大剂量粉防己对大鼠肾功能及肾组织形态影响的研究

目的:观察粉防己对大鼠肾脏的影响.方法:39只Sprague-dawley大鼠随机分为4个组:正常组给予自来水灌胃,小剂量粉防己组,中剂量粉防己组,大剂量粉防己组.分别以粉防己的水煎剂每天灌胃一次,分别持续60,45,30 d.结果:3组大鼠尿比重、pH、尿糖、尿N-乙酰-β-葡萄糖苷酶(尿NAG)、尿β2微球蛋白(尿β2MG)正常,大剂量组出现明显的蛋白尿(P＜0.01)以及尿Tamm-Horsfall蛋白(尿THP)升高(P＜0.05),中小剂量组尿THP与正常组对照无差异.大剂量应用30 d及小剂量应用60 d后,出现轻度的血清肌酐升高(P＜0.05),3组大鼠肝功能基本正常.病理结果显示3组大鼠的肾小球形态正常,肾小管上皮细胞有轻度空泡变性,肾间质有灶性炎细胞浸润及少量纤维组织增生.结论:粉防己可引起肾小管间质损害,并随着用量的增大,损害呈加重趋势.     

甲基莲心碱对大鼠心电图和蟾蜍坐骨神经动作电位的影响

在麻醉大鼠观察了恒速iv甲基莲心碱对ECG的影响,并与奎尼丁和粉防己碱进行了对比性研究。甲基莲心碱对大鼠ECG的影响与奎尼丁相似,剂量(2～40mg/kg)依赖性地延长P-R和Q-T,使QRS增宽;粉防己碱不增宽QRS。结果显示甲基莲心碱作用机理与奎尼丁相似,不同于粉防己碱。甲基莲心碱与奎尼丁都可依浓度抑制蟾蜍坐骨神经APA,二者作用强度相近。     

粉防己碱对分离大鼠神经垂体末梢及Y1小鼠肾上腺皮质瘤细胞离子通道作用之比

本文采用标准及穿孔膜片钳技术研究了粉防己碱对分离大鼠神经垂体末梢及Y1小鼠肾上腺皮质瘤细胞的电压控门的钙道，钾道及钠道的作用，细胞外的低浓度粉防己碱（13umol.L^-1)可强抑制Y1细胞株上的T型钙道电流52．9％，粉防己碱33umol.L^-1细胞外使用时亦可强抑制分离的神经垂体末梢L型下道电流54．2％，但对其N－型钙道电流无明显影响，1umol.L^-1粉防己碱在细胞外可阻断神经垂体末梢的慢控门，大电导，钙激活的钾道，使其开放概率降低84．4％，同一剂量的粉防己碱不影响快瞬时的钾道，本文结论为（1）离子通道对粉防己碱敏感性的顺序是：钙激活钾道＞T型钙道＞L型钙道＞N型钙道＞钠道；（2）粉防己碱可为一慢控门的钙激活钾道的特异性阻断剂。     

粉防己碱和甲基莲心碱对大鼠离体胸主动脉环收缩的影响

以苯肾上腺素为激动剂,在无钙K-H液中诱发大鼠去内皮主动脉环收缩并随后复钙的方法,观察粉防已碱和甲基莲心碱对主动脉环Ⅰ相收缩(胞内Ca~(++)释放)和Ⅱ相收缩(胞外Ca~(++)内流)的效应。结果表明,2×10~(-5)mol/L粉防己碱或甲基莲心碱预孵20min后,对Ⅰ、Ⅱ相的收缩均有非常明显的抑制作用(抑制率>92%,P<0.001);而短时预孵(5～10min)两药仅对Ⅲ相收缩有抑制作用,甲基莲心碱比粉防己碱起效慢、作用弱。     

高效液相色谱法测定防己药材中粉防己碱与防己诺林碱的含量

目的用高效液相色谱法测定不同产地防己药材中粉防己碱与防己诺林碱的含量。方法色谱柱为Kromasil ODS-1（4．6mm×200mm，5μm），流动相为乙腈0．01％三乙胺水溶液，梯度洗脱，流速为1．0mL·min^-1，检测波长为280nm。结果粉防己碱在474-4740ng内线性关系良好，r=0．9999，平均回收率为99．3％，RSD=1．5％；防己诺林碱在238-2380ng线性关系良好，r=0．9998，平均回收率为99．5％，RSD=1．1％。结论建立了高效液相色谱法同时测定防己药材中粉防己碱与防己诺林碱的含量，比较了不同产地防己药材中粉防己碱与防己诺林碱的含量。     

RP-HPLC测定舒肝饮丸中粉防己碱与防己诺林碱的含量

舒肝饮丸为我院自制制剂，由防己等9味中药组成，临床用于治疗慢性肝炎、肝硬化等疗效确切。防己为该制剂主要药物之一，含有粉防己碱（tetrandrine）和防己诺林碱（fangchinoline）等有效成分，高效液相色谱法测定防己及其他制剂中粉防己碱和防己诺林碱含量已有报道田。笔者用甲醇超声提取，采用高效液相色谱法测定舒肝饮丸中粉防己碱和防己诺林碱的含量，为该制剂的质量控制提供简便、快速、准确的测定方法。     

HPLC法测定风痛安胶囊中粉防己碱和防己诺林碱的含量

目的建立风痛安胶囊中粉防己碱和防己诺林碱含量的测定方法。方法采用高效液相色谱法。色谱柱为Diamonsil C18;流动相：甲醇-乙腈-0.03mol·L^-1磷酸二氢钾溶液-二乙胺（55∶25∶20∶0.05）;检测波长230nm。结果粉防己碱、防己诺林碱进样量分别在0.1355～2.7100μg（r=0.9999）,0.1033～2.0660μg（r=0.9999）范围内线性关系良好。粉防己碱、防己诺林碱平均回收率分别为98.02%（RSD=1.31%）,97.11%（RSD=1.42%）。结论本方法简便,专属,重复性好,可用于该制剂的质量控制。     

3，4—二甲氧基—5—羟基芪对HL—60细胞凋亡的诱导作用

肝癌是一种常见的致死性肿瘤,不易进行外科手术。作者研究了粉防己碱对人肝胚细胞癌 HepG2细胞增殖的抑制作用及诱导该细胞凋亡的作用。在抗细胞增殖实验中,HepG2细胞与8～24μm0l/L 的粉防己碱培养48 h,在24和48 h 用 MTT 法检测细胞活力。结果显示,     

HPLC法同时测定湿热痹胶囊中粉防己碱及防己诺林碱的含量

目的建立同时测定湿热痹胶囊中粉防己碱及防己诺林碱的HPLC法。方法采用高效液相色谱法测定湿热痹胶囊中粉防己碱及防己诺林碱的含量。结果粉防己碱在0.04322~0.8644μg范围内呈良好线性关系,平均回收率为99.2%(RSD=1.3%,n=6);防己诺林碱在0.04406~0.8812μg范围内线性关系良好,平均回收率为98.2%(RSD=1.7%,n=6)。结论该方法重复性、准确性良好,可作为标准用于湿热痹胶囊的质量控制。     

粉防己碱的提取和含量的测定

目的提取分离粉防己碱并测定其含量。方法自提粉防己碱,用反相高效液相色谱法是测定其含量,采用Lichrospher C18（250mmx4.6mm,5μm）色谱柱;流动相为乙腈-20mmol/L磷酸二氢钾-三乙胺（80-20-0.06）;柱温25℃;检测波长为282nm;流速1.0ml/min。结果粉防己碱在0.5～10μg/ml范围内与峰面积呈良好的线性关系（r=0.9998）;平均回收率为102%（RSD=1.5%）。结论建立的方法能很好的分离并准确测定粉防己碱。     

Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords.

In July 5, 2002 fishermen working in harvesting sea urchin (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated by consumption of filter-feeder bivalve Aulacomya ater. After the ingestion of 7-9 ribbed mussel, two fishermen died 3-4 h after shellfish consumption. The forensic examination in both victims did not show pathological abnormalities with the exception of the lungs conditions, crackling to the touch, pulmonary congestion and edema. The toxic mussel sample showed a toxicity measured by mouse bioassay of 8575 microg of STX (saxitoxin) equivalent by 100 g of shellfish meat. Using post-column derivatization HPLC method with fluorescent on line detection was possible to measure mass amount of each paralytic shellfish poisoning (PSP) toxin yielding individual toxin concentrations. These PSP toxins were identified in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The toxin profiles of each body fluid and tissue samples and the amount of each PSP toxin detected are reported. The PSP toxins found in the gastric content, were STX and the gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2) which showed to be the major amount of PSP toxins found in the victims biological samples. The PSP toxin composition in urine and bile showed as major PSP toxins neoSaxitoxin (neoSTX) and GTX4/GTX1 epimers, both STX analogues with an hydroxyl group (-OH) in the N(1) of the tetrahydropurine nucleus. The neoSTX was not present in the gastric content sample, indicating that the oxidation of N(1) in the STX tetrahydropurine nucleus resulted neoSTX, in a similar way that GTX3/GTX2 epimers were transformed in GTX4/GTX1 epimers. Beside this metabolic transformation, also the hydrolysis of carbamoyl group from STX to form its decarbomoyl analogue decarbamoylsaxitoxin was detected in liver, kidney and lung. These two findings show that PSP toxins went under metabolic transformation during the 3-4 h of human intoxication period, in which PSP toxins showed enzymatic oxidation of N(1) in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. This study conclude, that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound.     

乙氧苯柳胺在家兔体内的主要代谢产物

采用高效液相色谱法对家兔口服乙氧苯柳胺（Ｎ－（４－乙氧苯基）－２－羟基苯甲酰胺）后在尿中的代谢物进行了分离与检测．用β－Ｄ－葡糖苷酸酶和该酶加专属性抑制剂葡糖二酸－１，４－内酯分别对尿样进行水解处理，发现该药的主要代谢物为β－Ｄ－葡糖醛酸结合物，其含量超过尿中代谢物总量的８０％．利用色谱保留值和紫外双波长吸收比对酶解后的主要生成物进行定性分析，证明该代谢物为乙氧苯柳胺葡糖醛酸结合物（Ｎ－（４－乙氧苯基）－苯甲酰胺－２－Ｏ－葡糖苷酸）．此外，在家兔给药后０～１２ｈ尿样中未检测出乙氧苯柳胺原形药物及其可能的代谢产物水杨酸．     

Synthesis and antitumor activity of a series of ftorafur analogues: the effect of varying electronegativity at the 1'-position

To test the effect of changes in electronegativity within the alicyclic N-1 substituent 5-fluorouracil analogues on cytotoxic activity, a series of derivatives of ftorafur, 1-(2'-tetrahydrofuranyl)-5-fluorouracil, was synthesized and tested for antitumor activity in the P388 lymphocytic leukemia screen and cytotoxic activity in the L1210 cell culture screen. Two compounds of N-1 substituent with high electronegativity, the 2'-tetrahydrothiophene 1'-oxide and the 2'-tetrahydrothiophene 1',1'-dioxide derivatives, demonstrated the highest in vitro L1210 cell inhibition (84.5% and 92.0%, respectively). Furthermore, against P388 lymphocytic leukemia in vivo, the 2'-tetrahydrothiophene 1'-oxide derivative showed significant activity (T/C = 143). Other compounds of similar or lower electronegativity within the N-1 cyclic substituent were inactive against P388 lymphocytic leukemia and less active against L1210 cells.     

Characterization of metabolites of xylazine produced in vivo and in vitro by LC/MS/MS and by GC/MS.

The metabolic fate of xylazine, 2-(2,6-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazine, in horses is described. The major metabolites identified in the hydrolyzed horse urine were 2-(4'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, 2-(3'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, N-(2,6-dimethylphenyl)thiourea, and 2-(2',6'-dimethylphenylamino)-4-oxo-5,6-dihydro-1,3-thiazine. These metabolites were also produced by incubating xylazine with rat liver microsomes. The major metabolite produced in vitro by rat liver preparations was found to be the ring opened N-(2,6-dimethylphenyl)thiourea. The identities of these metabolites were confirmed by spectroscopic comparisons with synthetic standards. Phenolic metabolic standards were synthesized efficiently by the use of Fenton's reagent. This reagent was used to monohydroxylate multiply substituted aromatic ring systems. LC/MS/MS, with an atmospheric pressure chemical ionization source, was found to be particularly useful in confirming the presence of phenolic metabolites in hydrolyzed equine urine and microsomal extracts. These phenolic metabolites could not be analyzed by GC/MS even after derivatization with silylating agents. The advantage of LC/MS/MS was that no or little sample preparation of urine or microsomal extract was necessary prior to the analysis. A mechanism is also proposed for the formation of the major metabolite, N-(2,6-dimethylphenyl)thiourea, from xylazine.     

Formation of [20R]-dihydrodigoxin from digoxin in humans

The objective of this research was to determine the stereochemical identity of dihydrodigoxin (DHD3), a metabolite of digoxin excreted in urine after digoxin administration in man. Separation of the individual epimers in reference DHD3 was effected by a chemical derivatization-HPLC procedure. Comparison of individual derivatized epimers of DHD3 with the known 20R and 20S epimers of derivatized dihydrodigoxigenin using chromatographic data and NMR spectroscopy permitted identification of the 20R and 20S epimers in reference DHD3. Material corresponding chromatographically to R-DHD3 was isolated from urine of a volunteer taking oral digoxin daily. The NMR spectrum of the chromatographically pure, derivatized urinary isolate was identical to the NMR spectrum of derivatized R-DHD3. Urine samples from 20 patients on chronic digoxin therapy and from two volunteers were surveyed for chromatographic evidence of R- or S-DHD3 using HPLC of a fluorescent derivative as well as HPLC of a UV-absorbing derivative. The more sensitive fluorescence procedure yielded evidence for R-DHD3 in eight patients and both volunteers. There was a sufficient concentration in four patients and both volunteers for independent evidence of R-DHD3 using the UV-absorbing derivative. Chromatographic evidence (fluorescent derivative) for S-DHD3 was found in urine from three patients. However, this evidence for S-DHD3 was demonstrated to be an artifact by the absence of the peak expected for S-DHD3 in the chromatogram of the UV-absorbing derivative as well as by the inability of fecal incubates to convert digoxin to S-DHD3. The results of the investigation indicate that the DHD3 metabolite formed in humans is the 20R epimer.     

Detection of a novel reactive metabolite of diclofenac: evidence for CYP2C9-mediated bioactivation via arene oxides.

A new glutathione adduct (M4) was tentatively identified, likely as 2'-hydroxy-3'-(glutathione-S-yl)-monoclofenac, using liquid chromatography-tandem mass spectrometry analysis of incubations of diclofenac with human liver microsomes. The same conjugate was not detected in incubations with either rat or monkey liver microsomes. Formation of M4 was mediated specifically by CYP2C9 in human liver microsomes, as evidenced by the following observations: 1) cDNA-expressed CYP2C9-catalyzing formation of M4; 2) inhibition of M4 formation by sulfaphenazole, a CYP2C9-selective inhibitor; and 3) strong correlation between the production of M4 and CYP2C9-mediated tolbutamide 4-hydroxylase activities in a panel of human liver microsome samples. Formation of M4 suggests the existence of a new reactive intermediate as diclofenac-2',3'-oxide. A tentative pathway states that diclofenac is oxidized to diclofenac-2',3'-oxide that reacts with glutathione (GSH) to form a thioether conjugate at the C-3' position, followed by a concomitant loss of chlorine to give rise to M4. Furthermore, a likely mechanism leading to the formation of diclofenac oxides is rationalized: CYP2C9-catalyzed oxidation at the C-3' position of the dichlorophenyl ring to form a cationic sigma-complex that subsequently results in diclofenac-3',4'-oxide and diclofenac-2',3'-oxide; the former oxide is converted to 4'-hydroxy-diclofenac as a major metabolite and can be trapped by GSH to produce 4'-hydroxy-3'-glutathione-S-yl diclofenac (M2), whereas the latter oxide forms 3'-hydroxy-diclofenac and can be trapped by GSH to produce M4. This mechanism is consistent with the structural modeling of the CYP2C9-diclofenac complex, which reveals that both the C-3' and C-4' of the dichlorophenyl ring are proximate to the heme group.     

Formation and reversibility of S-linked conjugates of N-(1-methyl-3,3-diphenylpropyl)isocyanate, an in vivo metabolite of N-(1-methyl-3,3-diphenylpropyl)formamaide, in rats

The metabolic disposition of N-(1-methyl-3,3-diphenylpropyl) formamide was studied in rats. The water-soluble metabolites, N-acetyl-S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]cysteine and S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]glutathione, were identified in urine and bile, respectively, of rats doses with the secondary formamide. The structures of these metabolites were confirmed by comparison with synthetic standards and by using liquid chromatography mass spectrometry and fast atom bombardment mass spectrometry. Synthetic standards of these metabolites were obtained by reacting the N-(1-methyl-3,3-diphenylpropyl)isocyanate with glutathione or N-acetylcysteine in methanolic solutions. The isocyanate was obtained in high yield by reacting 1-methyl-3,3-diphenylpropylamine with trichloromethyl chloroformate. The S-linked conjugates released the isocyanate in mild alkali, but were stable under acidic conditions. The released isocyanate was characterized by comparison with the synthetic standard using GC/MS and HPLC. A mechanism is proposed for the base-catalyzed elimination of the isocyanate from the thiol conjugates.     

汉防己甲素对低温灌洗保存大鼠供肝的肝功能影响的实验研究

目的:观察汉防己甲素(TET)在低温灌洗保存供肝时对肝细胞的钙超载及肝功能的影响。方法:低温灌洗保存大鼠肝脏4 h,按保存液的不同分为4组:A组(对照组):乳酸林格液灌洗保存;B组(TET注射组):腹腔内注射TET,乳酸林格液灌洗保存;C组(TET灌洗组):腹腔内注射TET,乳酸林格液中加入TET灌洗保存;D组(Collins组):Collins液灌洗保存,测定供肝低温灌洗保存后肝细胞内钙浓度和肝功能,并在光镜和电镜下观察肝脏的结构。结果:细胞内钙浓度和肝功能测定,各实验组与对照组、C组与B组比较,差异有统计学意义(P<0.05);组织学观察支持以上结果。结论:TET能显著减轻低温灌洗保存下肝细胞的钙超载并起到保护肝功能作用;TET溶液直接灌洗保存供肝比TET腹腔注射效果好;用TET溶液灌洗并保存供肝4 h与Collins液灌洗保存组效果相近;TET对供肝的灌洗保存有积极的作用,因此推断该药有望用于临床供肝的保存。     

Route of metabolization and detoxication of paralytic shellfish toxins in humans

Paralytic shellfish toxins (PST) are a collection of over 26 structurally related imidazoline guanidinium derivatives produced by marine dinoflagellates and freshwater cyanobacteria.Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is the major phase II conjugation reaction in mammalian liver.In this study, using human liver microsomes, the in vitro paralytic shellfish toxins oxidation and sequential glucuronidation are achieved. Neosaxitoxin (neoSTX), Gonyautoxin 3/2 epimers (GTX3/GTX2) and Saxitoxin (STX) are used as starting enzymatic substrates. The enzymatic reaction final product metabolites are identified by using HPLC-FLD and HPLC/ESI-IT/MS. Four metabolites from GTX3/GTX2 epimers precursors, three of neoSTX and two of STX are clearly identified after incubating with UDPGA/NADPH and fresh liver microsomes. The glucuronic-Paralytic Shellfish Toxins were completely hydrolysed by treatment with β-glucuronidase. All toxin analogs were identified comparing their HPLC retention time with those of analytical standard reference samples and further confirmed by HPLC/ESI-IT/MS. Paralytic Shellfish Toxins (PST) were widely metabolized by human microsomes and less than 15% of the original PST, incubated as substrate, stayed behind at the end of the incubation.The apparent V_max and Km formation values for the respective glucuronides of neoSTX, GTX3/GTX2 epimers and STX were determined. The V_max formation values for Glucuronic-GTX3 and Glucuronic-GTX2 were lower than Glucuronic-neoSTX and Glucuronic-STX (6.8 ± 1.9 × 10⁻³; 8.3 ± 2.8 × 10⁻³ and 9.7 ± 2.8 × 10⁻³ pmol/min/mg protein respectively). Km of the glucuronidation reaction for GTX3/GTX2 epimers was less than that of glucuronidation of neoSTX and STX (20.2 ± 0.12; 27.06 ± 0.23 and 32.02 ± 0.64 μM respectively).In conclusion, these data show for the first time, direct evidence for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. The PST oxidation and glucuronidation pathway showed here, is the hepatic conversion of its properly glucuronic-PST synthesized, and the sequential route of PST detoxication in human.