柠檬醛对黄曲霉质膜损伤机制的初步研究

与正常生长的黄曲霉对照 ,通过测定经柠檬醛毒化的菌丝体对还原糖和蛋白质利用率、[Na+ ,K+ ] ATPase分解ATP活性、细胞电解质渗出率、并结合扫描电镜和快速显微多道分光光度法观察菌丝体细胞及孢子形态变化 ,结果表明经该醛MIC毒化后 ,菌丝体细胞及孢子表面疏松而粗糙 ;隘痕缩小并关闭 ;电导率增加 5 2 8% ;对还原糖和蛋白质的利用速率分别下降 6 1 5 %和 44 3 % ;孢子萌发率下降至 6 1 4% ;该醛能明显改变细胞质膜的分子结构 ,使其失去选择通透性而抑制菌丝体生长和孢子萌发率。     

柠檬醛抗黄曲霉作用的分子机理

以多组分山苍子[Litsea cubeba(Lour)Per]香精油作为复合中药模型。以该香精油中主要抗菌成分柠檬醛为中药靶部位,以能分泌致癌毒素的黄曲霉单细胞作为药物作用对象,吸收当今医学影像领域先进的科学技术,采用多学科交叉策略,将多维显微、瑞利光散射(Rayleigh scattering)、电镜与生化分析4项技术构筑平台, 从细胞、亚细胞和生物大分子三个水平,研究柠檬醛作用于黄曲霉的动静态过程,阐明模拟的中药方剂靶部位对细胞整体的作用规律.发现该醛不仅能改变黄曲霉细胞膜的形态结构、物理学特性及其生物学功能(如对物质吸收的选择通透性,细胞体积调节机制等),而且使细胞膜产生脂质过氧化损伤;进入细胞后,既作用于细胞器(如线粒体、细胞核等),使其产生损伤及区域性分布;又通过干扰细胞内大分子拥挤状态,导致细胞内生物大分子构象的改变、高含量类大分子缔合反应不可逆增强以及因生化反应区域效应丧失而产生的新陈代谢紊乱,揭示该醛能使黄曲霉孢子失去萌发力、菌丝体生长被抑制及产生孢子的能力,在于黄曲霉细胞膜、细胞器及大分子失去了正常结构、功能及相关的调节机制.在实现对柠檬醛抗黄曲霉机理阐明的过程中,在研究思路和方法上进行全新的探索.     

微量液基稀释法测定中药活性成分的体外抗曲霉菌活性*

通过测定中药活性成分肉桂醛和柠檬醛对常见深部条件致病性真菌黄曲霉、烟曲霉的抗菌活性,为建立中药抗曲霉菌药敏试验标准提供参考依据。参照美国国家临床试验标准化委员会(National Committee for Clinical Laboratory Standards,NCCLS)提出的标准,用微量液基稀释法分别测定肉桂醛和柠檬醛对黄曲霉、烟曲霉的抗菌活性。肉桂醛对黄曲霉、烟曲霉最低抑菌浓度(Minimal inhibitory concentration,MIC)分别为：0.100μg/mL,0.050μg/mL,柠檬醛对黄曲霉、烟曲霉的MIC分别为：2．600μg/mL、0．650μg/mL。中药活性成分肉桂醛和柠檬醛具有高效抗曲霉作用。该研究可为制定中药抗曲霉作用评价标准提供参考依据。     

云南野香橼叶油的化学成分

云南省盈江县产野香橼(Citrus medica L.)叶油,用Finnigan-4510型毛细管气相色谱/质谱/计算机联用方法进行了化学成分分析、共检出了31个成分,鉴定了其中22个成分,占全精油的98.5％,主要成分为柠檬烯(56.63％),橙花醛(8.18％),香叶醛(13.52％),对一聚伞花素(3.92％),乙酸香叶酯(2.34％),甲基庚烯酮和月桂烯(3.26％)等。该油具有特征的柠檬—柑桔香气,适宜于调配果香和化妆香精。     

木里柠檬叶精油化学成份的研究

用毛细管气相色谱-质谱-计算机数据联用技术、标准品叠加法和毛细管保留指数定性法分析了木里柠檬(Citrus Liilion(L.)Burm.f.)的叶精油化学成分。从水蒸汽蒸馏叶精油被分离的110多个色谱峰中鉴定了41种化合物,并测定了其相对含量。其主要成分是香叶醛(21.3058％)、橙花醛(13.9580)、d-柠檬烯(9.8359)、β-水芹烯(9.3297)、橙花醇(4.4657)、乙酸橙花酯(2.8162)、香茅醛(2.6338)、乙酸芳樟酯(2.2179)、α-水芹烯(2.0390)、香芹酮(2.0213)、6-甲基-5-庚烯-2-酮(1.7250)和乙酸香叶酯(1.6586)。被鉴定的成份占总成份的96.6652％     

显微多道分光光度法研究受柠檬醛损伤后黄曲霉细胞形态变化

采用从山苍籽油分离出的柠檬醛作为抗菌药物,产毒和无毒的黄曲霉细胞（Aspergillus flavus cell, AFC）作为药物作用的靶,以显微多道分光光度法和显微图像分析法对被该醛所损伤的AFC进行图像捕获,测定受损伤后细胞内吸收光谱、截面积、周长、长轴长及短轴长所发生的变化。发现在410nm和665nm处产毒AFC存在特征吸收峰,受该醛损伤后产毒和无毒AFC的吸收光谱波峰均发生迁移,且峰面积增大;截面积等4类形态参数的数值随柠檬醛浓度升高而减少;为质膜物理化学及细胞内生理生化指标变化提供了理论依据。表明柠檬醛不仅破坏质膜的选择性通透性,而且使细胞质膜结构改变并进入细胞,与靶分子及靶细胞器作用而引发一系列新的生理生化现象的出现。实现对活态细胞受药物作用后形态及生物大分子动态变化的快速、实时、在位的测定,对抗菌药物作用于细胞并使其发生形态结构及靶分子的变化提供了必要的物理参数,在药物抗菌理论及方法研究上具有重要意义。     

柠檬醛损伤黄曲霉线粒体生化机理的研究

应用生物化学方法并结合扫描电镜,研究柠檬醛掺入黄曲霉细胞,并通过损伤线粒体(Mt),导致抑制其生长的机理。结果表明,在药物致敏浓度时,菌丝体经该醛作用后,胞内Mt呈不规则增多,氧化还原反应系统受到破坏,与对照组相比,柠檬醛组的琥珀酸脱氢酶(Succinate Dehydrogenase,SDH)、苹果酸脱氢酶(Malate Dehydrogenase,MDH)活性分别呈不可逆下降271%和242%,随着药物浓度的升高,SDH、MDH的活性直至消失;以琥珀酸、α酮戊二酸和丙酮酸为底物时,线粒体呼吸速率分别下降24.1%、14.3%和36.1%,提示柠檬醛能使菌丝体DNA、RNA、脂类和蛋白质等生物合成受到抑制,促进细胞死亡。     

阴香叶精油化学成分及抗菌活性

研究阴香叶精油的化学成分及体外抗真菌活性,以期为开发绿色、安全、高效的抑菌剂提供参考。水蒸气蒸馏法提取阴香叶精油,气相色谱-质谱法（GC-MS）分析精油成分;熏蒸法研究阴香叶精油抗黄曲霉菌和黑曲霉菌的活性;超高效液相色谱-串联质谱（UPLC-MS/MS）分析精油对黄曲霉毒素B₁（AFB₁）和赭曲霉毒素A（OTA）的抑制作用。从阴香叶精油中鉴定出38种化学成分,主要成分有龙脑、桉油精、α-水芹烯、p-伞花烃、α-萜品醇、乙酸龙脑酯等。阴香叶精油用量为40 μL时,对黄曲霉菌丝体的抑制率为92.59%,对AFB₁的抑制率为47.05%;精油用量为20 μL时,黑曲霉菌丝体的抑制率为67.18%,对OTA的抑制率为100%。阴香叶精油的化学成分丰富,具有显著抑制黄曲霉菌,黑曲霉菌及毒素产生的作用,具有开发为抑菌剂的应用价值。     

木里柠檬果皮精油化学成分的研究

用毛细管气相色谱-质谱-计算机数据联用枝术、标准品叠加法和毛细管保留指数定性法分析了木里柠檬(Citrus Limun(L.)Burm.f.)的果皮精油化学成分。从蒸馏精油被分离出的78个色谱峰中鉴定了39种化合物,并测定了其相对含量。其主要成分是d-柠檬烯(69.4406％);;γ-萜品烯(8.3169);β-水芹烯(6.5947);芳樟醇(3.2172);香叶醛(1.7618);橙花醛(1.3422);α-蒎烯(1.3488);香叶烯(1.2371)和α-水芹烯(1.5753)。被鉴定的成分占总成分的99.1646％。比较了压榨法、蒸馏法和蒸馏-萃取法所得精油的化学成分,并与市售冷磨柠檬油和尤力克蒸馏油的主要成分进行了比较。     

柠檬醛致黄曲霉孢子丧失萌发力的机制

通过由倒置显微镜、衍射光栅和线阵光电偶合器件CCD(chargecoupleddevice)等构成的显微多道分光光度系统及由计算机DEPHI编程工具编制的单细胞凝胶电泳SCGE(single cellgelelectro phoresis)图像分析系统 ,摄取荧光显微镜所呈图像 ,再由图像捕捉卡将CCD产生的图像信号送入计算机 ,将柠檬醛对黄曲霉质膜和核DNA损伤的图像进行显示存储和分析处理 ,测定彗星长度、荧光强度、矩类及头尾DNA含量比等彗星参数指标 .结果发现Olive尾矩、尾长、尾分布矩等彗星尾参数指标与柠檬醛致黄曲霉损伤浓度呈正相关性 ,当致损浓度达到 1 5mg L以上时 ,DNA损伤为致死性损伤 ,不能被细胞内修复系统所修复 .揭示柠檬醛通过损伤质膜而进入细胞 ,对DNA产生不可逆损伤 ,使孢子失去萌发力的机制 .实现将DNA损伤的生化定性检测推进到数值化研究范围 ,为柠檬醛的开发应用提供了重要理论依据 .与国内外同类技术相比 ,本检测观察系统还具有高灵敏度、快速、无扰、多光谱显微测定之特点 .     

Effects of citral on Aspergillus flavus spores by quasi-elastic light scattering and multiplex microanalysis techniques

Litsea cubeba (Lour.) Pers. is a kind of medicinal plantin China. The first report about the antibacterial and anti-phlogistic function of Litsea cubeba (Lour.) Pers. and itsoil appeared in the Zhong Yao Da Ci Dian [1]. Since 1980s,many studies showed that Litsea cubeba oil had wideantibacterial and antifungal activity [2–4]. The antibioticfunctions of Litsea cubeba oil are attributable mainly tocitral [5–7], which amounts to 60%–80% of the essentialoil [8]. Pattnaik [9] reported that…     

Mechanism of inhibition of aldehyde dehydrogenase by citral, a retinoid antagonist.

Low concentrations of citral (3,7-dimethyl-2,6-octadienal), an inhibitor of retinoic acid biosynthesis, inhibited E1, E2 and E3 isozymes of human aldehyde dehydrogenase (EC1.2.1.3). The inhibition was reversible on dilution and upon long incubation in the presence of NAD+; it occurred with simultaneous formation of NADH and of geranic acid. Thus, citral is an inhibitor and also a substrate. Km values for citral were 4 microM for E1, 1 microM for E2 and 0.1 microM for E3; Vmax values were highest for E1 (73 nmol x min-1 x mg-1), intermediate for E2 (17 nmol x min-1 x mg-1) and lowest (0.07 nmol x min-1 x mg-1) for the E3 isozyme. Citral is a 1 : 2 mixture of isomers: cis isomer neral and trans isomer, geranial; the latter structurally resembles physiologically important retinoids. Both were utilized by all three isozymes; a preference for the trans isomer, geranial, was observed by HPLC and by enzyme kinetics. With the E1 isozyme, both geranial and neral, and with the E2 isozyme, only neral obeyed Michaelis-Menten kinetics. With the E2 isozyme and geranial sigmoidal saturation curves were observed with S0.5 of approximately 50 nM; the n-values of 2-2.5 indicated positive cooperativity. Geranial was a better substrate and a better inhibitor than neral. The low Vmax, which appeared to be controlled by either the slow formation, or decomposition via the hydride transfer, of the thiohemiacetal reaction intermediate, makes citral an excellent inhibitor whose selectivity is enhanced by low Km values. The Vmax for citral with the E1 isozyme was higher than those of the E2 and E3 isozymes which explains its fast recovery following inhibition by citral and suggests that E1 may be the enzyme involved in vivo citral metabolism.     

肉桂醛、柠檬醛抑制黑曲霉生长的比较研究

黑曲霉是一类极易通过饲料、食品、粮油霉变而具致病性的有害真菌。与物理和化学方法抑制黑曲霉生长相比,生物抑菌剂抗黑曲霉生长具有药效久、无抗药性并安全健康的优点。本实验采用天然肉桂醛、柠檬醛作为抑菌剂,以正常生长的黑曲霉为对照,分别采用牛津杯法、气体扩散法比较对黑曲霉生长效果的影响。结果表明,柠檬醛作用所形成的抑菌圈显著大于肉桂醛作用所形成的抑菌圈,且在同一浓度下柠檬醛对菌丝体形态和孢子囊形态的抑制比肉桂醛显著,而气体扩散法抗黑曲霉效果优于牛津杯法。     

柠檬醛胁迫环境下黄曲霉线粒体的畸变

通过对黄曲霉细胞受柠檬醛损伤后线粒体形态畸变的透射电镜观察,发现柠檬醛所产生胁迫环境影响线粒体DNA复制系统,产生增生变异的巨型线粒体而与之应答。丙二醛法测定黄曲霉细胞内自由基,结果表明药物进入细胞后还通过诱发自由基使线粒体损伤,致使氧化还原系统及细胞能量代谢途径受到影响。     

The mechanism of antifungal action of essential oil from dill (Anethum graveolens L.) on Aspergillus flavus

The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 μl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus.     

Chromatographic separation and spectroscopic characterization of the e/z isomers of acrivastine

A reverse phase high performance liquid chromatography (HPLC) method has been developed for the separation of two geometric isomers of Acrivastine using crude reaction mixture. The resolution between two isomers was found more than 2.9. The geometric isomers have been isolated by preparative HPLC and characterized by spectroscopic techniques, such as NMR, infrared, and MS. The developed method has been validated for the determination of Z‐isomer in Acrivastine. The limit of detection and limit of quantification of the Z‐isomer were 0.05 and 0.2 μg/ml, respectively. The developed method is precise, linear, accurate, rugged and robust for its intended use. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Interaction between platelet receptor and iloprost isomers

Iloprost, a stable analog of prostacyclin, has been used for studying the interaction between prostacyclin and its effector cells such as platelets and vascular cells. The compound is usually prepared as a mixture of 16(S) and 16(R) stereoisomers. In this work, we compared the biological activity and platelet receptor binding characteristics between the two isomers. The 16(S) isomer was 20-times more potent than the 16(R) in inhibiting collagen-induced platelet aggregation. Equilibrium binding of iloprost isomers to platelet membrane receptors measured by rapid filtration method revealed that the specific binding data of 16(S) isomer was fit for a single binding species with Kd of 13.4 nM and Bmax 665 fmol/mg protein. By contrast, the Kd and Bmax of 16(R) isomer were 288 nM and 425 fmol/mg, respectively. To further assess different binding behavior of these two isomers, association rate was measured. The observed association rate of the S isomer was 0.036 s-1 and 0.001 s-1 for the R isomer at 15 nM iloprost and 2 mg/ml platelet membrane proteins. We postulate that the striking difference in the association rate with resultant difference in binding affinity and biologic activity between the two isomers was due to fitting of the molecule to the receptor channel. The 16(S) form has a more favorable orientation for fitting into the receptor. We conclude that the two iloprost isomers must be considered as two entirely different compounds when iloprost is used as the ligand for quantifying prostacyclin receptor binding.