续随二萜酯药代动力学研究
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摘要
研究背景和目的
大戟属(Euphorbia Linn)是大戟科(Euphorbiaceae)植物中最大的一个属。大戟属的很多植物是药用植物,大戟属植物的生物活性也多与其所含的二萜酯类(diterpenoids)密切相关。续随子(Euphorbia Lathyris Linn)为大戟属植物,在中国分布广泛,民间常用作除疣、抗肿瘤等。从续随子分离出一系列二萜类化合物,主要是续随烷型二萜,有较强的抗肿瘤和多药耐药抑制(multi-drug resistance, MDR)活性。
续随二萜酯(euphorbiasteroid),又名大戟因子LI(Euphorbia factor L1)是从续随子分离得到的天然续随烷型二萜,对人宫颈癌HeLa细胞的增殖有显著抑制活性,对多种肿瘤耐药细胞株MCF-7/ADR、MES-SA/Dx5、KBV200等具有P-gp介导的多药耐药抑制活性。
续随二萜酯在体外具有一定的抗HIV活性。本课题组的前期研究发现续随二萜酯对HIV感染的慢性H9细胞(H9/HIV-1ⅢB)与靶细胞MT2的融合有一定的抑制作用,测得半数抑制浓度IC5。和治疗指数T1分别为0.8μM、600。
艾可清2号是广州中医药大学热带医学研究所在艾可清基础上研制的创新中药,由复方中药与续随二萜酯组合而成。实验研究表明艾可清2号能显著提升艾滋病恒河猴模型的CD4+T细胞比率及CD4+/CD8比值。创新中药复方艾可清2号显示了作为人类艾滋病治疗中药新药的应用前景。基于此,续随二萜酯有望成为既具有多药耐药抑制,又具有一定抗病毒能力的新型抗HIV先导化合物。
到目前为止,有关续随二萜酯在体内的吸收、代谢等相关药代动力学特征尚不清楚。为满足新药的研究与开发需要,本课题对续随二萜酯在大鼠、中国恒河猴及猴艾滋病模型体开展了较为系统的药代动力学探索性研究。
研究方法
1.采用高效液相色谱/串联质谱联用(HPLC-MS/MS)定量分析方法,建立了生物样品中续随二萜酯的检测,用于续随二萜酯在大鼠、恒河猴、猴艾滋病模型体内药代动力学研究。
2.通过Waters2767制备液相色谱仪分离,从续随二萜酯在碱作用下水解液得到环氧续随子醇。采用MS、1H-NMR、13C-NM等波谱学分析方法,鉴定了该化合物的结构。
3.采用液相色谱-离子阱质谱联用(LC/MS”)技术,在多级质谱扫描模式下,分析了续随二萜酯在大鼠体内的代谢产物。
4.采用超滤法进行了续随二萜酯与不同种属血浆蛋白结合率的研究。
研究结果
1.建立和确证了大鼠血浆、恒河猴血浆、猴艾滋病模型血浆等生物样品中续随二萜酯的液质联用(HPLC-MS/MS)定量分析方法。
生物样品预处理采用乙酸乙酯液-液萃取,经Waters Symmetry Shield C8(4.6mm×150mm,5.0μm)色谱柱,流动相甲醇(A)-10mM含0.1%甲酸的甲酸铵水溶液(B)梯度洗脱。三重四极杆质谱仪进行多反应监测(MRM)模式下的电喷雾离子源正离子检测(ESI+),续随二萜酯和内标汉黄芩素用于定量分析离子对分别为m/z553→m/z297、m/z285→m/z270.标准曲线在1～10000μg·L-1范围内线性关系良好,线性相关系数r为0.9975；定量下限为1μg·L-1,续随二萜酯保留时间(tR)为4.0min,内标保留时间(tR)为4.3min,单个样品分析时间7.0min。日内、日间精密度(RSD)在2.6%-10.7%,准确度在1.6%～5.1%,符合生物样品分析的要求。
2.考察了续随二萜酯在大鼠、恒河猴、猴艾滋病模型体内药动学特征。
应用生物样品中续随二萜酯的HPLCMS/MS定量分析方法,考察了续随二萜酯大鼠单次口服给药和静注给药、恒河猴单次口服给药和静注给药及猴艾滋病模型口服给药的体内药物动力学特征,应用DAS2.1药动学对数据进行处理,得到非室模型药代动力学参数。
续随二萜酯大鼠静脉注射10mg·kg-1,血药浓度极大值(Cmax)平均为3418.6μg·L-1,续随二萜酯大鼠口服给药100mg·kg-1,Cmax平均为87±32.4μg·L大鼠口服给药达峰时间平均为0.33h.
续随二萜酯恒河猴静脉注射给药5mg·kg-1,Cmax平均为1359.5μg.L-1;口服给药30mg·kg-1,Cmax平均为139.1μg·L-1。恒河猴口服给药,达峰时间平均为0.31h。
大鼠静和恒河猴静脉注射和口服给药后,续随二萜酯在体内均迅速衰减。药物在大鼠和恒河猴中都存在明显的个体差异。
续随二萜酯猴艾滋病模型口服给药相同剂量,与正常恒河猴相比,血药浓度达到极大值为56.9μg.L-1,达峰时间平均为0.50h。续随二萜酯在恒河猴和猴艾滋病模型体内都存在明显的吸收差异性。
大鼠、恒河猴、猴艾滋病模型口服给药绝对生物利用度分别为2.0%、4.6%、1.8%。相对于健康猴口服给药,猴艾滋病模型口服利用度约为1/3。
3.续随二萜酯在THF中和50℃水浴温度下,经1.25mol·L-1NaOH作用48h,水解液经HPLC-DAD检测,液质联用多级质谱分析,Waters液相制备柱分离,得到一化合物纯品(得率5.42%),经电喷雾离子源正负离子质谱(ESI-MS)、13C-NMR,1H-NMR分析,鉴定为环氧续随子醇(epoxylathyrol)。
4.应用电喷雾离子源多级质谱法分析了续随二萜酯的结构,并对它的裂解规律进行分析归纳。
在正离子模式,利用离子阱质谱多级质谱扫描技术,对续随二萜酯在大鼠血液和尿液中的16个未知代谢产物进行了合理的推测。
从续随二萜酯大鼠静注给药血样中推测了7个代谢产物可能的结构,代谢产物涉及酯酶水解成羟基,有的伴随羟基的甲基化,甲基加氧(0)氧化或者C-6环氧加氢(H)还原。从续随二萜酯大鼠口服给药血样中推测了5个代谢产物可能的结构,其中4个与静注给药血样中代谢产物相同。大鼠血样中的代谢产物以Ⅰ相代谢为主,而尿液中的代谢产以Ⅱ相代谢为主。从续随二萜酯大鼠口服给药尿样中推测了4个代谢产物结构。尿样中的代谢产物除少量酯酶水解产物,大部分是与葡萄糖醛酸结合产物,有些还伴有甲基化。
5.采用超滤法、Amicon Ultra-0.5超滤离心装置(截留分子量10K)对续随二萜酯与三种属(SD大鼠、恒河猴、人)血浆蛋白结合程度进行研究。续随二萜酯磷酸缓冲盐溶液非特性结合率平均为60.5%。在三个浓度水平(1000、100、20μg·L-1)条件下,续随二萜酯与大鼠、猴、人血浆蛋白结合率均较高(76.0%、98.0%),与血浆蛋白的结合不存在浓度依赖性。续随二萜酯与大鼠血浆蛋白结合率相对较低(76.0%～79.8%),与恒河猴、人血浆蛋白结合率较高(94.6%～98.0%),续随二萜酯与人和猴血浆蛋白结合率无统计学差异(p>0.05)。考虑到高非特异性结合率,对血浆蛋白结合率进行校正后,续随二萜酯与人和猴血浆蛋白结合率均高于86%。
结论和创新点
1.建立和确证了生物样品中续随二萜酯在ESI电离源、MRM模式下液质联用定量分析方法。血样最低定量限1μg·L-1,线性范围1-10000μg· L-1,适用于动物口服给药和静注给药后血浆样品中药物的检测。
应用液质联用方法,首次研究比较了续随二萜酯在大鼠、健康及猴艾滋病模型中国恒河猴体内的药代动力学特征。发现大鼠与恒河猴口服给药后,血药浓度达峰时间均约0.3-0.5h。给药后,续随二萜酯血药浓度在体内均发生迅速衰减；生物利用度均很低,大鼠、恒河猴或其艾滋病模型的绝对生物利用度均低于5.0%,提示提高生物利用度对于给药可能具有关键意义。
2.首次从续随二萜酯制备环氧续随子醇。水解过程采用液质谱联用(LC-DAD/ESI-MS")技术监控。
3.利用液相色谱-离子阱质谱联用(LC/MSn)技术,对续随二萜酯在大鼠血液和尿液中,16个未知代谢产物的结构进行了合理的推测。
4.首次应用超滤法测定了续随二萜酯与大鼠、恒河猴、人血浆蛋白结合率。续随二萜酯与恒河猴、人血浆蛋白结合率(94.6%、98.0%)高于与大鼠血浆蛋白结合率(76.0%～79.8%)。续随二萜酯对人和猴血浆蛋白的结合率无统计学差异(p>0.05)。对非特异性结合进行校正后,续随二萜酯与恒河猴、人血浆蛋白结合率仍大于86%。
Background and Objective
The genus Euphorbia is the largest one in Euphorbiaceae. Many species of Euphorbia are used as medicinal plants. Euphorbia plants are rich resources in R&D of bioactive diterpenoids. Euphorbia Lathyris Linn belonging to Euphorbia is widely distributed in China and has been used in the treatment of cancer and warts as Chinese traditional medicine. A series of diterpenoids based on the lathyrane skeleton have been isolated from Euphorbia Lathyris Linn. Biological activity of these lathyrane-type diterpenoids have been carried out showing powerful anti-cancer activity and ability of reversing multi-drug resistance (MDR)。
Euphorbiasteroid(Euphorbia factor L1) is one of the main constituents isolated from Euphorbia lathyris. It was reported to have significant growth-inhibitory effects on human cervical cancer HeLa cells in vitro, reversal activity against P-glycoprotein (P-gp) mediated MDR in resistant cells such as KBv200, MCF-7/adr and MES-SA/Dx5cell lines. In our preceding researches, we found that euphorbiasteroid was potential against HIV in vitro, for its inhibitory activity in the fusion between H9cells chronically infected by HIV-1ⅢB with the target MT-2cells with a half inhibitory concentration (IC50) and therapeutic index (TI)0.8μM, and600respectively. Aikeqing-2, developed by the Tropical Medicine Institute of Guangzhou University of Traditional Chinese Medicine, is a combinatorial medicine of compound Chinese herbal medicine Aikeqing with euphorbiasteroid for the treatment of acquired immunodeficiency syndrome (AIDS). In our previous researches, Aikeqing-2exerted certain effect on increasing peripheral CD4+T lymphocytes count and the ratio of CD4+/CD8+So euphorbiasteroid is expected to be developed as a lead compound for the treatment of AIDS and cancer. Profiles including pharmacological activities mentioned above and results related to content determination, toxic action were reported, as far as we know the knowledge regard to pharmacokinetics and metabolism of euphorbiasteroid is not clear. In order to provide a meaningful basis in pharmacokinetics for the new drug development we carried out a pharmacokinetic study on the compound euphobiasteroid. This paper describes the establishment and validation of a method based on high performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of euphorbiasteroid in biological sample, which was used subsequently to determine the pharmacokinetics of euphorbiasteroid in rats, healthy rhesus macaques and rhesus macaques infected with simian immunodeficiency virus (SIV), namely SAIDS model monkeys.
Methods
1. A high performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of euphorbiasteroid in biological sample was established and validated, which was used subsequently to determine the pharmacokinetics of euphorbiasteroid in rats, rhesus macaques and SAIDS rhesus macaques.
2. Epoxylathyrol was isolated from alkaline hydrolysis of euphorbiasteroid by Waters2767Sample Manager. On the basis of spectroscopic data including those of MS,1H-NMR, and13C-NMR spectra, the structure was confirmed.
3. Liquid chromatography-ion trap mass spectrometry in the multi-stage MS full scan mode (LC/MSn) was used to analyze metabolites of euphorbiasteroid in rats.
4. Study on the binding of euphorbiasteroid to plasma proteins from different biological species by ultrafiltration.
Results
1. An HPLC-MS/MS method for the determination of euphorbiasteroid in plasma of rats, healthy and SAIDS monkeys was established and validated. Eupborbiasteroid was extracted with ethyl acetate from biological samples. A Waters Symmetry Shield C8(4.6mm×150mm,5.0μm) column was used with a mobile phase consisting of methanol (A)-lOmM ammonium formate containing0.1%formic acid (B) gradient elution. The determination was performed with electrospray ionization (ESI) source in the positive mode by a triple quadrupole tandem mass spectrometer. Quantification was performed by multiple reaction monitoring (MRM) of the transitions of m/z553～m/2297for euphorbiasteroid and m/z285→m/270for IS(wogonin). The linear calibration curves were obtained in the concentration range of1～10000μg·L-1(r=0.9975). The lower limit of determination (LLOQ) was1μg·L-1. The run time for each sample was7. Omin. The intra-and inter-day precision at three quality control (QC) levels were within2.6%-10.7%. The accuracy of the assay was within1.6%-5.1%.The method was proved suitable for the preclinical pharmacokinetics of euphorbiasteroid, which offered advantages of high sensitivity and selectivity.
2. The pharmacokinetics of euphorbiasteroid in rats, healthy and SAIDS monkeys were investigated using the established HPLC-MS/MS method.
An HPLC-MS/MS method for the determination of euphorbiasteroid in biological sample was used to determine the pharmacokinetics of euphorbiasteroid in rats and rhesus macaques following a single oral dose and intravenous administration, the pharmacokinetics of euphorbiasteroid in SAIDS monkeys following an oral administration. The non-compartmental pharmacokinetics parameters were processed by DAS2.1software program (Chinese Pharmacological Society).
The mean maximum concentration of drug (Cmax) was3418.6μg· L-1after intravenous administration at a dose of10mg·kg-'and87μg· L-1after an oral administration of euphorbiasteroid to SD rats at a dose of100mg· kg-1respectively.
The mean values of Cmax were1359.5μg·L-1after intravenous administration at a dose of5mg· kg-1and139.1μg· L-1after an oral administration of euphorbiasteroid to rhesus macaques at a dose of30mg·kg-1respectively.
After oral and intravenous administration, the elimination of euphorbiasteroid in rats and rhesus macaques were both rapid. Apparently individual difference in the absorption of euphorbiasteroid existed both in rats and rhesus macaques.
The mean value of Cmax was56.9μg· L-1to SAIDS rhesus macaques after oral administration at the same dose with rhesus macaques. Apparently differences were found existing in the absorption of euphorbiasteroid in both healthy and SAIDS rhesus macaques.
The mean bioavailability of euphorbiasteroid in rats, rhesus macaques and SAIDS rhesus macaques were2.0%,4.6%and1.8%, respectively.
Z. Under a waler bath at50℃, euphorbiasteroid was dissolved in THF and hydrolyzed with mol· L-1NaOH for48h. The hydrolysate was detected by photodiode array detector (DAD), analyzed by Liquid chromatography electrospray ion trap mass spectrometry in the multi-stage MS full scan mode(LC/MS"), and isolated by preparative HPLC to give a pure product with a yield rate of5.42%. The chemical structure was confirmed as epoxylathyrol on the basis of its spectroscopic data including those of MS,1H-NMR and13C-NMR spectra.
4. Electrospray ionization mass spectrometry method was applied to analyze the possible structures of metabolites of euphorbiasteroid in plasma and urine of rats.
In positive mode, a total of sixteen euphorbiasteroid derivatives were detected by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode (LC/MS") in rats plasma and urine after oral or intravenous administration. Seven euphorbiasteroid derivatives in the plasma were deduced as products related esterases hydrolysis, methylation, oxidation and reduction after intravenous administration. Four of the five metabolites in oral administration were the same as intravenous administration. The main metabolites in rat plasma was phase Ⅰ metabolism of euphorbiasteroid, whereas phase Ⅱ metabolites was found in urine. Four euphorbiasteroid derivatives were deduced as glucuronidated and methyl-glucuronidated metabolites in rat urine after oral administration.
5. The plasma protein binding (PPB) of euphorbiasteroid in rat, rhesus macaque, human plasma were studied by ultrafiltration. Amicon Ultra-0.5centrifugal filter devices(Nominal Molecular Weight Limit, NMWL10K) were used for the nonspecific binding(NSB) and PPB measurements. The mean NSB of euphorbiasteroid in PBS is60.5%. All of the mean PPB in rat, rhesus macaque, human plasma under the three levels of concentration (1000,100,20μg·L-1) were as high as76.0%~98.0%, and independent of the concentration. The PPB values were independent of the concentration. The PPB of euphorbiasteroid in human and rhesus macaque plasma are higher(94.6%~98.0%) than in rat plasma (76.0%~79.8%).There was no statistical difference between the PPB of euphorbiasteroid in human plasma and the PPB in rhesus macaque plasma at three dose levels. The PPB values with NSB correction in human and rhesus macaque plasma were still more than86%.
Conelusion
1. An HPLC-MS/MS method with ESI source in MRM mode was developed and validated for determination of euphorbiasteroid in biological sample. The lower limit of determination (LLOQ) was1μg· L-1The linear calibration curves were obtained in the concentration range of1～10000μg·L-1. The established method was proved suitable for preclinical pharmacokinetics of euphorbiasteroid. The pharmacokinetics of euphorbiasteroid in rhesus macaques and SAIDS rhesus macaques were investigated by an HPLC-MS/MS method for the first time. The non-compartmental pharmacokinetics parameters in rats, healthy rhesus macaques and SAIDS rhesus macaques were determined and compared after oral and intravenous administrations. The results showed that times to reach the peaks of blood concentrations for rats, healthy or SIV-infectious rhesus macaques in oral route were measured as0.3-0.5h. The elimination of euphorbiasteroid in rats or monkeys after oral administration were both rapid. The oral bioavailabilities of euphorbiasteroid in rats, healthy or SIV-infectious rhesus macaques were as low as2.0%,4.6%and1.8%respectively implying procedures which are capable of increasing bioavailability may be crucial for the oral administration.
2.Epoxylathyrol was prepared from alkaline hydrolysates of euphorbiasteroid for the first time. The LC-DAD/MSn method was used in monitoring the process of hydrolysis.
3. Electrospray ionization mass spectrometry method was applied to analyze the possible structures of metabolites of euphorbiasteroid in plasma and urine of rats. More than ten euphorbiasteroid derivatives in plasma or urine were deduced as metabolites of euphorbiasteroid related the products of esterase hydrolysis, methylation, oxidation and reduction.
4.The plasma protein binding (PPB) of euphorbiasteroid in rat, rhesus macaque, and human plasma were studied by ultrafiltration for the first time. The PPB of euphorbiasteroid in human and rhesus macaque plasma are higher (94.6%~98.0%) than those in rat plasma (6.0%~79.8%). There was no statistical difference for PPB of euphorbiasteroid in human and rhesus macaque at the three dose levels. The PPB values with NSB correction in human and rhesus macaque plasma were still more than86%.
大戟属(Euphorbia Linn)是大戟科(Euphorbiaceae)植物中最大的一个属。大戟属的很多植物是药用植物,大戟属植物的生物活性也多与其所含的二萜酯类(diterpenoids)密切相关。续随子(Euphorbia Lathyris Linn)为大戟属植物,在中国分布广泛,民间常用作除疣、抗肿瘤等。从续随子分离出一系列二萜类化合物,主要是续随烷型二萜,有较强的抗肿瘤和多药耐药抑制(multi-drug resistance, MDR)活性。
续随二萜酯(euphorbiasteroid),又名大戟因子LI(Euphorbia factor L1)是从续随子分离得到的天然续随烷型二萜,对人宫颈癌HeLa细胞的增殖有显著抑制活性,对多种肿瘤耐药细胞株MCF-7/ADR、MES-SA/Dx5、KBV200等具有P-gp介导的多药耐药抑制活性。
续随二萜酯在体外具有一定的抗HIV活性。本课题组的前期研究发现续随二萜酯对HIV感染的慢性H9细胞(H9/HIV-1ⅢB)与靶细胞MT2的融合有一定的抑制作用,测得半数抑制浓度IC5。和治疗指数T1分别为0.8μM、600。
艾可清2号是广州中医药大学热带医学研究所在艾可清基础上研制的创新中药,由复方中药与续随二萜酯组合而成。实验研究表明艾可清2号能显著提升艾滋病恒河猴模型的CD4+T细胞比率及CD4+/CD8比值。创新中药复方艾可清2号显示了作为人类艾滋病治疗中药新药的应用前景。基于此,续随二萜酯有望成为既具有多药耐药抑制,又具有一定抗病毒能力的新型抗HIV先导化合物。
到目前为止,有关续随二萜酯在体内的吸收、代谢等相关药代动力学特征尚不清楚。为满足新药的研究与开发需要,本课题对续随二萜酯在大鼠、中国恒河猴及猴艾滋病模型体开展了较为系统的药代动力学探索性研究。
研究方法
1.采用高效液相色谱/串联质谱联用(HPLC-MS/MS)定量分析方法,建立了生物样品中续随二萜酯的检测,用于续随二萜酯在大鼠、恒河猴、猴艾滋病模型体内药代动力学研究。
2.通过Waters2767制备液相色谱仪分离,从续随二萜酯在碱作用下水解液得到环氧续随子醇。采用MS、1H-NMR、13C-NM等波谱学分析方法,鉴定了该化合物的结构。
3.采用液相色谱-离子阱质谱联用(LC/MS”)技术,在多级质谱扫描模式下,分析了续随二萜酯在大鼠体内的代谢产物。
4.采用超滤法进行了续随二萜酯与不同种属血浆蛋白结合率的研究。
研究结果
1.建立和确证了大鼠血浆、恒河猴血浆、猴艾滋病模型血浆等生物样品中续随二萜酯的液质联用(HPLC-MS/MS)定量分析方法。
生物样品预处理采用乙酸乙酯液-液萃取,经Waters Symmetry Shield C8(4.6mm×150mm,5.0μm)色谱柱,流动相甲醇(A)-10mM含0.1%甲酸的甲酸铵水溶液(B)梯度洗脱。三重四极杆质谱仪进行多反应监测(MRM)模式下的电喷雾离子源正离子检测(ESI+),续随二萜酯和内标汉黄芩素用于定量分析离子对分别为m/z553→m/z297、m/z285→m/z270.标准曲线在1～10000μg·L-1范围内线性关系良好,线性相关系数r为0.9975；定量下限为1μg·L-1,续随二萜酯保留时间(tR)为4.0min,内标保留时间(tR)为4.3min,单个样品分析时间7.0min。日内、日间精密度(RSD)在2.6%-10.7%,准确度在1.6%～5.1%,符合生物样品分析的要求。
2.考察了续随二萜酯在大鼠、恒河猴、猴艾滋病模型体内药动学特征。
应用生物样品中续随二萜酯的HPLCMS/MS定量分析方法,考察了续随二萜酯大鼠单次口服给药和静注给药、恒河猴单次口服给药和静注给药及猴艾滋病模型口服给药的体内药物动力学特征,应用DAS2.1药动学对数据进行处理,得到非室模型药代动力学参数。
续随二萜酯大鼠静脉注射10mg·kg-1,血药浓度极大值(Cmax)平均为3418.6μg·L-1,续随二萜酯大鼠口服给药100mg·kg-1,Cmax平均为87±32.4μg·L大鼠口服给药达峰时间平均为0.33h.
续随二萜酯恒河猴静脉注射给药5mg·kg-1,Cmax平均为1359.5μg.L-1;口服给药30mg·kg-1,Cmax平均为139.1μg·L-1。恒河猴口服给药,达峰时间平均为0.31h。
大鼠静和恒河猴静脉注射和口服给药后,续随二萜酯在体内均迅速衰减。药物在大鼠和恒河猴中都存在明显的个体差异。
续随二萜酯猴艾滋病模型口服给药相同剂量,与正常恒河猴相比,血药浓度达到极大值为56.9μg.L-1,达峰时间平均为0.50h。续随二萜酯在恒河猴和猴艾滋病模型体内都存在明显的吸收差异性。
大鼠、恒河猴、猴艾滋病模型口服给药绝对生物利用度分别为2.0%、4.6%、1.8%。相对于健康猴口服给药,猴艾滋病模型口服利用度约为1/3。
3.续随二萜酯在THF中和50℃水浴温度下,经1.25mol·L-1NaOH作用48h,水解液经HPLC-DAD检测,液质联用多级质谱分析,Waters液相制备柱分离,得到一化合物纯品(得率5.42%),经电喷雾离子源正负离子质谱(ESI-MS)、13C-NMR,1H-NMR分析,鉴定为环氧续随子醇(epoxylathyrol)。
4.应用电喷雾离子源多级质谱法分析了续随二萜酯的结构,并对它的裂解规律进行分析归纳。
在正离子模式,利用离子阱质谱多级质谱扫描技术,对续随二萜酯在大鼠血液和尿液中的16个未知代谢产物进行了合理的推测。
从续随二萜酯大鼠静注给药血样中推测了7个代谢产物可能的结构,代谢产物涉及酯酶水解成羟基,有的伴随羟基的甲基化,甲基加氧(0)氧化或者C-6环氧加氢(H)还原。从续随二萜酯大鼠口服给药血样中推测了5个代谢产物可能的结构,其中4个与静注给药血样中代谢产物相同。大鼠血样中的代谢产物以Ⅰ相代谢为主,而尿液中的代谢产以Ⅱ相代谢为主。从续随二萜酯大鼠口服给药尿样中推测了4个代谢产物结构。尿样中的代谢产物除少量酯酶水解产物,大部分是与葡萄糖醛酸结合产物,有些还伴有甲基化。
5.采用超滤法、Amicon Ultra-0.5超滤离心装置(截留分子量10K)对续随二萜酯与三种属(SD大鼠、恒河猴、人)血浆蛋白结合程度进行研究。续随二萜酯磷酸缓冲盐溶液非特性结合率平均为60.5%。在三个浓度水平(1000、100、20μg·L-1)条件下,续随二萜酯与大鼠、猴、人血浆蛋白结合率均较高(76.0%、98.0%),与血浆蛋白的结合不存在浓度依赖性。续随二萜酯与大鼠血浆蛋白结合率相对较低(76.0%～79.8%),与恒河猴、人血浆蛋白结合率较高(94.6%～98.0%),续随二萜酯与人和猴血浆蛋白结合率无统计学差异(p>0.05)。考虑到高非特异性结合率,对血浆蛋白结合率进行校正后,续随二萜酯与人和猴血浆蛋白结合率均高于86%。
结论和创新点
1.建立和确证了生物样品中续随二萜酯在ESI电离源、MRM模式下液质联用定量分析方法。血样最低定量限1μg·L-1,线性范围1-10000μg· L-1,适用于动物口服给药和静注给药后血浆样品中药物的检测。
应用液质联用方法,首次研究比较了续随二萜酯在大鼠、健康及猴艾滋病模型中国恒河猴体内的药代动力学特征。发现大鼠与恒河猴口服给药后,血药浓度达峰时间均约0.3-0.5h。给药后,续随二萜酯血药浓度在体内均发生迅速衰减；生物利用度均很低,大鼠、恒河猴或其艾滋病模型的绝对生物利用度均低于5.0%,提示提高生物利用度对于给药可能具有关键意义。
2.首次从续随二萜酯制备环氧续随子醇。水解过程采用液质谱联用(LC-DAD/ESI-MS")技术监控。
3.利用液相色谱-离子阱质谱联用(LC/MSn)技术,对续随二萜酯在大鼠血液和尿液中,16个未知代谢产物的结构进行了合理的推测。
4.首次应用超滤法测定了续随二萜酯与大鼠、恒河猴、人血浆蛋白结合率。续随二萜酯与恒河猴、人血浆蛋白结合率(94.6%、98.0%)高于与大鼠血浆蛋白结合率(76.0%～79.8%)。续随二萜酯对人和猴血浆蛋白的结合率无统计学差异(p>0.05)。对非特异性结合进行校正后,续随二萜酯与恒河猴、人血浆蛋白结合率仍大于86%。
Background and Objective
The genus Euphorbia is the largest one in Euphorbiaceae. Many species of Euphorbia are used as medicinal plants. Euphorbia plants are rich resources in R&D of bioactive diterpenoids. Euphorbia Lathyris Linn belonging to Euphorbia is widely distributed in China and has been used in the treatment of cancer and warts as Chinese traditional medicine. A series of diterpenoids based on the lathyrane skeleton have been isolated from Euphorbia Lathyris Linn. Biological activity of these lathyrane-type diterpenoids have been carried out showing powerful anti-cancer activity and ability of reversing multi-drug resistance (MDR)。
Euphorbiasteroid(Euphorbia factor L1) is one of the main constituents isolated from Euphorbia lathyris. It was reported to have significant growth-inhibitory effects on human cervical cancer HeLa cells in vitro, reversal activity against P-glycoprotein (P-gp) mediated MDR in resistant cells such as KBv200, MCF-7/adr and MES-SA/Dx5cell lines. In our preceding researches, we found that euphorbiasteroid was potential against HIV in vitro, for its inhibitory activity in the fusion between H9cells chronically infected by HIV-1ⅢB with the target MT-2cells with a half inhibitory concentration (IC50) and therapeutic index (TI)0.8μM, and600respectively. Aikeqing-2, developed by the Tropical Medicine Institute of Guangzhou University of Traditional Chinese Medicine, is a combinatorial medicine of compound Chinese herbal medicine Aikeqing with euphorbiasteroid for the treatment of acquired immunodeficiency syndrome (AIDS). In our previous researches, Aikeqing-2exerted certain effect on increasing peripheral CD4+T lymphocytes count and the ratio of CD4+/CD8+So euphorbiasteroid is expected to be developed as a lead compound for the treatment of AIDS and cancer. Profiles including pharmacological activities mentioned above and results related to content determination, toxic action were reported, as far as we know the knowledge regard to pharmacokinetics and metabolism of euphorbiasteroid is not clear. In order to provide a meaningful basis in pharmacokinetics for the new drug development we carried out a pharmacokinetic study on the compound euphobiasteroid. This paper describes the establishment and validation of a method based on high performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of euphorbiasteroid in biological sample, which was used subsequently to determine the pharmacokinetics of euphorbiasteroid in rats, healthy rhesus macaques and rhesus macaques infected with simian immunodeficiency virus (SIV), namely SAIDS model monkeys.
Methods
1. A high performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of euphorbiasteroid in biological sample was established and validated, which was used subsequently to determine the pharmacokinetics of euphorbiasteroid in rats, rhesus macaques and SAIDS rhesus macaques.
2. Epoxylathyrol was isolated from alkaline hydrolysis of euphorbiasteroid by Waters2767Sample Manager. On the basis of spectroscopic data including those of MS,1H-NMR, and13C-NMR spectra, the structure was confirmed.
3. Liquid chromatography-ion trap mass spectrometry in the multi-stage MS full scan mode (LC/MSn) was used to analyze metabolites of euphorbiasteroid in rats.
4. Study on the binding of euphorbiasteroid to plasma proteins from different biological species by ultrafiltration.
Results
1. An HPLC-MS/MS method for the determination of euphorbiasteroid in plasma of rats, healthy and SAIDS monkeys was established and validated. Eupborbiasteroid was extracted with ethyl acetate from biological samples. A Waters Symmetry Shield C8(4.6mm×150mm,5.0μm) column was used with a mobile phase consisting of methanol (A)-lOmM ammonium formate containing0.1%formic acid (B) gradient elution. The determination was performed with electrospray ionization (ESI) source in the positive mode by a triple quadrupole tandem mass spectrometer. Quantification was performed by multiple reaction monitoring (MRM) of the transitions of m/z553～m/2297for euphorbiasteroid and m/z285→m/270for IS(wogonin). The linear calibration curves were obtained in the concentration range of1～10000μg·L-1(r=0.9975). The lower limit of determination (LLOQ) was1μg·L-1. The run time for each sample was7. Omin. The intra-and inter-day precision at three quality control (QC) levels were within2.6%-10.7%. The accuracy of the assay was within1.6%-5.1%.The method was proved suitable for the preclinical pharmacokinetics of euphorbiasteroid, which offered advantages of high sensitivity and selectivity.
2. The pharmacokinetics of euphorbiasteroid in rats, healthy and SAIDS monkeys were investigated using the established HPLC-MS/MS method.
An HPLC-MS/MS method for the determination of euphorbiasteroid in biological sample was used to determine the pharmacokinetics of euphorbiasteroid in rats and rhesus macaques following a single oral dose and intravenous administration, the pharmacokinetics of euphorbiasteroid in SAIDS monkeys following an oral administration. The non-compartmental pharmacokinetics parameters were processed by DAS2.1software program (Chinese Pharmacological Society).
The mean maximum concentration of drug (Cmax) was3418.6μg· L-1after intravenous administration at a dose of10mg·kg-'and87μg· L-1after an oral administration of euphorbiasteroid to SD rats at a dose of100mg· kg-1respectively.
The mean values of Cmax were1359.5μg·L-1after intravenous administration at a dose of5mg· kg-1and139.1μg· L-1after an oral administration of euphorbiasteroid to rhesus macaques at a dose of30mg·kg-1respectively.
After oral and intravenous administration, the elimination of euphorbiasteroid in rats and rhesus macaques were both rapid. Apparently individual difference in the absorption of euphorbiasteroid existed both in rats and rhesus macaques.
The mean value of Cmax was56.9μg· L-1to SAIDS rhesus macaques after oral administration at the same dose with rhesus macaques. Apparently differences were found existing in the absorption of euphorbiasteroid in both healthy and SAIDS rhesus macaques.
The mean bioavailability of euphorbiasteroid in rats, rhesus macaques and SAIDS rhesus macaques were2.0%,4.6%and1.8%, respectively.
Z. Under a waler bath at50℃, euphorbiasteroid was dissolved in THF and hydrolyzed with mol· L-1NaOH for48h. The hydrolysate was detected by photodiode array detector (DAD), analyzed by Liquid chromatography electrospray ion trap mass spectrometry in the multi-stage MS full scan mode(LC/MS"), and isolated by preparative HPLC to give a pure product with a yield rate of5.42%. The chemical structure was confirmed as epoxylathyrol on the basis of its spectroscopic data including those of MS,1H-NMR and13C-NMR spectra.
4. Electrospray ionization mass spectrometry method was applied to analyze the possible structures of metabolites of euphorbiasteroid in plasma and urine of rats.
In positive mode, a total of sixteen euphorbiasteroid derivatives were detected by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode (LC/MS") in rats plasma and urine after oral or intravenous administration. Seven euphorbiasteroid derivatives in the plasma were deduced as products related esterases hydrolysis, methylation, oxidation and reduction after intravenous administration. Four of the five metabolites in oral administration were the same as intravenous administration. The main metabolites in rat plasma was phase Ⅰ metabolism of euphorbiasteroid, whereas phase Ⅱ metabolites was found in urine. Four euphorbiasteroid derivatives were deduced as glucuronidated and methyl-glucuronidated metabolites in rat urine after oral administration.
5. The plasma protein binding (PPB) of euphorbiasteroid in rat, rhesus macaque, human plasma were studied by ultrafiltration. Amicon Ultra-0.5centrifugal filter devices(Nominal Molecular Weight Limit, NMWL10K) were used for the nonspecific binding(NSB) and PPB measurements. The mean NSB of euphorbiasteroid in PBS is60.5%. All of the mean PPB in rat, rhesus macaque, human plasma under the three levels of concentration (1000,100,20μg·L-1) were as high as76.0%~98.0%, and independent of the concentration. The PPB values were independent of the concentration. The PPB of euphorbiasteroid in human and rhesus macaque plasma are higher(94.6%~98.0%) than in rat plasma (76.0%~79.8%).There was no statistical difference between the PPB of euphorbiasteroid in human plasma and the PPB in rhesus macaque plasma at three dose levels. The PPB values with NSB correction in human and rhesus macaque plasma were still more than86%.
Conelusion
1. An HPLC-MS/MS method with ESI source in MRM mode was developed and validated for determination of euphorbiasteroid in biological sample. The lower limit of determination (LLOQ) was1μg· L-1The linear calibration curves were obtained in the concentration range of1～10000μg·L-1. The established method was proved suitable for preclinical pharmacokinetics of euphorbiasteroid. The pharmacokinetics of euphorbiasteroid in rhesus macaques and SAIDS rhesus macaques were investigated by an HPLC-MS/MS method for the first time. The non-compartmental pharmacokinetics parameters in rats, healthy rhesus macaques and SAIDS rhesus macaques were determined and compared after oral and intravenous administrations. The results showed that times to reach the peaks of blood concentrations for rats, healthy or SIV-infectious rhesus macaques in oral route were measured as0.3-0.5h. The elimination of euphorbiasteroid in rats or monkeys after oral administration were both rapid. The oral bioavailabilities of euphorbiasteroid in rats, healthy or SIV-infectious rhesus macaques were as low as2.0%,4.6%and1.8%respectively implying procedures which are capable of increasing bioavailability may be crucial for the oral administration.
2.Epoxylathyrol was prepared from alkaline hydrolysates of euphorbiasteroid for the first time. The LC-DAD/MSn method was used in monitoring the process of hydrolysis.
3. Electrospray ionization mass spectrometry method was applied to analyze the possible structures of metabolites of euphorbiasteroid in plasma and urine of rats. More than ten euphorbiasteroid derivatives in plasma or urine were deduced as metabolites of euphorbiasteroid related the products of esterase hydrolysis, methylation, oxidation and reduction.
4.The plasma protein binding (PPB) of euphorbiasteroid in rat, rhesus macaque, and human plasma were studied by ultrafiltration for the first time. The PPB of euphorbiasteroid in human and rhesus macaque plasma are higher (94.6%~98.0%) than those in rat plasma (6.0%~79.8%). There was no statistical difference for PPB of euphorbiasteroid in human and rhesus macaque at the three dose levels. The PPB values with NSB correction in human and rhesus macaque plasma were still more than86%.
引文
[1]曾毅.艾滋病的流行趋势、研究进展及遏制策略[J].中国公共卫生,2001,17(12)
[2]UNAIDS World AIDS Day report 2012 http://www. unaids. org/en/resources/campaigns/20121120_globalreport2012/re sults/
[3]Antiretroviral drugs used in the treatment of HIV infection http://www. fda. gov/ForConsumers/byAudience/ForPatientAdvocates/HIVandAIDS
[4]张清仲,符林春,芩玉文,等.艾可清治疗HIV/AIDS的研究进展[J].中药新药与临床药理,2010,21(1)：98-100
[5]姜海鸥,李汝润,陈太刚,等.复方三黄散胶囊体外抗艾滋病病毒的实验研究[J].山东中医杂志,2005,24(8)：295-296.
[6]郭会军,刘学伟,王丹妮.爱康1号治疗艾滋病相关综合征疗效观察[J].河南中医学院学报,2005,11(6)：6-7.
[7]吕维柏.新世纪康保治疗43例艾滋病病人的临床报告[J].中国性病艾滋病防治,2000,6(3)：168.
[8]张妍玲,张涛源,陈集.双黄连粉针剂治疗艾滋病的临床观察[J].山西医科大学学报,1999,3(2)：177.
[9]黄卫平,王健,于智敏,等.艾通治疗艾滋病病毒感染及艾滋病患者15例[J].中国中医药信息杂志,2002,9(11)：49-50.
[10]孙阳,伍治平,倪燕萍,等.“艾泰定”治疗艾滋病31例临床观察[J].中国民族民间医药杂志,1999,(40)：258-260.
[11]李平,王慧,关崇芬.中研Ⅱ号抗猴免疫缺陷病毒SIVmac251感染的实验研究[J].中国中医基础医学杂志,1998,4(8)：26-28.
[12]张奉学,张俊丽,符林春,等.淫羊藿苷体外抑制S1V感染诱导的CEMxl74细胞凋亡[J].中国药理学通报,2008,24(5)：684-687
[13]Fujioka T, Kashiwada Y, Kikushie R E, et al. Anti AIDS agents,11. Betulinic acid and plantanic acid as anti-HIVprinciples from Syzigium claviflorum, and the anti-HIVactivity of structurally related triterpenoids[J].J Nat Prod, 1994,57(2):243-247.
[14]Poujade C, Soler F, Ribeill Y, et al. Betulinic acid derivatives:a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action. [J]. J Med Chem,1996,39(5):1056-1068.
[15]Kitamura K, Honda. M, Yoshizaki H, et al. Baicalin, an inhibitor of HIV-1 production in vitro[J]. Antiviral Res,1998,37(2):131-140.
[16]Li B Q, Fu T, Yan Y D, et al. Inhibition of HIV infection by baicalin-a f lavonoid compound purified from Chinese herbal medicine[J]. Cell Mol Biol Res, 1993,39(2):119-124.
[17]Zhang X, Tang X, Chen H. Inhibition of HIV replication by baicalin and S. baicalensis extracts in H9 cell culture[J]. Chin Med Sci J.1991,6(4): 230-232.
[18]Ono K, Nakane H, Fukushima M, et al. Inhibition of reverse transcriptase activity by a flavonoid compound,5.6,7-trihydroxyflavone[J]. Biochem Biophys Res Commun,1989,160(3):982-987.
[19]Ito M, Sato A, Hirabayashi K, et al. Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV) [J]. Antiviral Res,1988,10(6):289-298.
[20]Zhang Z F, Peng Z G, Gao L, et al. Three new derivatives of anti-HIV-1 polyphenols isolated from Salvia yunnanensis [J]. J Asian Nat Prod Res,2008, 10(5-6):391-396.
[21]张奉学,邓文娣,胡英杰,等.中药艾可清体外抑制猴免疫缺陷病毒活性的观察[J].广州中医药大学学报,1999,16(2)：127-129
[22]马伯艳,符林春,蔡卫平,等.艾可清胶囊联合高效抗病毒逆转录疗法治疗艾滋病临床观察[J].广州中医药大学学报,2007,24(6)：466-470.
[23]马伯艳,符林春,蔡卫平,等.艾可清胶囊对高效抗病毒逆转录疗法的增效减毒作用[J].中国实验方剂学杂志,2007,13(8)：6063.
[24]张苗苗,符林春,蔡卫平,等.艾可清胶囊对HIV感染者的疗效观察[J].中华中医药学刊,2008,26(10)：2233-2236
[25]马伯艳,符林春,陈谐婕,等.艾可清胶囊治疗获得性免疫缺陷综合征疗效分析[J].中医杂志,2007,48(12)：1092-1094
[26]王思明,王溪,苏晓会,等.续随子中千金二萜烷化合物抑制人妇科肿瘤细胞增殖活性的研究[J].中国药理学通报,2011,27(6)：774-776
[27]Appendino G, Tron G C, Jarevang T, et al. Unnatural natural products from the transannular cyclization of lathyarae diterpenes[J]. Org Lett,2001,7(11): 1609-1612
[28]Choi J S, Kang N S, Min Y K, et al. Euphorbiasteroid reverses P-glycoprotein mediated multidrug resistance in human sarcoma cell line MES-SA/Dx5[J]. Phytother Res.,2010,24(7):1042-1066
[29]Zhang J Y, Mi Y J, Chen S P et al. Euphorbia Factor L1 Reverses ABCBl-Mediated Multidrug Resistance Involving Interaction With ABCB1 Independent of ABCB1 Downregualtion[J]. J. Cellular Biochem,2011,112: 1076-1083
[30]Qian K, Kuo R Y, Chen C H, et al. Anti-AIDS agents 81. Design, synthesis and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors [J].J Med Chem,2010,53(8): 3133-3141.
[31]Sato N, Zhang Q, Ma C M, et al. Anti-human immunodeficiency virus-1 protease activity of new lanostane-type triterpenoids from Ganoderma sinense[J].Chem Pharm Bull,2009,57(10):1076-1080.
[32]Kuo R Y, Qian K, Morris-Natschke S L, et al. Plant-derived triterpenoids and analogues as antitumor and anti-HIV agents [J]. Nat Prod Rep,2009,26(10): 1321-1344.
[33]Wei Y, Ma C M, Hattori M. Synthesis and evaluation of A seco type triterpenoids for anti-HIV-lprotease activity[J].Eur J Med Chem.,2009 44(10):4112-4120.
[34]Wei Y, Ma C M, Chen D Y, et al. Anti-HIV-1 protease triterpenoids from Stauntonia obovatifoliola Hayata subsp. intermedia [J]. Phytochemistry.,2008, 69(9):1875-1879.
[35]Tuchinda P, Kornsakulkarn J, Pohmakotr M, et al. Dichapetalin-type triterpenoids and lignans from the aerial parts of Phyllanthus acutissima[J].J Nat Prod,2008,71(4):655-663.
[36]Chen J C, Zhang G H, Zhang Z Q, et al. Octanorcucurbitane and cucurbitane triterpenoids from the tubers of Hemsleya endecaphylla with HIV-1 inhibitory activity[J]. J Nat Prod,2008,71(1):153-155.
[37]Yu Y B, Miyashiro H, Nakamura N, et al. Effects of triterpenoids and flavonoids isolated from Alnus firma on HIV-1 viral enzymes[J].Arch Pharm Res,2007,30(7):820-826.
[38]Xiao W L, Tian R R, Pu J X, et al. Triterpenoids from Schisandra lancifolia with anti-HIV-1 activity[J]. J Nat Prod,2006,69(2):277-279.
[39]FujiokaT, Kashiwada Y,Kikushie R E, et al. Anti AIDSagents,11. betulinic acid and plantanic acid as anti-HIVprinciples fromSyzigium claviflorum, and the anti-HIVactivity of structurally related triterpenoids [J]. J Nat Prod, 1994,57(2):243-247.
[40]Poujade C, Soler F, Ribeill Y, et al. Betulinic acid derivatives:a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action. [J]. J Med Chem,1996,39(5):1056-68.
[41]Ahmed N, Brahmbhatt K G, Sabde S, et al. Synthesis and anti-HIV activity of alkylated quinoline 2,4-diols[J]. Bioorg Med Chem,2010,18(8):2872-2879.
[42]He J, Chen X Q, Li M M, et al. Lycojapodine A, a novel alkaloid from Lycopodium japonicum[J]. Org Lett,2009,11(6):1397-1400.
[43]YanMH, Cheng P, Jiang Z Y, et al. Periglaucines A-D, anti-HBV and -HIV-1 alkaloids from Pericampylus glaucus[J]. J Nat Prod,2008,71(5):760-763.
[44]Ernst J, Dahl R, Lum C, et al. Anti-HIV-1 entry optimization of novel imidazopiperidine-tropane CCR5 antagonists[J]. Bioorg Med Chem Lett,2008 18(4):1498-1501.
[45]Abere T A, Agoreyo F O. Antimicrobial and toxicological evaluation of the leaves of Baissea axillaries Hua used in the management of HIV/AIDS patients[J]. BMC Complement Altern Med.,2006,6:22.
[46]Casano G, Dumetre A, Pannecouque C, et al. Anti-HIV and antiplasmodial activity of original flavonoid derivatives [J]. Bioorg Med Chem,2010,18(16); 6012-6023.
[47]Feng T, Wang R R, Cai XH, et al.Anti-human immunodeficiency virus-1 constituents of the bark of Poncirus trifoliata[J]. Chem Pharm Bull,2010, 58(7):971-975.
[48]Fink R C, Roschek B J, Alberte R S. HIV type-1 entry inhibitors with a new mode of action[J]. Antivir Chem Chemother.2009,19(6):243-255.
[49]Tanaka R, Tsujii H, Yamada T, et al. Novel 3alpha-methoxyserrat-14-en-21beta-ol (PJ-1) and 3beta-methoxyserrat-14-en-21beta-ol (PJ-2)-curcumin, kojic acid, quercetin, and baicalein conjugates as HIV agents[J]. Bioorg Med Chem.,2009,17(14): 5238-5246.
[50]Nance C L, Siwak E B, Shearer W T. Preclinical development of the green tea catechin, epigallocatechin gal late, as an HIV-1 therapy[J]. J Allergy Clin Immunol,2009,123(2):459-465.
[51]Zhang Z F, Chen H S, Peng Z G, et al.A potent anti-HIV polyphenol from Salvia yunnanensis[J L J Asian Nat Prod Res,2008,10(3-4):273-277.
[52]Yu Y B, Miyashiro H, Nakamura N, et al. Effects of triterpenoids and flavonoids isolated from Alnus firma on HIV-1 viral enzymes[J]. Arch Pharm Res,2007,30(7):820-826.
[53]Reutrakul V, Ningnuek N, Pohmakotr M, et al.Anti HIV-1 flavonoid glycosides from Ochna integerrima[J]. Planta Med,2007,73(7):683-688.
[54]Yang G X, Qi J B, Cheng K J, et al. Anti-HIV chemical constituents of aerial parts of Caragana rosea[J].Yao Xue Xue Bao,2007,42(2):179-82.
[55]Williamson M P, McCormick T G, Nance C L, et al. Epigallocatechin gal late, the main polyphenol in green tea, binds to the T-cell receptor, CD4:Potential for HIV-1 therapy[J]. J Allergy Clin Immunol,2006,118(6):1369-1374.
[56]Zhang G H, Zheng Y T. Anti-HIV-1 effect of compound K3 from flower of Japanese pagoda tree in vitro. Zhong Yao Cai.2006,9(4):355-358
[57]Cheenpracha S, Karalai C, Ponglimanont C, et al. Anti-HIV-1 protease activity of compounds from Boesenbergia pandurata[J]. Bioorg Med Chem,2006, 14(6):1710-1714.
[58]Yang G Y, Li Y K, Wang R R, et al. Dibenzocyclooctadiene lignans from the fruits of Schisandra wilsoniana and their anti-HIV-1 activities[J]. J Asian Nat Prod Res,2010,12(6):470-476.
[59]Xiao W L, Wang R R, Zhao W, et al. Anti-HIV-1 activity of lignans from the fruits of Schisandra rubriflora[J]. Arch Pharm Res,2010,33(5):697-701.
[60]Yang G Y, Li Y K, Wag R R, et al.Dibenzocyclooctadiene lignans from Schisandra wilsoniana and their anti-HIV-1 activities[J]. J Nat Prod,2010, 73(5):915-919.
[61]Yang G Y, Fan P, Wang R R, et al.Dibenzocyclooctadiene lignans from Schisandra lancifolia and their anti-human immunodeficiency virus-1 activities[J].Chem Pharm Bull,2010,58(5):734-737.
[62]Lee J, Huh MS, Kim Y C, et al.Lignan, sesquilignans and di lignans, novel HIV-1 protease and cytopathic effect inhibitors purified from the rhizomes of Saururus chinensis[J]. Antiviral Res,2010,85(2):425-428.
[63]Li X N, Pu J X, Du X, et al. Lignans with anti-HIV activity from Schisandra propinqua var. sinensis[J]. J Nat Prod.2009 Jun;72(6):1133-41.
[64]Chen M, Kilgore N, Lee K H et al. Rubrisandrins A and B, lignans and related anti-HIV compounds from Schisandra rubriflora[J]. J Nat Prod,2006,69(12): 1697-1701.
[65]Hwu J R, Hsu M H, Huang R C. New nordihydroguaiaretic acid derivatives as anti-HIV agents[J]. Bioorg Med Chem Lett,2008,18(6):1884-1888.
[66]Pu J X, Yang L M, Xiao W L, et al.Compounds from Kadsura heteroclita and related anti-HIV activity[J]. Phytochemistry,2008,69(5):1266-1272.
[67]Chen S W, Wang Y H, Jin Y, et al. Synthesis and anti-HIV-1 activities of novel podophyllotoxin derivatives [J]. Bioorg Med Chem Lett.,2007,17(7): 2091-2095.
[68]Dunlop D C, Bonomelli C, Man sab F, et al. Polysaccharide mimicry of the epitope of the broadly neutralizing anti-HIV antibody, induces enhanced antibody responses to self oligomannose glycans[J]. Glycobiology,2010, 20(7):812-823.
[69]Peng Z G, Chen H S, Guo Z M, et al. Anti-HIV activities of Achyranthes bidentata polysaccharide sulfate in vitro and in vivo [J].Yao Xue Xue Bao, 2008,43(7):702-706
[70]Wang S C, Bligh S W, Zhu C L, et al.Sulfated beta-glucan derived from oat bran with potent anti-HIV activity[J]. J Agric Food Chem,2008,56(8): 2624-2629.
[71]Stagliano K W, Emadi A, Lu Z, et al. Regiocontrolled synthesis and HIV inhibitory activity of unsymmetrical binaphthoquinone and trimeric naphthoquinone derivatives of conocurvone[J]. Bioorg Med Chem,2006,14(16): 5651-565.
[72]Garg H, Francella N, Tony KA, et al.Glycoside analogs of beta-galactosylceramide, a novel class of small molecule antiviral agents that inhibit HIV-1 entry[J]. Antiviral Res,2008,80(1):54-61.
[73]Yu H, Alf sen A, Tudor D, et al. The binding of HIV-1 gp41 membrane proximal domain to its mucosal receptor, galactosyl ceramide, is structure-dependent [J].Cell Calcium,2008,43(1):73-82.
[74]Sato Y, Okuyama S, HoriK. Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii[J]. J Biol Chem.,2007,282(15) 11021-11029.
[75]Appendino G, Tron G C, Cravotto G, et al.An expeditious procedure for the isolation of ingenol from the seeds of Euphorbia lathyris[J] ·J Nat Prod, 1999,62 (1):76-79
[76]Daoubi M, Marquez N, Mazoir N, et al. Isolation of new phenylacetylingol derivatives that reactivate HIV-1 latency and a novel spirotriterpenoid from Euphorbia officinarumlatex[J].J. Bioorg. Med. Chem.,2007,15(13):4577-4584
[77]Xu W, Zhu C, Cheng W et al. Chemical Constituents of the Roots of Euphorbia micractina. J Nat Prod,2009,72(9):1620-1626
[78]中国植物志编委会.中国植物志[M].北京：科学出版社,1979,44：26-29
[79]中国植物志编委会.中国植物志[M].北京：科学出版社,1979,44：69-71
[80]孙国君,张付玉,占扎君等.千金子化学成分和药理活性研究进展.中药材,2010,33(2)：308-312
[81]Dutta P K, Banerjee Dilip, Dutta N. L. Euphorbetin:a new bicoumarin from Euphorbia Lathyris L. [J]. Tetrahedron Letters,1972,13(7):601-604.
[82]郑飞龙,罗跃华,魏孝义等.千金子中非萜类化学成分的研究.热带亚热带植物学报,2009,17(3)：298-301
[83]焦威,鲁璐,邓美彩等,千金子化学成分的研究[J].中草药,2010,41(2)：181-7
[84]全国中草药汇编编写组,全国中草药汇编(上册)[M],人民卫生出版社,1975：807.
[85]Piazza G J, Saggese E J, Spletzer K M. Triterpene Biosynthesis in the Latex of Euphorbia lathyris. Plant Physio],1987,83:177-180
[86]肖崇厚.中药化学[M].上海：上海科学技术出版社,1997,第1版：422-464
[87]Adolf W, Hecker E. On the active principles of the spurge family. Ⅲ. Skin irritant and cocarcinogenic factors from the caper spurge[J].J. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol,1975,84(3):325-344.
[88]Shizuri Y, Kupchan S M, Haynes H R, et al. Isolation and structural elucidation of new potent antileukemic diterpenoid esters from Gnidia species[J].Tetrahedron Lett,1984,25(48):5547
[89]Miranda F J, Alabadi J A, Orti M, et al. Comparative analysis of the vascular actions of diterpenes isolated from euphorbia canariensis[J]. J Pharm Pharmacol,1998,50(2):237-241
[90]Shokoohinia Y, Chianese G, Zolfaghari B, et al. Macrocyclic diterpenoids from the Iranian plant Euphorbia bungei Boiss[J]. Fitoterapia,2011,82: 317-322
[91]帅彦平,贾忠建.我国大戟二萜酯及其生理活性研究新进展[J].高等学校化学学报,1997,18(7)：1107-12)
[92]Itokawa H, Ichihara Y, Watanabe K, et al.An antitumor principle from Euphorbia lathyris[J]·Planta Med,1989,55:271-272
[93]Fatope M O, Zeng L, Ohayagha J E, et al.New 19-acetoxyingol diterpenes from the latex of Euphorbia poisonii (Euphorbiaceae)[J]. Bioorg Med Chem, 1996,4(10):1679-1683
[94]Valente C, Pedro M, Ascenso J R, et al. Euphopubescenol and euphopubescene, two new jatrophane polyesters, and lathyrane-type diterpenes from Euphorbia pubescens[J].Planta Med,2004,70(3):244-249.
[95]Pusztai R, Ferreira M J, Duarte N, et al. Macrocyclic lathyrane diterpenes as antitumor promoters[J]. Anticancer Res.,2007,27(1):201-205.
[96]Lage H, Duarte N, Coburger C, et al. Antitumor activity of terpenoids against classical and atypical multidrug resistant cancer cells[J].Phytomedicine,2010,17(6):441-448.
[97]李忌,郑耘,郑荣梁等.天然二萜类化合物的抗肿瘤活性肿瘤防治研究[J].1995,22(5)：271-2
[98]Ravikanth V, Reddy L N, Reddy A V. Three New Ingol Diterpenes from Euphorbia nivulia evaluation of cytotoxic activity[J]. Chem. Pharm. Bull, 2003,51(4):431-434
[99]BeckW T · The cell biology ofmultiple drug resistance [J] · Biochem Pharmacol,1987,36(18):2879-2887
[100]TakaraK, SakaedaT, OkumuraK · An update on overcoming MDRl-mediated multidrug resistance in cancer chemotherapy [J] · Curr Pharm,2006,12(3): 273-286.
[101]徐珊,徐昌芬·肿瘤多药耐药性发生机制及中药逆转作用的研究进展[J]·中国肿瘤生物治疗杂志,2006,13(6)：404-411
[102]Appendino G, Della P C, Conseil G. et al. A new P-glycoprotein inhibitor from the caper spurge (Euphorbia lathyris) [J]. J Nat Prod,2003,66(1):140-142
[103]Vogg G, Mattes E, RothenburgerJ, et al. Tumor promoting diterpenes from Euphorbia leuconeura L. [J]. Phytochemistry,1999,51:289-295.
[104]郑维发.甘遂醇提物中4种二萜类化合物的体内抗病毒活性研究[J].中草药,2004,35(1)：65-68.
[105]黄晓桃,黄光英,薛存宽等.千金子甲醇提取物抗肿瘤作用的实验研究[J].肿瘤防治研究,2004,31(9)：5568
[106]杨珺,王世岭,付桂英等.千金子提取液对大鼠肺成纤维细胞增殖的影响及细胞毒性作用[J].中国临床康复,2005,9(27)：101-103
[107]Jiao W, Dong W W, Li Z F, et al. Lathyrane diterpenes from Euphorbia lathyris as modulators of multidrug resistance and their crystal structures[J]. Bioorganic & Medicinal Chemistry,2009,17:4786-4792
[108]窦志华,丁安伟.大戟属有毒中药毒性成分及炮制减毒研究进展[C].中华中 医药学会第六届中药炮制学术会议论文集,2006,226-232.
[109]Imam Bakbsh Baloch, Musa Kaleem Baloch Qazi Najam us Saqib. Cytotoxic Macrocylic Diaterpenoid Easters from Euphorbia cornigera[J].. Planta Med, 2006,72 (5):830-834
[110]江苏新医学院编·中药大辞典[M]·第二版·上海：上海科学技术出版社,1985
[111]李滨,刘石磊,邹存珍等.千金子急性毒性实验研究[J].黑龙江医药,2006,19(2)：96
[112]梁娅君,郑飞龙,唐大轩等.千金子不同提取物对小鼠的毒性及药效学的初步研究[J].华西药学杂志,2011,26(1)：027-029
[113]郑飞龙,宁火华,马双成等.HPLC法测定千金子中4个二萜类化合物[J].中草药2009,40(10)：
[114]Hou X R, Wan L L, Zhang Z J, et al. Analysis and determination of diterpenoids in unprocessed and processed euphorbia lathyris seeds byHPLC-ESI-MS[J]. Journal of Pharmaceutical Analysis,2011,1(3):197-202
[115]Meng X, Zhao X, Long Z M, et al. A sensitive liquid chromatography mass spectrometry method for simultaneous determination of three diterpenoid esters from Euphorbia lathyris L. in rat plasma[J].J Pharmaceutical and Biomedical Analysis,2013,72:299-305
[116]萧参,陈坚行.生物药剂分析方法的认证[J].中国药学杂志,1993,24：425-426
[117]Karnes H T, March C. Precision, acuuracy and data acceptance criteria in biopharmaceutical analysis[J]. Pharm. Res.1993,10:1420-1422
[118]姚新生主编.有机化合物波普分析[M].北京：中国医药科技出版社,2006,11：188-189
[119]常乘,吴松锋,马洁,等.基于质谱的选择反应监测技术相关策略和方法的研究进展[J].生物化学与生物物理进展,2012,39(11)：1118-1127
[120]顾景凯,钟大放LC-MS/MS技术在药代动力学研究中的应用[J].质谱学报,2003,24：61-62
[121]Matuszewski B K, Constanzer M. L, Chavez-Eng C.M, etal. Matrix effect in quantitative LC/MS/MS analyses of biological fluids:a method for determination of finasteride in human plasma at picogram per milliliter concentrations[J]. Anal. Chem,1998,70:882-887
[122]Shah V P, Midha K K, Findlay J W, etal. Bioanalytical method validation-A revist with a decade of prograss[J]. Pharm. Res.2000,17:1551-1557
[123]徐叔云,卞如濂,陈修主编.药理实验方法学[M].北京：人民卫生出版社,2006,3：175
[124]卢耀增,吴小闲,涂新明等.猴免疫缺陷病毒(SIV)猴模型的建立.中国实验动物学报,1994,2(2)：94-101
[125]Murray P D, Dabid J A, Patrick M C. Biovalability of syrup and tableformulations of cefet amet pivoxly[J]. Antimicrob Agents Chemother 1993,37:2706.
[126]李焕德,彭文兴,李阳,等.头孢他美酯人体内药动学研究[J].中国药学杂志,2001,36(7)：467-469
[127]强荣.盐酸头孢他美酯片在Beagle犬体内的药代动力学研究[J].中国药业,2011,20(7)：11-12
[128]Bullingham R, Monroe S, Nicholls A, et al. Pharmacokinetics and bioavailability of mycophenolate mofetil in healthy subjects after single□dose oral and intravenous administration [J]. J Clin Pharmacol,1996, 36(4):315-324.
[129]邱娟,杭太俊,沈建平,等LC-MS/MS法研究阿德福韦酯胶囊的人体药动学,中国临床药学杂志,2006,15(5)：291-293
[130]王广基主编.药物代谢动力学[M].北京：化学工业出版社,2005,1：27-29
[131]Bicchi C, Appendino G, Cordero C, et al ·. HPLC-UV and HPLC-positive-ESI-MS Analysisof the Diterpenoid Fraction from Caper Spurge (Euphorbia lathyris) Seed Oil [J]. J. Phytochem.2001,12:255-62
[132]Taylor S, Marker A. Modification of the ultrafiltration technique to overcome solubility and nonspecific binding challenges associated with the measurement of plasma protein binding of corticosteroids[J]. Pharm Biomed Anal. 2006,41(1):299-303.
[133]Wang C G, Williams N S. A mass balance approach for calculation of recovery and binding enables the use of ultrafiltration as a rapid method for measurement of plasma protein binding for even highly lipophilic compounds. Journal of Pharmaceutical and Biomedical Analysis,2013,75:112-117
[134]Lee K J, Mower R, Hollenbeck T, etlc. Modulation of nonspecific binding in ultrafiltration protein binding studies, Pharm. Res,2003,20 (7):1015-1021.
[135]徐叔云,卞如濂,陈修主编.药理实验方法学[M].北京：人民卫生出版社,2006,3：708-710
[2]UNAIDS World AIDS Day report 2012 http://www. unaids. org/en/resources/campaigns/20121120_globalreport2012/re sults/
[3]Antiretroviral drugs used in the treatment of HIV infection http://www. fda. gov/ForConsumers/byAudience/ForPatientAdvocates/HIVandAIDS
[4]张清仲,符林春,芩玉文,等.艾可清治疗HIV/AIDS的研究进展[J].中药新药与临床药理,2010,21(1)：98-100
[5]姜海鸥,李汝润,陈太刚,等.复方三黄散胶囊体外抗艾滋病病毒的实验研究[J].山东中医杂志,2005,24(8)：295-296.
[6]郭会军,刘学伟,王丹妮.爱康1号治疗艾滋病相关综合征疗效观察[J].河南中医学院学报,2005,11(6)：6-7.
[7]吕维柏.新世纪康保治疗43例艾滋病病人的临床报告[J].中国性病艾滋病防治,2000,6(3)：168.
[8]张妍玲,张涛源,陈集.双黄连粉针剂治疗艾滋病的临床观察[J].山西医科大学学报,1999,3(2)：177.
[9]黄卫平,王健,于智敏,等.艾通治疗艾滋病病毒感染及艾滋病患者15例[J].中国中医药信息杂志,2002,9(11)：49-50.
[10]孙阳,伍治平,倪燕萍,等.“艾泰定”治疗艾滋病31例临床观察[J].中国民族民间医药杂志,1999,(40)：258-260.
[11]李平,王慧,关崇芬.中研Ⅱ号抗猴免疫缺陷病毒SIVmac251感染的实验研究[J].中国中医基础医学杂志,1998,4(8)：26-28.
[12]张奉学,张俊丽,符林春,等.淫羊藿苷体外抑制S1V感染诱导的CEMxl74细胞凋亡[J].中国药理学通报,2008,24(5)：684-687
[13]Fujioka T, Kashiwada Y, Kikushie R E, et al. Anti AIDS agents,11. Betulinic acid and plantanic acid as anti-HIVprinciples from Syzigium claviflorum, and the anti-HIVactivity of structurally related triterpenoids[J].J Nat Prod, 1994,57(2):243-247.
[14]Poujade C, Soler F, Ribeill Y, et al. Betulinic acid derivatives:a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action. [J]. J Med Chem,1996,39(5):1056-1068.
[15]Kitamura K, Honda. M, Yoshizaki H, et al. Baicalin, an inhibitor of HIV-1 production in vitro[J]. Antiviral Res,1998,37(2):131-140.
[16]Li B Q, Fu T, Yan Y D, et al. Inhibition of HIV infection by baicalin-a f lavonoid compound purified from Chinese herbal medicine[J]. Cell Mol Biol Res, 1993,39(2):119-124.
[17]Zhang X, Tang X, Chen H. Inhibition of HIV replication by baicalin and S. baicalensis extracts in H9 cell culture[J]. Chin Med Sci J.1991,6(4): 230-232.
[18]Ono K, Nakane H, Fukushima M, et al. Inhibition of reverse transcriptase activity by a flavonoid compound,5.6,7-trihydroxyflavone[J]. Biochem Biophys Res Commun,1989,160(3):982-987.
[19]Ito M, Sato A, Hirabayashi K, et al. Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV) [J]. Antiviral Res,1988,10(6):289-298.
[20]Zhang Z F, Peng Z G, Gao L, et al. Three new derivatives of anti-HIV-1 polyphenols isolated from Salvia yunnanensis [J]. J Asian Nat Prod Res,2008, 10(5-6):391-396.
[21]张奉学,邓文娣,胡英杰,等.中药艾可清体外抑制猴免疫缺陷病毒活性的观察[J].广州中医药大学学报,1999,16(2)：127-129
[22]马伯艳,符林春,蔡卫平,等.艾可清胶囊联合高效抗病毒逆转录疗法治疗艾滋病临床观察[J].广州中医药大学学报,2007,24(6)：466-470.
[23]马伯艳,符林春,蔡卫平,等.艾可清胶囊对高效抗病毒逆转录疗法的增效减毒作用[J].中国实验方剂学杂志,2007,13(8)：6063.
[24]张苗苗,符林春,蔡卫平,等.艾可清胶囊对HIV感染者的疗效观察[J].中华中医药学刊,2008,26(10)：2233-2236
[25]马伯艳,符林春,陈谐婕,等.艾可清胶囊治疗获得性免疫缺陷综合征疗效分析[J].中医杂志,2007,48(12)：1092-1094
[26]王思明,王溪,苏晓会,等.续随子中千金二萜烷化合物抑制人妇科肿瘤细胞增殖活性的研究[J].中国药理学通报,2011,27(6)：774-776
[27]Appendino G, Tron G C, Jarevang T, et al. Unnatural natural products from the transannular cyclization of lathyarae diterpenes[J]. Org Lett,2001,7(11): 1609-1612
[28]Choi J S, Kang N S, Min Y K, et al. Euphorbiasteroid reverses P-glycoprotein mediated multidrug resistance in human sarcoma cell line MES-SA/Dx5[J]. Phytother Res.,2010,24(7):1042-1066
[29]Zhang J Y, Mi Y J, Chen S P et al. Euphorbia Factor L1 Reverses ABCBl-Mediated Multidrug Resistance Involving Interaction With ABCB1 Independent of ABCB1 Downregualtion[J]. J. Cellular Biochem,2011,112: 1076-1083
[30]Qian K, Kuo R Y, Chen C H, et al. Anti-AIDS agents 81. Design, synthesis and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors [J].J Med Chem,2010,53(8): 3133-3141.
[31]Sato N, Zhang Q, Ma C M, et al. Anti-human immunodeficiency virus-1 protease activity of new lanostane-type triterpenoids from Ganoderma sinense[J].Chem Pharm Bull,2009,57(10):1076-1080.
[32]Kuo R Y, Qian K, Morris-Natschke S L, et al. Plant-derived triterpenoids and analogues as antitumor and anti-HIV agents [J]. Nat Prod Rep,2009,26(10): 1321-1344.
[33]Wei Y, Ma C M, Hattori M. Synthesis and evaluation of A seco type triterpenoids for anti-HIV-lprotease activity[J].Eur J Med Chem.,2009 44(10):4112-4120.
[34]Wei Y, Ma C M, Chen D Y, et al. Anti-HIV-1 protease triterpenoids from Stauntonia obovatifoliola Hayata subsp. intermedia [J]. Phytochemistry.,2008, 69(9):1875-1879.
[35]Tuchinda P, Kornsakulkarn J, Pohmakotr M, et al. Dichapetalin-type triterpenoids and lignans from the aerial parts of Phyllanthus acutissima[J].J Nat Prod,2008,71(4):655-663.
[36]Chen J C, Zhang G H, Zhang Z Q, et al. Octanorcucurbitane and cucurbitane triterpenoids from the tubers of Hemsleya endecaphylla with HIV-1 inhibitory activity[J]. J Nat Prod,2008,71(1):153-155.
[37]Yu Y B, Miyashiro H, Nakamura N, et al. Effects of triterpenoids and flavonoids isolated from Alnus firma on HIV-1 viral enzymes[J].Arch Pharm Res,2007,30(7):820-826.
[38]Xiao W L, Tian R R, Pu J X, et al. Triterpenoids from Schisandra lancifolia with anti-HIV-1 activity[J]. J Nat Prod,2006,69(2):277-279.
[39]FujiokaT, Kashiwada Y,Kikushie R E, et al. Anti AIDSagents,11. betulinic acid and plantanic acid as anti-HIVprinciples fromSyzigium claviflorum, and the anti-HIVactivity of structurally related triterpenoids [J]. J Nat Prod, 1994,57(2):243-247.
[40]Poujade C, Soler F, Ribeill Y, et al. Betulinic acid derivatives:a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action. [J]. J Med Chem,1996,39(5):1056-68.
[41]Ahmed N, Brahmbhatt K G, Sabde S, et al. Synthesis and anti-HIV activity of alkylated quinoline 2,4-diols[J]. Bioorg Med Chem,2010,18(8):2872-2879.
[42]He J, Chen X Q, Li M M, et al. Lycojapodine A, a novel alkaloid from Lycopodium japonicum[J]. Org Lett,2009,11(6):1397-1400.
[43]YanMH, Cheng P, Jiang Z Y, et al. Periglaucines A-D, anti-HBV and -HIV-1 alkaloids from Pericampylus glaucus[J]. J Nat Prod,2008,71(5):760-763.
[44]Ernst J, Dahl R, Lum C, et al. Anti-HIV-1 entry optimization of novel imidazopiperidine-tropane CCR5 antagonists[J]. Bioorg Med Chem Lett,2008 18(4):1498-1501.
[45]Abere T A, Agoreyo F O. Antimicrobial and toxicological evaluation of the leaves of Baissea axillaries Hua used in the management of HIV/AIDS patients[J]. BMC Complement Altern Med.,2006,6:22.
[46]Casano G, Dumetre A, Pannecouque C, et al. Anti-HIV and antiplasmodial activity of original flavonoid derivatives [J]. Bioorg Med Chem,2010,18(16); 6012-6023.
[47]Feng T, Wang R R, Cai XH, et al.Anti-human immunodeficiency virus-1 constituents of the bark of Poncirus trifoliata[J]. Chem Pharm Bull,2010, 58(7):971-975.
[48]Fink R C, Roschek B J, Alberte R S. HIV type-1 entry inhibitors with a new mode of action[J]. Antivir Chem Chemother.2009,19(6):243-255.
[49]Tanaka R, Tsujii H, Yamada T, et al. Novel 3alpha-methoxyserrat-14-en-21beta-ol (PJ-1) and 3beta-methoxyserrat-14-en-21beta-ol (PJ-2)-curcumin, kojic acid, quercetin, and baicalein conjugates as HIV agents[J]. Bioorg Med Chem.,2009,17(14): 5238-5246.
[50]Nance C L, Siwak E B, Shearer W T. Preclinical development of the green tea catechin, epigallocatechin gal late, as an HIV-1 therapy[J]. J Allergy Clin Immunol,2009,123(2):459-465.
[51]Zhang Z F, Chen H S, Peng Z G, et al.A potent anti-HIV polyphenol from Salvia yunnanensis[J L J Asian Nat Prod Res,2008,10(3-4):273-277.
[52]Yu Y B, Miyashiro H, Nakamura N, et al. Effects of triterpenoids and flavonoids isolated from Alnus firma on HIV-1 viral enzymes[J]. Arch Pharm Res,2007,30(7):820-826.
[53]Reutrakul V, Ningnuek N, Pohmakotr M, et al.Anti HIV-1 flavonoid glycosides from Ochna integerrima[J]. Planta Med,2007,73(7):683-688.
[54]Yang G X, Qi J B, Cheng K J, et al. Anti-HIV chemical constituents of aerial parts of Caragana rosea[J].Yao Xue Xue Bao,2007,42(2):179-82.
[55]Williamson M P, McCormick T G, Nance C L, et al. Epigallocatechin gal late, the main polyphenol in green tea, binds to the T-cell receptor, CD4:Potential for HIV-1 therapy[J]. J Allergy Clin Immunol,2006,118(6):1369-1374.
[56]Zhang G H, Zheng Y T. Anti-HIV-1 effect of compound K3 from flower of Japanese pagoda tree in vitro. Zhong Yao Cai.2006,9(4):355-358
[57]Cheenpracha S, Karalai C, Ponglimanont C, et al. Anti-HIV-1 protease activity of compounds from Boesenbergia pandurata[J]. Bioorg Med Chem,2006, 14(6):1710-1714.
[58]Yang G Y, Li Y K, Wang R R, et al. Dibenzocyclooctadiene lignans from the fruits of Schisandra wilsoniana and their anti-HIV-1 activities[J]. J Asian Nat Prod Res,2010,12(6):470-476.
[59]Xiao W L, Wang R R, Zhao W, et al. Anti-HIV-1 activity of lignans from the fruits of Schisandra rubriflora[J]. Arch Pharm Res,2010,33(5):697-701.
[60]Yang G Y, Li Y K, Wag R R, et al.Dibenzocyclooctadiene lignans from Schisandra wilsoniana and their anti-HIV-1 activities[J]. J Nat Prod,2010, 73(5):915-919.
[61]Yang G Y, Fan P, Wang R R, et al.Dibenzocyclooctadiene lignans from Schisandra lancifolia and their anti-human immunodeficiency virus-1 activities[J].Chem Pharm Bull,2010,58(5):734-737.
[62]Lee J, Huh MS, Kim Y C, et al.Lignan, sesquilignans and di lignans, novel HIV-1 protease and cytopathic effect inhibitors purified from the rhizomes of Saururus chinensis[J]. Antiviral Res,2010,85(2):425-428.
[63]Li X N, Pu J X, Du X, et al. Lignans with anti-HIV activity from Schisandra propinqua var. sinensis[J]. J Nat Prod.2009 Jun;72(6):1133-41.
[64]Chen M, Kilgore N, Lee K H et al. Rubrisandrins A and B, lignans and related anti-HIV compounds from Schisandra rubriflora[J]. J Nat Prod,2006,69(12): 1697-1701.
[65]Hwu J R, Hsu M H, Huang R C. New nordihydroguaiaretic acid derivatives as anti-HIV agents[J]. Bioorg Med Chem Lett,2008,18(6):1884-1888.
[66]Pu J X, Yang L M, Xiao W L, et al.Compounds from Kadsura heteroclita and related anti-HIV activity[J]. Phytochemistry,2008,69(5):1266-1272.
[67]Chen S W, Wang Y H, Jin Y, et al. Synthesis and anti-HIV-1 activities of novel podophyllotoxin derivatives [J]. Bioorg Med Chem Lett.,2007,17(7): 2091-2095.
[68]Dunlop D C, Bonomelli C, Man sab F, et al. Polysaccharide mimicry of the epitope of the broadly neutralizing anti-HIV antibody, induces enhanced antibody responses to self oligomannose glycans[J]. Glycobiology,2010, 20(7):812-823.
[69]Peng Z G, Chen H S, Guo Z M, et al. Anti-HIV activities of Achyranthes bidentata polysaccharide sulfate in vitro and in vivo [J].Yao Xue Xue Bao, 2008,43(7):702-706
[70]Wang S C, Bligh S W, Zhu C L, et al.Sulfated beta-glucan derived from oat bran with potent anti-HIV activity[J]. J Agric Food Chem,2008,56(8): 2624-2629.
[71]Stagliano K W, Emadi A, Lu Z, et al. Regiocontrolled synthesis and HIV inhibitory activity of unsymmetrical binaphthoquinone and trimeric naphthoquinone derivatives of conocurvone[J]. Bioorg Med Chem,2006,14(16): 5651-565.
[72]Garg H, Francella N, Tony KA, et al.Glycoside analogs of beta-galactosylceramide, a novel class of small molecule antiviral agents that inhibit HIV-1 entry[J]. Antiviral Res,2008,80(1):54-61.
[73]Yu H, Alf sen A, Tudor D, et al. The binding of HIV-1 gp41 membrane proximal domain to its mucosal receptor, galactosyl ceramide, is structure-dependent [J].Cell Calcium,2008,43(1):73-82.
[74]Sato Y, Okuyama S, HoriK. Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii[J]. J Biol Chem.,2007,282(15) 11021-11029.
[75]Appendino G, Tron G C, Cravotto G, et al.An expeditious procedure for the isolation of ingenol from the seeds of Euphorbia lathyris[J] ·J Nat Prod, 1999,62 (1):76-79
[76]Daoubi M, Marquez N, Mazoir N, et al. Isolation of new phenylacetylingol derivatives that reactivate HIV-1 latency and a novel spirotriterpenoid from Euphorbia officinarumlatex[J].J. Bioorg. Med. Chem.,2007,15(13):4577-4584
[77]Xu W, Zhu C, Cheng W et al. Chemical Constituents of the Roots of Euphorbia micractina. J Nat Prod,2009,72(9):1620-1626
[78]中国植物志编委会.中国植物志[M].北京：科学出版社,1979,44：26-29
[79]中国植物志编委会.中国植物志[M].北京：科学出版社,1979,44：69-71
[80]孙国君,张付玉,占扎君等.千金子化学成分和药理活性研究进展.中药材,2010,33(2)：308-312
[81]Dutta P K, Banerjee Dilip, Dutta N. L. Euphorbetin:a new bicoumarin from Euphorbia Lathyris L. [J]. Tetrahedron Letters,1972,13(7):601-604.
[82]郑飞龙,罗跃华,魏孝义等.千金子中非萜类化学成分的研究.热带亚热带植物学报,2009,17(3)：298-301
[83]焦威,鲁璐,邓美彩等,千金子化学成分的研究[J].中草药,2010,41(2)：181-7
[84]全国中草药汇编编写组,全国中草药汇编(上册)[M],人民卫生出版社,1975：807.
[85]Piazza G J, Saggese E J, Spletzer K M. Triterpene Biosynthesis in the Latex of Euphorbia lathyris. Plant Physio],1987,83:177-180
[86]肖崇厚.中药化学[M].上海：上海科学技术出版社,1997,第1版：422-464
[87]Adolf W, Hecker E. On the active principles of the spurge family. Ⅲ. Skin irritant and cocarcinogenic factors from the caper spurge[J].J. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol,1975,84(3):325-344.
[88]Shizuri Y, Kupchan S M, Haynes H R, et al. Isolation and structural elucidation of new potent antileukemic diterpenoid esters from Gnidia species[J].Tetrahedron Lett,1984,25(48):5547
[89]Miranda F J, Alabadi J A, Orti M, et al. Comparative analysis of the vascular actions of diterpenes isolated from euphorbia canariensis[J]. J Pharm Pharmacol,1998,50(2):237-241
[90]Shokoohinia Y, Chianese G, Zolfaghari B, et al. Macrocyclic diterpenoids from the Iranian plant Euphorbia bungei Boiss[J]. Fitoterapia,2011,82: 317-322
[91]帅彦平,贾忠建.我国大戟二萜酯及其生理活性研究新进展[J].高等学校化学学报,1997,18(7)：1107-12)
[92]Itokawa H, Ichihara Y, Watanabe K, et al.An antitumor principle from Euphorbia lathyris[J]·Planta Med,1989,55:271-272
[93]Fatope M O, Zeng L, Ohayagha J E, et al.New 19-acetoxyingol diterpenes from the latex of Euphorbia poisonii (Euphorbiaceae)[J]. Bioorg Med Chem, 1996,4(10):1679-1683
[94]Valente C, Pedro M, Ascenso J R, et al. Euphopubescenol and euphopubescene, two new jatrophane polyesters, and lathyrane-type diterpenes from Euphorbia pubescens[J].Planta Med,2004,70(3):244-249.
[95]Pusztai R, Ferreira M J, Duarte N, et al. Macrocyclic lathyrane diterpenes as antitumor promoters[J]. Anticancer Res.,2007,27(1):201-205.
[96]Lage H, Duarte N, Coburger C, et al. Antitumor activity of terpenoids against classical and atypical multidrug resistant cancer cells[J].Phytomedicine,2010,17(6):441-448.
[97]李忌,郑耘,郑荣梁等.天然二萜类化合物的抗肿瘤活性肿瘤防治研究[J].1995,22(5)：271-2
[98]Ravikanth V, Reddy L N, Reddy A V. Three New Ingol Diterpenes from Euphorbia nivulia evaluation of cytotoxic activity[J]. Chem. Pharm. Bull, 2003,51(4):431-434
[99]BeckW T · The cell biology ofmultiple drug resistance [J] · Biochem Pharmacol,1987,36(18):2879-2887
[100]TakaraK, SakaedaT, OkumuraK · An update on overcoming MDRl-mediated multidrug resistance in cancer chemotherapy [J] · Curr Pharm,2006,12(3): 273-286.
[101]徐珊,徐昌芬·肿瘤多药耐药性发生机制及中药逆转作用的研究进展[J]·中国肿瘤生物治疗杂志,2006,13(6)：404-411
[102]Appendino G, Della P C, Conseil G. et al. A new P-glycoprotein inhibitor from the caper spurge (Euphorbia lathyris) [J]. J Nat Prod,2003,66(1):140-142
[103]Vogg G, Mattes E, RothenburgerJ, et al. Tumor promoting diterpenes from Euphorbia leuconeura L. [J]. Phytochemistry,1999,51:289-295.
[104]郑维发.甘遂醇提物中4种二萜类化合物的体内抗病毒活性研究[J].中草药,2004,35(1)：65-68.
[105]黄晓桃,黄光英,薛存宽等.千金子甲醇提取物抗肿瘤作用的实验研究[J].肿瘤防治研究,2004,31(9)：5568
[106]杨珺,王世岭,付桂英等.千金子提取液对大鼠肺成纤维细胞增殖的影响及细胞毒性作用[J].中国临床康复,2005,9(27)：101-103
[107]Jiao W, Dong W W, Li Z F, et al. Lathyrane diterpenes from Euphorbia lathyris as modulators of multidrug resistance and their crystal structures[J]. Bioorganic & Medicinal Chemistry,2009,17:4786-4792
[108]窦志华,丁安伟.大戟属有毒中药毒性成分及炮制减毒研究进展[C].中华中 医药学会第六届中药炮制学术会议论文集,2006,226-232.
[109]Imam Bakbsh Baloch, Musa Kaleem Baloch Qazi Najam us Saqib. Cytotoxic Macrocylic Diaterpenoid Easters from Euphorbia cornigera[J].. Planta Med, 2006,72 (5):830-834
[110]江苏新医学院编·中药大辞典[M]·第二版·上海：上海科学技术出版社,1985
[111]李滨,刘石磊,邹存珍等.千金子急性毒性实验研究[J].黑龙江医药,2006,19(2)：96
[112]梁娅君,郑飞龙,唐大轩等.千金子不同提取物对小鼠的毒性及药效学的初步研究[J].华西药学杂志,2011,26(1)：027-029
[113]郑飞龙,宁火华,马双成等.HPLC法测定千金子中4个二萜类化合物[J].中草药2009,40(10)：
[114]Hou X R, Wan L L, Zhang Z J, et al. Analysis and determination of diterpenoids in unprocessed and processed euphorbia lathyris seeds byHPLC-ESI-MS[J]. Journal of Pharmaceutical Analysis,2011,1(3):197-202
[115]Meng X, Zhao X, Long Z M, et al. A sensitive liquid chromatography mass spectrometry method for simultaneous determination of three diterpenoid esters from Euphorbia lathyris L. in rat plasma[J].J Pharmaceutical and Biomedical Analysis,2013,72:299-305
[116]萧参,陈坚行.生物药剂分析方法的认证[J].中国药学杂志,1993,24：425-426
[117]Karnes H T, March C. Precision, acuuracy and data acceptance criteria in biopharmaceutical analysis[J]. Pharm. Res.1993,10:1420-1422
[118]姚新生主编.有机化合物波普分析[M].北京：中国医药科技出版社,2006,11：188-189
[119]常乘,吴松锋,马洁,等.基于质谱的选择反应监测技术相关策略和方法的研究进展[J].生物化学与生物物理进展,2012,39(11)：1118-1127
[120]顾景凯,钟大放LC-MS/MS技术在药代动力学研究中的应用[J].质谱学报,2003,24：61-62
[121]Matuszewski B K, Constanzer M. L, Chavez-Eng C.M, etal. Matrix effect in quantitative LC/MS/MS analyses of biological fluids:a method for determination of finasteride in human plasma at picogram per milliliter concentrations[J]. Anal. Chem,1998,70:882-887
[122]Shah V P, Midha K K, Findlay J W, etal. Bioanalytical method validation-A revist with a decade of prograss[J]. Pharm. Res.2000,17:1551-1557
[123]徐叔云,卞如濂,陈修主编.药理实验方法学[M].北京：人民卫生出版社,2006,3：175
[124]卢耀增,吴小闲,涂新明等.猴免疫缺陷病毒(SIV)猴模型的建立.中国实验动物学报,1994,2(2)：94-101
[125]Murray P D, Dabid J A, Patrick M C. Biovalability of syrup and tableformulations of cefet amet pivoxly[J]. Antimicrob Agents Chemother 1993,37:2706.
[126]李焕德,彭文兴,李阳,等.头孢他美酯人体内药动学研究[J].中国药学杂志,2001,36(7)：467-469
[127]强荣.盐酸头孢他美酯片在Beagle犬体内的药代动力学研究[J].中国药业,2011,20(7)：11-12
[128]Bullingham R, Monroe S, Nicholls A, et al. Pharmacokinetics and bioavailability of mycophenolate mofetil in healthy subjects after single□dose oral and intravenous administration [J]. J Clin Pharmacol,1996, 36(4):315-324.
[129]邱娟,杭太俊,沈建平,等LC-MS/MS法研究阿德福韦酯胶囊的人体药动学,中国临床药学杂志,2006,15(5)：291-293
[130]王广基主编.药物代谢动力学[M].北京：化学工业出版社,2005,1：27-29
[131]Bicchi C, Appendino G, Cordero C, et al ·. HPLC-UV and HPLC-positive-ESI-MS Analysisof the Diterpenoid Fraction from Caper Spurge (Euphorbia lathyris) Seed Oil [J]. J. Phytochem.2001,12:255-62
[132]Taylor S, Marker A. Modification of the ultrafiltration technique to overcome solubility and nonspecific binding challenges associated with the measurement of plasma protein binding of corticosteroids[J]. Pharm Biomed Anal. 2006,41(1):299-303.
[133]Wang C G, Williams N S. A mass balance approach for calculation of recovery and binding enables the use of ultrafiltration as a rapid method for measurement of plasma protein binding for even highly lipophilic compounds. Journal of Pharmaceutical and Biomedical Analysis,2013,75:112-117
[134]Lee K J, Mower R, Hollenbeck T, etlc. Modulation of nonspecific binding in ultrafiltration protein binding studies, Pharm. Res,2003,20 (7):1015-1021.
[135]徐叔云,卞如濂,陈修主编.药理实验方法学[M].北京：人民卫生出版社,2006,3：708-710