两种中药中抗前列腺疾病活性成分的研究

【作者】 韩慧英；

【导师】 姚新生； 王云川；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2007， 博士

【摘要】 随着世界人口的老龄化趋势，前列腺疾病的发病率在不断升高，其预防与治疗成为老年医学领域的一项重要课题，从天然产物中发掘有效治疗药物日益引起了人们的关注。本文对市售抗前列腺增生的部分中药制剂，采用抑制前列腺癌细胞LNCaP分泌PSA的活性评价体系进行了筛选，发现其中舒列安的活性较为显著，因此选择舒列安为深入研究对象，对其原料药材毛杭子稍[Campylotropis hirtella（Franch．）Schindl．]的活性部位进行了确认，并对活性部位的活性成分进行了追踪分离。从毛杭子稍的活性部位乙酸乙酯层，共分离得到43个化合物，并通过理化性质和现代波谱学手段(UV、IR、MS、¹H-NMR、¹³C-NMR和2D-NMR)鉴定了它们的结构，分别为erythro-guaiacylglycerol-β-4′-（5′）-methoxylariciresinol（1*），4-[（6-hydroxy-2，3-dihydro-1-benzofuran-3-y1）methyl]-5-methoxybenzene-1，3-diol（2*），hedyotisol A（3），hedyotisol C（4），hedyotisol B（5），buddlenol B（6），sesquipinsapols B（7），sesquimarocanol B（8），5，5′-二甲氧基落叶松脂醇（5，5′-dimethoxylariciresinol）（9），erythro-guaiacylglycerol-β-O-4′-coniferyl ether（10），threo-guaiacylglycerol-β-O-4′-coniferyl ether（11），开环异落叶松脂素（secoisolariciresinol）（12），去氢双松柏醇（dehydrodiconiferyl alcohol）（13），（-）开环异落叶松脂素-9′-β-D-吡喃葡萄糖苷[（-）-secoisolariciresinol-9′-β-D-glucopyranoside]（14），7，2′，4′-三羟基-5-甲氧基-3-苯基香豆素（7，2′，4′-trihydroxy-5-methoxy-3-arylcoumarin）（15*），angelol M（16*），angelol A（17），angelol B（18），angelol G（19），花椒毒素（xathotoxin）（20），异虎耳草素（isopimpinellin）（21），佛手苷内酯（bergapten）（22），别异欧前胡素（alloimperatorin）（23），lomatin acetate（24），哥伦比亚苷元（columbianetin）（25），8-O-甲基蜂蜜曲菌素（8-O-methylmellein）（26），香豆雌酚（3，9-dihydroxy coumestan）（27），（-）表儿茶素[（-）epicatechin]（28），（+）儿茶素[（+）catechin]（29），7，2′-二甲氧基-5，4′-二羟基二氢异黄酮（isoferreirin）（30），染料木素（genistein）（31），5，7，4′-三羟基二氢黄酮（naringenin）（32），二氢山萘酚（dihydrokaempferol）（33），槲皮素（quercetin）（34），山萘酚（kaempferol）（35），异牡荆素（isovitexin）（36），异荭草苷（isoorientin）（37），槲皮素-3-β-D-吡喃葡萄糖苷（quercetin 3-β-D-glucopyranoside）（38），芦丁（rutin）（39），苄基-β-D-吡喃葡萄糖苷（benzyl-β-D-glucopyranoside）（40），9，10，13-三羟基-反-11-十八烯酸（9，10，13-trihydrosy-（E）-11-octadecenoic acid）（41），4-（2-乙醇基）苯酚[4-（2-hydroxyethyl）phenol]（42），胡萝卜苷（daucosterol）（43）。43个化合物中，木脂素类化合物14个，黄酮类化合物12个，香豆素类化合物13个，其它类型化合物4个。其中化合物1，2，15，16为新化合物，化合物3～14，17～27，29～43为首次从该属植物中分离得到。分离得到的43个化合物中，25个化合物显示了不同强度的抑制LNCaP细胞分泌PSA活性，活性化合物分为三种类型：木脂素类、黄酮类和香豆素类。木脂素和黄酮类化合物的总体活性强于香豆素类，前者的IC₅₀值多数小于150μM，后者的IC₅₀多数大于或接近150μM。化合物抑制PSA分泌活性的作用机制可能通过影响雄激素／雄激素受体（AR）信号通路。因此我们进一步分析了6个木脂素类活性成分对LNCaP细胞中AR和PSA表达的影响，结果显示，化合物13和2均显著降低了细胞内AR的表达，同时也降低了PSA的表达，并呈剂量依赖性。化合物10和11在最高浓度时降低了AR的表达，并呈剂量依赖性降低PSA的表达。化合物12则对AR的表达没有影响，但呈剂量依赖性降低PSA的表达。化合物9对AR和PSA的表达均未有明显影响。考虑到AR在前列腺增生和前列腺癌的发病机制中都具有关键作用，我们又对上述作用最为显著的木脂素类化合物13进一步研究了其对LNCaP细胞的增殖抑制作用以及诱导细胞凋亡作用。实验结果显示，化合物13在测试浓度范围0～100μg／ml时，可显著抑制LNCaP细胞增殖，并呈时间依赖性和剂量依赖性。免疫印迹分析实验表明化合物13降低了Bcl的表达，而对Bax和Bcl-xl的表达没有明显影响，同时激活了调亡执行蛋白酶Caspase 3以及其下游底物PARP。说明该化合物诱导了LNCaP细胞发生凋亡。以上结果说明该化合物可能具有预防或治疗前列腺癌的作用。在上述研究的基础上，我们收集了与上述活性化合物同类型和不同类型的天然成分，进行了抗前列腺癌活性成分的筛选，发现植物酸浆中的酸浆苦素A和B（Physalin A，B）活性显著，因此进一步研究了这两个化合物对非雄激素依赖型前列腺癌细胞CWR22Rv1和C42B的抑制增殖和诱导凋亡作用。研究结果显示：Physalin A和Physalin B可以显著抑制C42B和CWR22Rv1细胞的增殖，并呈时间依赖性和剂量依赖性。Physalin B对两种细胞株的增殖抑制作用均强于Physalin A。这两化合物可显著抑制CWR22Rv1细胞集落形成，并且Physalin B的活性显著强于Physalin A。免疫印迹实验分析表明：Physalin A和Physalin B均激活CWR22Rv1和C42B中的Caspase-3和其底物PARP，并呈时间依赖性和剂量依赖性，提示Physalin A和Physalin B可导致这两种细胞发生凋亡。在CWR22Rv1细胞中，Physalin A和B均显激活了JNK和ERK，致使其磷酸化形式p-JNK和p-ERK显著升高，并呈剂量依赖性。并且JNK的作用底物c-Jun的磷酸化形式p-c-Jun也显著升高；在C42B细胞中，PhysalinA和B则只激活了JNK，未有ERK的磷酸化活性形式发生；而P38则在两种细胞株里均未被激活。以上结果说明，Physalin A和B诱导C42B和CWR22Rv1细胞凋亡可能通过活化JNK通路实现。在CWR22Rv1中，p-ERK形式的显著升高，说明ERK很可能与JNK共同介导了该细胞的凋亡过程，而P38则没有参与。另外，Physalin A和Physalin B降低了C42B中AR及其功能蛋白PSA的表达，也降低了CWR22Rv1中AR的表达。C42B和CWR22Rv1均为非雄激素依赖生长的前列腺癌细胞，下调AR水平以降低其活性是目前研究治疗非激素依赖型前列腺癌的一个重要靶标。以上结果均表明Physalin A和Physalin B可能具有治疗非激素依赖型前列腺癌的作用。综上所述，本文在活性筛选的基础上对市售抗前列腺增生中药舒列安的原料药材毛杭子稍的活性成分进行了研究，并阐明了其主要活性物质为木脂素、黄酮和香豆素。初步研究了毛杭子稍中木脂素类活性成分抗前列腺疾病的作用机制，结果表明这类成分可能具有预防或治疗前列腺增生和前列腺癌的作用。本文又进一步对抑制前列腺癌细胞生长活性显著的化合物酸浆苦素A和B，进行了抑制细胞生长和诱导凋亡的作用研究，结果表明这两个化合物对非激素依赖型前列腺癌可能有治疗作用。更多还原

【Abstract】 With the global trends in aging, the incidence of prostate diseases is stably increasing. How to prevent and treat these diseases has been an important topic in the medical field for old men. To find safer and more effective agents from natural products attracts more and more attention.In this thesis, we tested parts of traditional Chinese agents on sale used for the treatment of benign prostate hyperplasia （BPH） by evaluating their inhibition effect on secreted prostate specific antigen （PSA） in LNCaP cells with ELISA method, and found ShuLieAn showed strong activity. The raw material plant of Shuliean is Campylotropis hirtella （Franch.） Schindl. We found that the ethyl acetate extract of Campylotropis hirtella is the active fraction of the whole plant, and further carried out active constitutes study on the fraction by bioassay-guided isolation. 43 compounds from the ethyl acetate extract of Campylotropis hirtella were isolated. Their structures were elucidated on the basis ofphysico-chemical property and spectroscopic analysis. The 43 compounds were erythro-Guaiacylglycerol-β-O-4’-（5’）-methoxylariciresinol （1*）, 4-[（-6-hydroxy-2, 3-dihydro-1-benzofuran-3-yl） methyl]-5-methoxybenzene-1, 3-diol （2*）, hedyotisol A （3）, hedyotisol C （4）, hedyotisol B （5）, buddlenol B （6）, sesquipinsapols B （7）, sesquimarocanol B （8）, 5, 5’-dimethoxylariciresinol （9）, erythro-guaiacylglycerol-β-O-4’-coniferyl ether （10）, threo-Guaiacylglycerol-β-O-4’-coniferyl ether （11）, secoisolariciresinol （12）, dehydrodiconiferyl alcohol) （13）, （-）-secoisolariciresinol-9’-β-D-glucopyranoside （14）, 7, 2’, 4’-trihydroxy-5-methoxy-3-arylcoumarin) （15*）, angelol-M （16*）, angelol A （17）, angelol B （18）, angelol-G （19）, xathotoxin （20）, isopimpinellin （21）, bergapten （22）, alloimperatorin （23）, lomatin acetate （24）, Columbianetin （25）, 8-O-Methylmellein （26）, 3, 9-dihydroxy coumestan （27）, （-） epicatechin （28）, （-） catechin （29）, isoferreirin （30）, genistein （31）, Naringenin （32）, dihydrokaempferol （33）, quercetin （34）, kaempferol （35）, isovitexin （36）, isoorientin （37）, quercetin 3-β-D-glucuropyranoside （38）, rutin （39）, benzyl-β-D-glucopyranoside （40）, 9, 10, 13-trihydrosy-（E）-11-octadecenoic acid （41）, 4-（2-hydroxyethyl）phenol（42）, daucosterol（43）, respectively. Compounds 1, 2, 15, 16 were new compounds, compounds 3-14, 17-27, 29-43 were isolated for the first time from the Campylotropisgenus.Among the 43 compounds, 25 compounds showed inhibition effect on PSA secretion in LNCaP cells at different levels. These 25 active compounds belong to three structural types, which were lignans, flavanoids and coumarins. Lignans and flavanoids were more effective than coumarins in general. The values of IC₅₀ for the most of lignans and flavanoids were smaller than 150μM, and for the most of coumarins, the values were more than 150μM.The mechanism of action on secreted PSA inhibition may be resulted from their interrupting androgen/androgen receptor （AR） functional axle. So we further studied the effects of six active lignans on AR and PSA proteins expression in LNCaP cells by western blotting assay. The results showed that compound 13 and 2 significantly down-regulated AR and PSA expression at dose-dependent manner. Compound 10 and 11 suppressed AR expression at the highest test dose, while down-regulated PSA expression at dose-dependent manner. Compound 12 had no obvious effect on AR expression, but dose-dependently down-regulated PSA expression. Compound 9 showed no influence both on AR and PSA expression.Given that AR plays an essential role in the pathogenesis of prostate cancer, and compound 13 exhibited most significant down-regulation effect on AR and PSA expression, we farther performed the study of growth inhibition and apoptosis induced on LNCaP cells by compound 13. The results showed that compound 13 inhibited proliferation of LNCaP cells by MTT assay at dose and time-dependent manner under the concentration range of 0～100μg/ml. Moreover, compound 13 activated Caspase 3 and its downstream substrate PARP, suppressed Bcl expression, and did not influence Bax and Bcl-xl expression. These results indicated that compound 13 inhibited LNcaP cell proliferate through, at least partly, inducing the cells apoptosis, and suggested that compound 13 also could have the potential to prevent or treat prostate cancer.Based on the above results, we further collected variety kinds of natural constitutes that were similar or different from the active compounds isolated from Campylotropis hirtella, and evaluated their activity on growth inhibition of prostate cancer cell lines to fred more potent compounds used for anti-prostate caner research. The results showed that Physalin A and B, from the Physalis alkekengi L. var. franchetii （Mast.） Makino, exhibited significant activity. Accordingly, we further investigated the mechanism of these two compounds involved in this action. The results showed that Physalin A and B significantly decreased the viability of androgen-independent prostate cell lines C42B and CWR22Rvl at time and dose-dependent manner, physalin B exhibited much stronger activity than phyaslin A. These two compounds reduced colony-forming ability of CWR22Rvl, and physalin B also was more effective than physalin A. Western blotting analysis showed that Physalin A and B activated Caspase 3 and its downstream substrate PARP, this result indicated these two compounds induced cells apoptosis. Further study indicated that physalin A and B activated c-Jun N-terminal kinase （JNK） and extracellular signal-regulated kinase （ERK） pathways in CWR22Rvl. The results were demonstrated by the experiments that these two compounds increased p-JNK and p-ERK levels, had no obvious effect on JNK and ERK expression, and activated the JNK substrate c-Jun. While in C42B, physalin A and B only activated JNK, and no obvious p-ERK was detected. In these two cell lines, P38 was not activated. These results suggested that physalin A and B induced C42B and CWR22Rvl apoptosis possibly by activating JNK pathway. In CWR22Rvl, p-ERK was also significantly activated, which indicated ERK and JNK both mediated this cell line undergoing apoptosis. P38 was not involved these two cell lines apoptosis process. In addition, physalin A and B down-regulated AR and PSA expression in C42B, and AR expression in CWR22Rvl at dose dependent-manner. To down-regulate AR expression levers is also a promising target for treatment of androgen-independent prostate caner. So these results collectively suggested physalin A and B could have the potential to treat androgen-independent prostate caner.In conclusion, based on the bioassay evaluation, this thesis studied active constitutes of the raw material plant of Shuliean, Campylotropis hirtella, and clarified the main active components of Campylotropis hirtella for treatment of BPH that were lignans, flavanoids and coumarins. The mechanism of action for active lignans on BPH was studied, and further suggested they could have the potential to prevent or treat BPH, even prostate cancer. Physalin A and B, from the Physalis alkekengi, showed strong effects on growth inhibition and induced apoptosis of cell lines C42B and CWR22Rvl. The results supported that physalin A and B could be used for treatment of androgen-independent prostate caner.更多还原