was inactive against all of the tested cancer cell lines. The biology results showed the purinyl moiety attached to the pyran ring of 6a-d, substituent at 6'-position of purine base and introduction of a halogen atom at 2'-position of 6'-chloropurine had obviously effect on the evaluated anticancer activity.     

Synthesis,cytotoxic activity and structure–activity relationships of hedychenone analogues

Hedychenone, a plant-derived labdane diterpenoid, showed potent in vitro cytotoxic activity against cancerous cells. In the present study, a series of analogues have been synthesized by modification of the furanoid ring, double bond and the vinylic methyl functionality of this natural product lead and evaluated for their cytotoxic activities against human cancer cell lines. The structures of the target compounds were established by IR, ¹H NMR and mass spectral analysis. Majority of the analogues displayed potent activity than the parent compound, hedychenone. Preliminary structure–activity relationship studies indicated that furanoid ring has a greater impact on cytotoxicity than that of the decalone nucleus. However, dimerization through C-8 significantly enhanced the cytotoxic activity of the hedychenone.     

Synthesis and structure-activity relationships of berberine analogues as a novel class of low-density-lipoprotein receptor up-regulators

Berberine (BBR, 1) is a novel cholesterol-lowering agent that up-regulates low-density-lipoprotein receptor (LDLR) expression through a mechanism different from that of statins. Because of the unique mode of action and good safety record, BBR provoked our interest to do structure modification at different domains for its cholesterol-lowering activity. Nineteen BBR analogues with substituents on the benzene ring D were synthesized in the present study. The analysis of structure-activity relationship (SAR) indicated that the two methoxyl groups in an ortho-distribution on this benzene ring afforded a good activity. Among the 19 analogues, compound 8j bearing a methoxyl at both 10- and 11-position showed an increased LDLR up-regulatory activity in respect to BBR, and therefore has been selected as a promising cholesterol-lowering drug candidate for further evaluation.     

Molecular docking and QSAR analyses of aromatic heterocycle thiosemicarbazone analogues for finding novel tyrosinase inhibitors

A collection of 36 thiosemicarbazone analogues possessed a broad span of tyrosinase inhibitory activities was designed and obtained. Robust and reliable CoMFA and CoMSIA models were gained to predict the structure–activity relationship and the new modifier direction. Inhibitory activities of the compounds were found to greatly depend upon molecular shape, size, and charge. The sterically bulky group at the C-4 position of the thiophene ring contributed a high capacity for biological activity. Some bulky substituents at the C1-position and C12-position, and electron-negative groups at the C3-position, helped to improve the activity of these analogues. The molecular docking results provided visual evidence for QSAR analysis and detailed information about binding mode, affinity, and the principal mechanism between the ligands and tyrosinase. Based on these, a prospective structure modification and optimization of the most potent compound, T32, was suggested for further research.     

Structural and stereospecific requirements for the nucleoside-triggered germination of Bacillus cereus spores

A selection of adenosine analogues was tested for their ability to trigger germination of Bacillus cereus NCIB 8122 spores. The germination-inducing activity was governed by the structural properties of the sugar rather than the base moieties of the nucleosides. Among the sugar-modified analogues, only those containing a 2'-deoxy-D-ribose moiety promoted spore germination. Requirements for a specific molecular structure of the base were not clearly identified, although the highest activity was observed when substituents were inserted at position 6 of the purine ring. All the base-modified analogues, even those such as coformycin and 2'-deoxycoformycin with an expanded base ring, retained the germination-inducing activity of adenosine. However, of the two 2'-deoxycoformycin diastereoisomers characterized by an asymmetric carbon atom at position 8 of the homopurine ring, only the 8S-isomer induced germination, thus indicating that stereospecific configuration of the inducer, at least in the case of 2'-deoxycoformycin, appears to be essential for the initiation of spore germination. The differences in the germination-inducing activity of the various analogues tested were not affected significantly by spore activation at different temperatures, although the higher the activation temperature, the lower was the concentration of each analogue required for maximum germination.     

Antinociceptive activity of structural analogues of rotundifolone: structure-activity relationship

Rotundifolone, a monoterpene isolated from the essential oil of the leaves of Mentha x villosa, is a constituent of several essential oils and known to have antinociceptive activity. Our recent study demonstrated that the analogues of rotundifolone showed also a significant antinociceptive effect. In the present report, to investigate the correlation between the structure and antinociceptive activity, rotundifolone and its analogues were evaluated in the acetic acid-induced writhing test in mice. All compounds showed to be more antinociceptive than rotundifolone against the pain response induced by acetic acid. Comparing the antinociceptive effect of rotundifolone with limonene oxide and (+)-pulegone, the results demonstrated that the epoxide group contributes as much as the ketone group to the antinociceptive activity of rotundifolone. Similarly, pulegone oxide and carvone epoxide were more antinociceptive than rotundifolone, thereby suggesting that the position of the functional group on the ring also influences the antinociceptive activity. (-)-Carvone produced maximal inhibition of the writhing response and was slightly more active than (+)-carvone. The study showed that by appropriate structural modification it may be possible to develop novel antinociceptive agents.     

Rational design and synthesis of highly potent anti-acetylcholinesterase activity huperzine A derivatives

By targeting multi-active sites of acetylcholinesterase (AChE), a series of huperzine A (Hup A) derivatives with various aromatic ring groups were designed and synthesized by Schiff reaction. They were evaluated as AChE and butyrylcholinesterase (BChE) inhibitors. Results showed very significant specificity that the group of imine derivatives could inhibit TcAChE and hAChE, but no inhibitory effect on hBChE was detected. The experiment was explained by a docking study. In the docking model, we confirmed that aromatic ring of Hup A derivatives played the π–π stacking against aminophenol residues of AChE, and the structure–activity relationship (SAR) was discussed.