石果衣菌藻次级代谢产物的初步研究

荒漠地衣石果衣 Endocarpon pusillum中未能检测出任何次级代谢产物,然而,其共生菌基因组中却含有14个PKS基因和2个NRPS基因。通过激活培养后从中分离出3个次级代谢产物,4,6,9-三羟基-7-甲基-1H,3H-萘并[1,8-cd]吡喃-1,3-二酮（lamellicolic anhydride）、石果衣菌素A（endocarpin A）及石果衣菌素B（endocarpin B）,后二者为新结构化合物。从其共生藻柯氏复球藻 Diplosphaera chodatii中分离出3个脱镁叶绿素类化合物,即脱镁叶绿素甲酯酸b（phaeophorbide b）、13-表-脱镁叶绿素甲酯酸a（13- epi-phaeophorbide a）及脱镁叶绿素甲酯酸a（phaeophorbide a）。其中脱镁叶绿素甲酯酸b具有中等的清除DPPH活性。     

植物脱镁叶绿素替代RS601反应中心中的细菌脱镁叶绿素的研究

在一定的温度和丙酮的协同作用下,植物脱镁叶绿素a可置换紫细菌RS601光合反应中心的细菌脱镁叶绿素而形成含有植物脱镁叶绿素的紫细菌光合反应中心(简称Phe a RC).当协同作用15和60min时,反应中心的细菌脱镁叶绿素分别被替代了50%和71%.在Phe a RC中,细菌脱镁叶绿素的QX(537nm)和QY(758nm)特征峰显著下降,而出现植物脱镁叶绿素的QX(509/542nm)和QY(674nm)特征峰.排除温度和丙酮的影响,替代时间为15或60min的Phe a RC的光化学活性分别为对照的78%或71%,其电化学特性也有所变化.     

东海赤潮高发区沉积物中叶绿素的分析

研究分析了2002年8～9月“赤潮973”航次所采集的沉积物中叶绿素a及其降解产物脱镁叶绿酸含量与分布．结果表明。叶绿素a和脱镁叶绿酸是同源的;随深度增加叶绿素a和脱镁叶绿酸的含量呈递减趋势,到一定深度后含量不再变化。个别站位叶绿素a和脱镁叶绿酸的垂直分布出现了许多小突跃,可能是由生物扰动引起;表层沉积物(0～0．5cm)中的叶绿素a和脱镁叶绿酸的变化幅度分别为0．14～1．17和0．83～5．58μg·g-1。平均值分别为0．54和2．45μg·g-1;并初步探讨水深(光强)、盐度、温度、含水量对叶绿素水平分布的影响;脱镁叶绿酸作为叶绿素的主要降解产物,到一定深度后,成为叶绿素的主要存在形式,约占80％～90％;由于水温的不同,夏季沉积物中的叶绿素a和脱镁叶绿酸含量是春季的3倍之多;比较了上层水柱中的叶绿素和沉积物中的叶绿素的相对含量,平均而言,沉积物中叶绿素占上层水柱中叶绿素的31％．     

SO_2对叶片中脱镁叶绿素 a 形成的影响

高等植物在正常生长情况下,一般叶组织是没有或具有少量脱镁叶绿素 a 的。当植物受至 SO_2污染后,叶组织脱镁叶绿素 a 含量增加,色带增浓;其变化规律是:在一定接触量范围内,与熏气时间和作用浓度呈正相关;与可见伤害呈现相一致;与植物的抗性呈负相关。实验表明,脱镁叶绿素 a 的变化是 SO_2污染的伤害指标。     

通过分子的修饰进行Chla分子功能基团的研究

众所周知,叶绿素A（Chla）是植物的最重要的光合作用色素.Chla的分子结构主要是由卟啉环和叶绿醇组成的,在卟啉环的中心络合了镁离子.在光合作用过程中Chla的哪个基团起到光合功能的作用？为此我们从海带里提取Chla,然后采用分子修饰的方法对Chla分子进行一步步修饰分别生成脱镁叶绿素A和脱镁叶绿素甲酯一酸A（Pha）.通过对新鲜菜叶和在实验室制备的相关样品溶液包括Chla、脱镁叶绿素A和脱镁叶绿素甲酯一酸A（Pha）的激发谱和荧光谱进行探测分析,并与原卟啉水溶液中卟啉的光谱进行比较.结果发现这些样品的光谱均与卟啉的光谱相似,在红光波段有较强的发射带,从而表明光合作用过程中Chla的功能团是卟啉环.     

叶绿素比吸收系数的测定

本实验用柱状层析分离和纯化叶绿素a和叶绿素b。叶绿素的无水乙醚溶液用盐酸进行酸化处理。从叶绿素分子释放出的镁用原子吸收分光光度法测定,从而推算出相应的叶绿素的量。并根据叶绿素a和叶绿素b无水乙醚溶液的光密度计算了它们的比吸收系数。测定范围为每毫升含镁0.01～0.1微克。本实验还检验了脱镁的完全程度和去镁叶绿素对原子吸收分光光度法测镁的干扰作用。     

金属叶绿素a配位结构的研究

在丙酮溶液中合成得到镧-叶绿素a、钐-叶绿素a和铜-叶绿素a复合物,并研究了它们的紫外可见光谱（UV-Vis）、Fourier红外光谱（FT-IR）和EXAFS结构.镧-叶绿素a、钐-叶绿素a和铜-叶绿素a的UV-Vis谱、FT-IR谱与叶绿素a（含镁）的光谱性质相似,但与脱镁叶绿素a的光谱性质差异很大.证明了La³⁺、Sm³⁺、Cu²⁺已配位到脱镁叶绿素的卟啉环上,形成了镧-叶绿素a、钐-叶绿素a和铜-叶绿素a复合物.通过扩展X射线吸收精细结构谱(EXAFS)研究表明：合成镧-叶绿素a、钐-叶绿素a具有双层夹心结构.La(Ⅲ)、Sm(Ⅲ)夹于两个卟啉环之间, 与上下卟啉环上共八个N原子配位, La-N平均键长0.261 nm,Sm-N平均键长0.243 nm, 而铜-叶绿素a的EXAFS表明为一单层结构,Cu(Ⅱ)与卟啉环中的四个N原子配位,Cu-N平均键长0.197 nm.元素分析也证明镧-叶绿素a、钐-叶绿素a为双层结构,铜-叶绿素a为单层结构.     

银杏叶中叶绿素衍生物的反相高效液相色谱分析

以检测植物色素降解过程中叶绿素衍生物的变化为主要目的,采用反相高效液相色谱,结合光电二极管阵列和荧光检测技术,分析了银杏叶中叶绿素衍生物的组成和含量变化,发现在衰老的银杏叶中主要存在叶绿素a、叶绿素b和脱镁叶绿素a,而其他降解产物并未大量累积。     

武汉东湖光合色素与叶绿素a代谢产物的HPLC研究

用高效液相色谱（HPLC）法研究了武汉东湖周年及围隔实验水柱颗粒物色素的组成及变化。共检测到约20种色素,类胡萝卜素含量较高的有硅藻的标志色素岩藻黄素,隐藻的异黄素,蓝绿藻的黄体素,玉米黄素及胡萝卜素。东湖叶绿素a的代谢产物主要为脱植基叶绿素a(全湖年均约占叶绿素a的5%）,而非脱镁叶绿素a或脱镁叶绿酸a。围隔实验结果表明：叶绿素a与总浮游植物（r=0.84)。叶绿素b与绿藻（r=0.77)。岩藻黄素与硅藻（r=0.68),异黄素与隐藻生物量（r=0.83)之间具显著的相关性。表明用HPLC分析色素快速,简捷,是研究浮游植物群落组成及动态变化的有力辅助工具。     

紫细菌光合反应中心中细菌脱镁叶绿素被置换后的光 …

采用新型表面活性剂ＬＤＡＯ,结合ＤＥＡＥ－纤维素层析法,我们提纯了紫细菌６０１的光合反应中心,在一定温度和丙酮的协同作用下,外加的植物脱镁叶绿素ａ可取代反应中心细菌脱镁叶绿素,形成含有脱镁叶绿素ａ的紫细菌光合反应中心。     

The synthesis of galactopyranosyl-substituted derivatives of pheophorbide

New pheophorbide and pyropheophorbide derivatives containing carbohydrate fragments, derivatives of galactopyranose, were synthesized. Galactopyranosylpheophorbide and galactopyranosylpyropheophorbide with free hydroxyl groups were found to be water-soluble and useful as sensitizers in photodynamic therapy of cancer.     

The synthesis of galactopyranosyl-substituted derivatives of pheophorbide

New pheophorbide and pyropheophorbide derivatives containing carbohydrate fragments, derivatives of galactopyranose, were synthesized. Galactopyranosylpheophorbide and galactopyranosylpyropheophorbide with free hydroxyl groups were found to be water-soluble and useful as sensitizers in photodynamic therapy of cancer. This article is dedicated to the 25th Anniversary of the journal Bioorganicheskaya Khimiya     

Potential anticancer activity of young Carpinus betulus leaves

As part of our continuing research for anticancer compounds from the Walloon Region forest, EtOAc extract from Carpinus betulus leaves was phytochemically studied, leading to the bioguided isolation of pheophorbide a, which is responsible of anticancer properties of C. betulus young leaves. This compound was identified using nuclear magnetic resonance and mass spectrophotometric data and comparison with a commercial standard. Evaluation of the growth inhibitory activities of pheophorbide a using MTT colorimetric assay and phase-contrast microscopy in various human cancer cell lines confirmed the photoactivable properties of this compound. Our research showed, for the first time, the presence of pheophorbide a, a chlorophyll derived compound, which we quantified in high quantities in young leaves of C. betulus. This is in contrast with the literature which generally describes pheophorbide a as a catabolic product of chlorophyll, then preferentially present in old leaves.     

Photophysical properties and photodynamic activity in vivo of some tetrapyrroles

Some of the photophysical properties (stationary absorbance and fluorescence, fluorescence decay times and singlet oxygen quantum yields) of pheophorbide a, metal-free, ClAl-, Cu- and Mg-t-butyl-substituted phthalocyanines, metal-free, ClAl- and Cu-t-butyl-substituted naphthalocyanines and of a number of tetraphenylporphyrins (5,10,15,20-tetraphenylporphyrin, 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin) have been studied in comparison with hematoporphyrin IX in order to select potent photosensitizers for the photodynamic treatment of cancer. The photodynamic activity of these compounds was investigated using Lewis lung carcinoma in mice. As a consequence of the photophysical parameters (relatively short singlet state lifetimes, and high singlet oxygen quantum yields) the photodynamic activities of pheophorbide a, t-butyl-substituted ClAl-phthalocyanine and ClAl-naphthalocyanine were selected for study in greater detail. Under the conditions employed in the present study, pheophorbide a was found to be the most effective sensitizer, as judged from its strong absorption at the excitation wavelength as compared with the hematoporphyrin derivative and greater singlet oxygen quantum yield relative to the phthalocyanines and naphthalocyanines. The photodynamic activity was observed to be strongly dependent on the photophysical parameters of the compounds. The primary mechanism underlying the photodynamic activity of these sensitizers probably consists of energy transfer from the lowest triplet state of the dyes to molecular oxygen, resulting in the formation of singlet oxygen (type II of photosensitization).     

Preparation and in vitro biological evaluation of tetrapyrrole ethanolamide derivatives as potential anticancer agents

Tetrapyrrole ethanolamide derivatives, 1 and 2, were prepared from hematoporphyrin IX (HPIX, 3) and methyl pheophorbide a (mPheo, 6). These were evaluated for their dual action as chemotherapeutics and photosensitizers in treatment of cancer. The novel compounds showed significant in vitro anticancer activity as measured in different cell lines using the MTT assay and photodynamic activity measured by erythrocytes' photohemolysis.     

Evaluation of human erythrocytes as model cells in photodynamic therapy

The role of erythrocytes as targets in photodynamic therapy is a controversially discussed topic in the literature. Therefore five different, but well known photosensitisers (three zinc phthalocyanines, tetrabenzoporphine and pheophorbide a delivered in liposomes were used for photodynamic treatment of human erythrocytes. The phototoxic effect on these cells showed pronounced differences. It was in the range: zinc phthalocyanine = pheophorbide a > tetrabenzoporphine > zinc octa-n-alkyl phthalocyanines. Data from the zinc octa-n-alkyl phthalocyanines were compared with photodynamic effects within cutaneous cell lines, treated under the same experimental conditions. The results show that erythrocytes are unlikely to make good models for predicting the efficiency of the photosensitiser in general, and the same applies to cells other than erythrocytes and in vivo. Possible reasons could be differences in dye accumulation. However, erythrocytes may well serve as model cells to explore the cellular and molecular mechanisms of photodynamic treatment.     

Effective photocleavage of DNA by pheophorbide a.

Remarkably effective photocleavage of plasmid DNA with pheophorbide a, as a visible light sensitizer, has been found for the first time. DNA is photocleaved even in the absence of oxygen. The novel oxygen-independent scission is ascribed to specific photodynamic function of the ring V in pheophorbide a.     

Chlorophyll breakdown in senescent leaves: demonstration of Mg-dechelatase activity

The action of Mg-dechelatase was brought to light by incubating senescent rape cotyledons or chloroplasts under conditions which prevented the oxidative cleavage of chlorophyll-porphyrin. The accumulation of chlorophyllide and pheophorbide taking place under such conditions was considered as a measure of apparent activities of chlorophyllase and dechelatase, respectively. In excised cotyledons metal chelators such as 2,2'-dipyridyl and o -phenanthroline caused a marked accumulation of pheophorbide a , without affecting the apparent activity of chlorophyllase. Treatment of cotyledons with an inhibitor of cytoplasmic protein synthesis d -2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide ( d -MDMP) caused a reduced accumulation of pheophorbide a in the presence of dipyridyl, suggesting that the appearance and maintenance of Mg-dechelatase activity in senescent cotyledons requires continuous cytoplasmic protein synthesis. In isolated senescent chloroplasts (gerontoplasts) the cleavage of chlorophyll-porphyrin requires the supplementation with glucose-6-phosphate (Glc6P). Upon the incubation of gerontoplasts in the absence of Glc6P, a conspicuous accumulation of pheophorbide a occurred. Much smaller pools of pheophorbide a were produced when porphyrin cleavage was allowed in the presence of Glc6P. These phenomena were not observed in pre-senescent chloroplasts. In contrast to the apparent Mg-dechelatase activity, chlorophyllase activity did not change in a senescent-specific fashion. The lysis of gerontoplasts by freezing and thawing caused an enhancement of apparent chlorophyllase activity whereas the activity of Mg-dechelatase was lower than in the intact organelles. In the pre-senescent chloroplasts, lysis evoked a small apparent Mg-dechelatase activity, suggesting that in a latent form this enzyme may be present even before the onset of foliar senescence.     

Peppermint (Mentha piperita L.) extract effectively inhibits cytochrome P450 3A4 (CYP3A4) mRNA induction in rifampicin-treated HepG2 cells

Interaction between foods and drugs is an important consideration in pharmaceutical therapy. Therefore, here, we examined the suppressive effects of the extracts from seven edible herbs on the induction of CYP3A4 gene expression in rifampicin-treated HepG2 cells. We evaluated the structure and suppressive activity of the most effective active compound isolated from dried peppermint (Mentha piperita L.). The structure of the compound was identified as that of pheophorbide a based on spectroscopic data. It suppressed the induction of CYP3A4 mRNA expression by rifampicin in a dose-dependent manner. Quantitative high-performance liquid chromatography showed that 2 g of dry leaves 0.43 mg in one cup of peppermint tea. These findings demonstrate that pheophorbide a suppresses the induction of CYP3A4 mRNA expression in rifampicin-treated HepG2 cells. Pheophorbide is known to cause photosensitivity. However, the effective dose of pheophorbide a that had a suppressive effect was very low, indicating a high safety margin.

Abbreviations: DAD: diode array detector; DMEM: Dulbecco’s modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; HPLC: high-performance liquid chromatography; PCR: polymerase chain reaction; PXR: pregnane X receptor; CAR: constitutive androstane receptor; AHR: aryl hydrocarbon receptor; TLC: thin-layer chromatography     

Impact of genetic variability in the ABCG2 gene on ABCG2 expression,function, and interaction with AT1 receptor antagonist telmisartan

The ATP-binding cassette transporter ABCG2 plays a prominent role in cardiovascular and cancer pathophysiology, is involved in the pathogenesis of gout, and affects pharmacokinetics of numerous drugs. Telmisartan, a widely used AT1 receptor antagonist, inhibits the transport capacity of ABCG2 and may cause drug–drug interactions, especially in individuals carrying polymorphism that facilitate the telmisartan–ABCG2 interaction. Thus, the aim of this study was to identify ABCG2 polymorphisms and somatic mutations with relevance for the telmisartan–ABCG2 interaction. For this purpose, a cellular system for the conditional expression of ABCG2 was established. ABCG2 variants were generated via site-directed mutagenesis. Interaction of telmisartan with these ABCG2 variants was investigated in HEK293-Tet-On cells using the pheophorbide A efflux assay. Moreover, expression of ABCG2 variants was studied in these cells. Importantly, protein levels of the Q141K and F489L variant were significantly reduced, a phenomenon that was partly reversed by pharmacological proteasome inhibition. Moreover, basal pheophorbide A efflux capacity of S248P, F431L, and F489L variants was significantly impaired. Interestingly, inhibition of ABCG2-mediated pheophorbide A transport by telmisartan was almost abolished in cells expressing the R482G variant, whereas it was largely increased in cells expressing the F489L variant. We conclude that the arginine residue at position 482 of the ABCG2 molecule is of major importance for the interaction of telmisartan with this ABC transporter. Furthermore, individuals carrying the F489L polymorphism may be at increased risk of developing adverse drug reactions in multi-drug regimens involving ABCG2 substrates and telmisartan.