两种中药中抗前列腺疾病活性成分的研究
|
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、~(13)C-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IC_(50)值多数小于150μM，后者的IC_(50)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。
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 IC_(50) 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.
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、~(13)C-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IC_(50)值多数小于150μM，后者的IC_(50)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。
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 IC_(50) 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.
引文
[1] Berry S J, Coffey DS, Walsh PC, et al. The development of human benign prostatic hyperplasia with age. J Urol, 1984, 132, 474-479.
[2] Roehrborn CG, McConnell JD. Etiology, pathophysiology, epidemiology and natural history of benign prostatic hyperplasia. In: Walsh PC, Retik AB, Vaughn EB Jr, et al, editors. Campbell's urology. 8th edition. Philadelphia: WB Saunders Co. 2002, p38, 1297-1330.
[3] Nakajima H. In: Denis L, Griffiths K, Khoury S, et al, editors. 4th International Consultation on Benign Prostatic Hyperplasia, Pads, July 2-5, 1997. Plymoth, UK: Plymbridge Distributors Ltd., 1998, p20.
[4] Gopal GARG, Deependra SINGH, Swamlata SARAF, et al. Management of Benign Prostate Hyperplasia: An Overview of a-Adrenergic Antagonist, Biol. Pharm. Bull, 2006, 29(8), 1554—1558.
[5] Sarma AV et al. A population based study of incidence and treatment of benign prostatic hyperplasia among residents of Olmsted County, Minnesota: 1987 to 1997. J Urol, 2005, 173, 2048-2053.
[6] William D. Steers. 5α-reductase activity in the prostate, Urology), 2001, 58(Supplement 6A), 17-24.
[7] Michael Marberger. Drug Insight: 5α-reductase inhibitors for the treatment of benign prostatic hyperplasia Nature clinical practice urology, 2006, 3(9), 495-503.
[8] DJ Carbone Jr, S Hodges. Medical therapy for benign prostatic hyperplasia: sexual dysfunction and impact on quality of life, International Journal of Impotence Research, 2003, 15, 299-306.
[9] Roehrborn CG, Schwinn DA. Alphal-adrenergic receptors and their inhibitors in lower urinary tract symptoms and benign prostate hyperplasia. J Urol, 2004, 171(3), 1029-1035.
[10] Dvorkin, L., Song, K. Y. Herbs for benign prostatic hyperplasia, The Annals of Pharmacotherapy, 2002, 36, 1443-1452.
[11] V. Steenkamp, M. C. Gouwsa, M. Gulumian, et al. Studies on antibacterial, anti-inflammatory and antioxidant activity of herbal remedies used in the treatment of benign prostatic hyperplasia and prostatitis, Journal of Ethnopharmacology, 2006, 103, 71-75.
[12] Timothy J Wilt, Areef Ishani, Indulis Rutks, et al. Phytotherapy for benign prostatic hyperplasia, Public Health Nutrition, 2000, 3(4A), 459-472.
[13] Yan Kit Fong, Sibylle Marihart, Mike Harik, et al. Preventing progression in men with mild symptoms of benign prostatic hyperplasia: A Potential Role for Phytotherapy, Reviews in urology, 2004, 6(4), 187-191.
[14] 薛蔚．良性前列腺增生的治疗与预后，中华老年医学杂志，2006，25(9)，716-718
[15] 阳诺，王志华(综述)，胡志全，叶章群(审校)．激素难治性前列腺癌的化疗进展，国际泌尿系统杂志 2006, 26(5), 625-629.
[16] Willian R, Berry, The evolving role of chemotherapy in Androgen-Independent(Homrmone—Refractory) prostate cancer, Urology, 2005, 65, 2-7.
[17] 吴晁(综述)，陈同钰(审校)．前列腺癌相关分子标志物研究进展，国际泌尿系统杂志，2006，26(5)，641-644．
[18] S. H. Landis, T. Murray, S. Bolden, P. A. Wingo. Cancer statistics, Ca Cancer J. Clin, 1999, 49, 8-31.
[19] M. R. Smith, J. B. Nelson. Future therapies in hormone-refractory prostate cancer, Urology, 2005, 65(5 Suppl.), 9-16.
[20] A. S. Kibel, An interdisciplinary approach to treating prostate cancer, Urology, 2005, 65(6 Suppl.), 13-18.
[21] 赵洪，张琚，冯剑利等．血清PSA水平与前列腺癌的检测，Chin J Urol，1998，19(10)，638-640．
[22] Lilja HA. kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein. J Clin Invest, 1985, 76, 1899-1903.
[23] Gittes RF. Carcinoma of the prostate, N Engl J Med, 1991, 324, 236-245.
[24] Kelly WK, Scher HI, Mazumdar M, et al. Prostate specific antigen as a measure of disease outcome in hormone-refractory prostatic cancer. J Clin Oncol, 1993 11, 607-615.
[25] Leonard S. Marks, Claus G. Roehrborn, Gerald L. Andriole, Prevention of Benign Prostatic Hyperplasia Disease, The journal of urology, 2006, 176, 1299-1306.
[26] 彭明强，张钊，鲍镇美(审校)．前列腺特异抗原临床研究进展，《国外医学》泌尿系统分，1999，19(2)，49-51．
[27] Horoszewicz, J. S., Leong, S. S., Kawinski, E., et al. LNCaP model of human prostatic carcinoma. Cancer research, 1983, 43, 1809-1818.
[28] Chiu K. Y., Yong C. R. Effects of finasteride on prostate volume and prostate-specific antigen. J Chin Med Assoc, 2004, 67, 571-574.
[29] Bandana Chatterjee. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer Molecular and Cellular Biochemistry, 2003, 253, 89-101.
[30] Wu CP, Gu FL. The prostate in eunuchs, Prog Clin Biol Res 1991, 370, 249-255.
[31] Culig Z, Klocker H, Bartsch G, et al. Androgen receptors in prostate cancer endocrine-related, Cancer, 2002, 9, 155-170.
[32] Grossmann ME, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer, J Nat Cancer Inst, 2001, 93, 1687-1697.
[33] 韩慧英前列康及其原料油菜花粉(Brassica napus L．pollen)活性成分的研究，沈阳药科大学硕士学位论文，2001．
[34] 专利申请：云南白药实业股份有限公司，一种治疗前列腺疾病的药物，1996，授权号：96121261 公告号：1183291．
[35] 江苏中医学院，中药大辞典，下册，上海人民出版社．
[36] 傅沛云．中国杭子稍属植物研究，植物研究，1987，7(4)，11-55．
[37] 中华人民共和国药典，1977年版，一部，民卫生出版社．
[38] 毛友昌．消乳癖丸及制备方法，专利申请，申请号CN03124461.0 公开号CN1548118
[39] 泰利，陈新民，陈维新等．大红袍中单宁化学成分的研究，天然产物研究与开发1991，3(4)，31-34
[40] 鲁学照，徐文豪，沈家祥等．大红袍化学成分的研究，中国中药杂志，1997，22(11)，681-682
[41] L. Denisa M. S, Mortonb K. Griffithsb. Diet and Its Preventive Role in Prostatic Disease. Eur Urol 1999, 35, 377-387.
[42] Colm Mordssey, R. William G. Watson. Phytoestrogens and Prostate Cancer, Current Drug Targets, 2003, 4, 231-241.
[43] Pamela J. Magee, Ian R. Rowland. Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer, British Journal of Nutrition, 2004, 91, 513-531.
[44] Adlercreutz H, Bannwart C, Wahala K, et al. Inhibitionof human aromatase by mammalian lignans and isoflavonoid phytoestrogens, J Steroid Biochem Mol Biol, 1993a, 44, 147-153.
[45] Morton MS, Chan PS, Cheng C et al. Lignans and isoflavonoids in plasma and prostatic fluid in men: samples from Portugal Hong Kong and the United Kingdom, Prostate, 1997, 32, 122-128.
[46] Evans BA, Griffiths K, Morton MS. Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids, J Endocrinol, 1995, 147(2), 295-302.
[47] Geller J, Sionit L, Partido C, et al. Genistein inhibits the growth of human-patient BPH and prostate cancer in histoculture, Prostate, 1998, 34, 75-79.
[48] Kyle E, Neckers L, Takimoto C, et al. Genistein-induced apoptosis of prostate cancer cells is preceded by a specific decrease in focal adhesion kinase activity, Mol Pharmaco, 1997, 151, 193-200.
[49] Xing N, Chen Y, Mitchell SH, et al. Quercetin inhibits the expression and function of the androgen receptor in LNCaP prostate cancer cells, Carcinogenesis, 2001, 22(3), 409-414.
[50] Thelen P, Seseke F, Ringert RH, et al. Pharmacological potential of phytoestrogens in the treatment of prostate cancer, Urologe A, 2006, 45(2), 195-201.
[51] Joanne N. Davis, Omer Kucuk, Fazlul H. Sarkar. Expression of prostate-specific antigen is transcriptionally regulated by genistein in Prostate Cancer Cells. Molecular carcinogenesis, 2002, 34, 91-101.
[52] 王明东，杨松松．锦灯笼化学成分及药理作用综述，2005，7(4)，341-342．
[53] Kawai M, Yamamoto T, Makino B, et al. The structure of physalin T from Physalis alkekengi var. franchetti, J Asian Nat Prod Res, 2001, 3(3), 199-205.
[54] Antoun MD, Abramson D, Tyson RL, et aL Potential antitumor agents. ⅩⅦ. physalin B and 25, 26-epidihydrophysalin C from Witheringia coccoloboides, JNat Prod, 1981, 44(5), 579-85.
[55] Chiang HC, Jaw SM, Chen CF, et al. Antitumor agent, physalin F from Physalis angulata L, Anticancer Res, 1992, 12(3), 837-43.
[56] Chiang HC, Jaw SM, Chen PM. Inhibitory effects ofphysalin B and physalin F on various human leukemia cells in vitro, Anticancer Res, 1992, 12(4), 1155-62.
[57] Chai HB, Castillo JJ, Soejarto DD, et al. Cytotoxic constituents of Brachistus stramoniifolius Fang L, Phytother Res. 2003, 17(5), 520-3.
[58] Lee CC, Houghton P. Cytotoxicity of plants from Malaysia and Thailand used traditionally to treat cancer, J Ethnopharmacol. 2005, 100(3), 237-43.
[59] Magalhaes HI, Veras ML, Torres MR, et al. In-vitro and in-vivo antitumour activity of physalins B and D from Physalis angulata. J Pharm Pharmacol. 2006, 58(2), 235-41.
[60] Grorge N. Thalmann, Ploutarchos E. Anezinis, Shi-Ming Chang, et al. Androgen-independent cancer progression and bone metastasis in the LNCaP model of Human prostate cancer. Cancer research, 1994, 54, 2577-2581.
[61] Sramkoski, R. M., Pretlow, T. G., Giaconia, J. M., Pretlow, T. P., et al. A new human prostate carcinoma cell line, 22Rvl. In Vitro Cell. Dev. Biol. Anim., 35, 403-409, 1999.
[62] Clifford G. Tepper, David L. Boucher, Philip E. Ryan, et al. Characterization of a Novel Androgen Receptor Mutation in a Relapsed CWR22 Prostate Cancer Xenograft and Cell Line, Cancer research, 2002, 62, 6606-6614.
[63] Horoszewicz, J. S., Leong, S. S., Kawinski, E., et al. LNCaP model of human prostatic carcinoma. Cancer research, 1983, 43, 1809-1818.
[64] Kaighn, M. E., Narayan, K. S., Ohnuki, Y., et al. Establishment and characterization of a human prostatic carcinoma cell line(PC-3). Investig. Urol, 1979, 17, 16-23.
[65] Stone, K. R., Mickey, D. D., Wunderli, H., et al. Isolation of a human prostate carcinoma cell line(DU 145). Int. J. Cancer, 1978, 21, 274-281.
[66] 娥登高．细胞调亡与谷光甘肽-s-转移酶关系的研究．解剖学报，1995，26(4)，32．
[67] Thompson CB. Apoptosis in the pathogenesis and treatment of disease, Science, 1995, 267, 1456.
[68] Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKs, TIBS, 1995, 20, 117
[69] Minden A, Lin A, Claret FX, et al. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTP ases Rac and Cdc42Hs. Cell, 1995, 81, 1147.
[70] Toumier C, Whitmarsh AJ, Cavanagh J, et al. Mitogen-aetivated protein kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc Natl Acad Sci USA, 1997, 94(14), 7337
[71] Minden A, Lin A, Mcmahon M et al. Differential activation of ERK and JNK mitogen-activated protein kinase by Raf-1 and MEKK, Science, 1994, 266, 1719
[72] Minden A, Lin A, Smeal T, et aL c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinase, Mol Cell Biol, 1994, 14, 6683.
[73] Raingeaud J, Whitmarsh AJ, Barrett T, et al. MKK3 and MKK6 regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transducion pathway, Mol Cell Biol, 1996, 16(3), 1247.
[74] Pages G, Lenormand P, L Allemain G et al. Mitogen-activated proein kinase p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA, 1993, 90: 8319
[75] Xia Z, Dickens M, Raingeand J, et al. Opposing effects of ERK and JNK-p38 MAP kinase on apoptosis, Science, 1995, 270, 13326.
[76] Chen YR; Wan XP, Templeton D, et al. The role of c-Jun terminal kinase(JNK) in apoptosis induced by ltraviolet C and γ radiation, J Biol Chem, 1996, 271(50), 31929.
[77] Chen YR, Meyer CF, Tan TH. Persistent activation of c-Jun N-terminal kinasel(JNK1) in γ radiation-induced apoptosis, J Biol Chem, 1996, 271, 631.
[78] Cohen GM. Caspase: the executioners ofapoptosis. Biochem J. 1997, 326, 1-16.
[79] Villa P, Kaufmann SH, Eamshaw WC. Caspase and caspase inhibitors. Trend Biochem Sci. 1997, 22, 388-393.
[80] Stennicke HR, Salvesen S. Properties of the caspases. Biochimica et Biophysica Acta. 1998, 1387, 17-31.
[81] Salvesen GS, Dixit VM. Caspase: intracellular signaling by proteolysis. Cell. 1997, 91, 443-446.
[82] Takahashi A, Eamshaw WC. ICE-related proteases in apoptosis. Curt Opin Gene Dev. 1996, 6, 50-55.
[83] Enari M, Sakahira H, Yokoyama H, et al. A caspase-activated Dnase that degrades DNA during apoptois, and inhibitor ICAD. Nature. 1998, 391, 43-50.
[84] Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med. 1997, 3, 614-619.
[85] Hengartner MO, Horvitz HR. C. elegans cell survival gene ted-9 encodes a functional homolog of the mammalian proto-oncogenebcl-2. Cell, 1994, 76, 665-676.
[86] Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998, 281, 1309-1311.
[87] Grooss A, McDonnell JM, Korsmeyer SJ. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev, 1999, 13, 1899-1911.
[88] Joza N. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 2001, 410, 549-554.
[89] Harris MH, Thompson CB. The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability, Cell Death Differ, 2000, 7, 1182-1191.
[90] Shi Y. A structural view of mitochondria-mediated apoptosis. Nat Struct Biol, 2001, 8, 394-401.
[91] Korsmeyer SJ. Bcl-2 gene family and the regulation of programmed cell death. Cancer Res. 1999, 59, 1693-1700.
[92] Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998, 281, 1312-1316.
[93] Haldar S, Basu A, Croce CM. Bcl-2 is the guardian of microtubule integrity. Cancer Res. 1997, 57, 229-233.
[94] Chan Zeng Wang, De Quan Yu. Lignan and acetylenic glycosides from Aster auriculatus, Phytochemistry, 1998, 48(4), 711-717.
[95] 原忠，李铣．8-O-4′型异木脂素立体构型的测定方法，波谱学杂志，2003，20(3)，307-314．
[96] Barbara vermes, Otto seligmann, Hildebert Wagner. Synthesis of biologically active terahydro-furofuranlignan-(syrining, pinoresinol)-mono-and bis-glucosides, Phytochemistry, 1991, 30(9), 3087-3089.
[97] Malada M. A., Rahman, Paul M., Dewick, David E., et al. Lignans of Forsythia intermedia, Phytochemistry, 1990, 29(6), 1971-1980.
[98] S. Matsuda, S. Kadota, T. Tai, T. Kikuchi. Isolation and structures ofhedyotisol-A, -B, and -C novel diligans from Hedyotis Lawsoniae, Chem. Pharm. Bull., 1984, 32, 5066-5069.
[99] Kaori Kijima, Hideaki Otsuka, Toshinori Ide, Choei Ogimi, et al. Sesquiterpene glycosides and sesquilignans glycosides from stems of Alangium premnifolium, Phytochemistry, 1998, 48(4), 669-676.
[100] Barrero, Alejandro F., Haidour, Ali, Munoz Dorado, M. Sesquipinsapols A and B: two sesquilignans from Abies pinsapo, Natural Product Letters, 1993), 2(4), 255-62.
[101] HANS ACHENBACH, MARKUS STOCKER, MANUEL A. Flavonoid and other constituents of Bauhinia Manca, Phytochemistry, 1988, 27(6), 1835-1841.
[102] Sebastiao F. Fonseca, Lawrence T. Nielsen, Ecmundo A. Ruveda. Lignans ofAraucaria angustifolia and ~(13)C NMR analysis of some phenyltetralin lignans, Phytochemirrry, 1979, 18, 1703-1708.
[103] 张梅，董小萍，邓赞等．藏药波棱瓜子中一个新的倍半降木脂素，药学学报，2006，41(7)，659-661．
[104] Osama Salama, Ratan K. Chaudhuri, Otto sticher. A lignan glucoside from Euphrasia rostkoviana, Phytochemistry, 1978, 20(11), 2603-2604.
[105] Shogo Inoshiri, Manami Sasaki, Hiroshi Kohda, et al. Aromatic glycosides from Berchemia racemosa, Phytochemistry, 1987, 26(10), 2811-2814.
[106] Tsutomu Hatano, Yasuhiro Aga, Yasushi Shintani, et al. Minor favonoids from licorice, Phytochemistry, 2000, 55, 959-963.
[107] Kimiye Baba, Youko Matsuyama, Mitsugi Koazwa. Studies on coumarins from the root of Angelica pubescens maxim. Ⅳ. structures of angelol-type prenylcoumadns, Chem. Phare. Bull., 1982, 30(6), 2025-2035.
[108] Jiang-Hua Liu, Sui-Xu Xu, Xin-sheng Yao, et al. Angelol-type coumarins from Angelica pubescence F. Biserrata and their inhibitory effect on platelet aggregation, Phytochemistry, 1995, 39(5), 1099-1101.
[109] 刘元艳，张村，李丽，肖永庆．白云花根的化学成分研究Ⅰ．中国中药杂志，2006，31(4)，309-311．
[110] 孙视，刘斌，孔令义，张涵庆，贺善安．福参化学成分研究，中国药科大学学报，2002，33(3)，181-183．
[111] 孙友福，肖永庆，刘晓宏，羌活化学成分的研究Ⅲ．中国中药杂志，1994，19(2)，99-100．
[112] E. Wong, G. C. M. Latch. Leguminosae Coumestans in diseased white clover, Phytochemistry, 1971, 10, 466-468.
[113] LIU Yi, MA Xiao xia, CHEN Hu biao. Chemical Constituents of Hedysamm gmelinii, Journal. of Chinese Pharmaceutical Sciences, 2005, 14(2), 75-78.
[114] Oldˇrich Lapˇc'lk, Jan Stursa, Tereza Kleinova, et al. Synthesis of hapten and conjugates ofcoumestrol and development of immunoassay, Steroids, 2003, 68, 1147-1155.
[115] 张雯洁，刘玉清，李兴从等．云南“生态茶”的化学成分，云南植物研究，1995，17(2)，204-208．
[116] 秦力，陈新民，陈维新等．大红袍中单宁化学成分的研究，天然产物研究与开发，1991，3(4)，31-34．
[117] 贾志胜，周波，杨立等．绿茶中茶多酚的2D NMR研究，波谱学杂志，1998，15(1)，23-30．
[118] Chien-Chang Shen, Yuan-Shiun Chang, Li-Kang Ho. Nuclera magnetic resonance studies of 5, 7-dihydroxyflavonoids, Phytochemistry, 1993, 34(3), 843-845.
[119] 徐德平，江汉湖，庞自洁．丹贝异黄酮的提取分离与结构鉴定，食品与发酵工业，27(3)，1-4．
[120] MA Chunhui, Li Bogang, XU Qing, et al. Chemical study on the stem ofLonicera landeolata, Chin J Appl Environ Biol, 2006, 12(4), 487-495.
[121] Beate Baderschneider, Peter Winterhalter. Isolation and Characterization of Novel Benzoates, Cinnamates, Flavonoids, and Lignans from Riesling Wine and Screening for Antioxidant Activity. J. Agric. Food Chem, 2001, 49, 2788-2798.
[122] 李霄，石任兵，刘斌等．清脑宣窍方有效部位的化学成分研究(Ⅱ)，北京中医药大学学报，2006，29(8)，545-550．
[123] 魏玉辉，武新安，张承忠等．光茎大黄化学成分研究(Ⅱ)，中国药学杂志，2006，41(4)，253-254．
[124] Ramarathnam, N., Osawa, T., Namiki, M., et al. Chemical studies on novel rice hull antioxidants. 2. Identification of isovitexin, a C-glycosyl flavonoid. Journal of the Agricultural and Food Chemistry, 1989, 37, 316-319.
[125] 李玉林，丁晨旭，刘健全等．红直獐牙菜的苷类成分，中草药，2002，33(2)，104-106．
[126] Yinrong Lu, L. Yeap Foo. The polyphenol constituents of grape pomace, Food Chemistry, 1999, 65, 1-8.
[127] 王景华，王亚琳，楼凤昌．槐树种子的化学成分研究，2001，32(6)，471-473．
[128] Salvatore De Rosa, Alfonso De Giulio, Giuseppina Tommonaro. Aliphatie and aromatic glycosides from the cell cultures of Lycopersicon esculentum, Phytochemistry, 1996, 42(40), 1031-1034.
[129] 王建忠，王峰鹏．川党参的化学成分研究，天然产物研究与开发，1996，8(2)，8-12．
[130] Leonard G. Gomella. Contemporary use of hormonal therapy in prostate cancer: managing complications and addressing quality-of-life issues, BJU international, 2007, 99, Supplement 1, 25-29.
[131] Richard A. Dixon, M. S. Srinivasa Reddy. Biosynthesis of monolignols. Genomic and reverse genetic approaches, Phytochernistry Reviews 2, 2003, 289-306.
[132] Brenda Winkel-Shirley. Flavonoid Biosynthesis. A Colorful Model for Genetics, Biochemistry, Cell Biology, and Biotechnology, Plant Physiology, 2001, 126, 485-493.
[133] P. M. Dewick, W. Barz, H. Grisebach. Biosynthesis of coumestrol in Phaseolus aureus, Phytochemistry, 1970, 9, 775-783.
[134] David R Gang, Michael A Costa, Masayuki Fuijita et al. Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis, chemistry and Biology, 1999, 6(3), 143-151.
[135] Sylvain Tranchimand, Thierry Tron, Christian Gaudin, et al. Synthesis of bis-lactone lignans through laccase catalysis, Journal of Molecular Catalysis B: Enzymatic, 2006, 42, 27-31.
[136] Joshua D. Ford, Kai-sheng Huang, Huai-Bin Wang, et al. Biosynthetic Pathway to the Cancer Chemopreventive Secoisolariciresinol Diglucoside-Hydroxymethyl Glutaryl Ester-Linked Lignan Oligomers in Flax (Linum usitatissimum) Seed, J. Nat. Prod, 2001, 64, 1388-1397.
[137] F. Bourgaud, A. Hehn, R. Larbat, et al. Biosynthesis of coumafins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes, Phytochem Rev, 2006, 5, 293-308.
[138] Setchell KDR, Adlercrentz H. Mammalian lignans and phytoestrogens. Recent studies on their formation metabolism and biological role in health and disease. In: Rowland I(ed) Role of the gut flora in toxicity and cancer. Academic Press, London, UK, 1998, 315-345
[139] Evans BA, Griffiths K, Morton MS. Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids. J Endocrinol. 1995,147(2), 295-302.
[140] Bylund A, Zhang JX, Sergh A, et al, Rye bran and soy protein delay growth and increase apoptosis of human LNCaP prostate adenocarcinoma in nude mice, Prostate, 2000,42,304-314.
[141]Dalu A, Haskel JF, Coward L, et al. Genistein, a component of soy. inhibits the expression of the EGF and ErbB2/Neu receptors in the rat dorsolated prostate. Prostate, 1998, 37(1): 36—43.
[142] Fotsis F, Pepper M, Adlercreutz H, et al. Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis. J Nutr, 1995,125(Suppl), 790-797.
[143] Davis J, Muqim N, Bhuiyan M, et al. Inhibition of prostate specific antigen expression by genistein in prostate cancer cells. Int J Oncol, 2000, 16, 1091-1097.
[144] Rosenberg Zand RS, Jenkins DJ, et al. Flavonoids can block PSA production by breast and prostate cancer cell lines, Clin Chim Acta, 2002,317, 17-26.
[145] Pages G, Lenormand P, L Allemain G, et al. Mitogen-activated proein kinase p42mapk and p44mapk are required for fibroblast proliferation. Proc NatlAcadSci USA, 1993, 90, 8319.
[146] Xia Z, Dickens M, Raingeand J, et al. Opposing effects of ERK and JNK-p38 MAP kinase on apoptosis, Science, 1995,270, 13326
[147] Chen YR, Wan XP, Templeton D, et al. The role of c-Jun terminal kinase (JNK) in apoptosis induced by ultraviolet C and γ radiation, J Biol Chem, 1996,271(50), 31929
[148] Li W, Whaley CD, Mondino A. Blocked signal transduction to the ERK and JNK protein kinase in anergrc CD4T cells, Science, 1996,271(5253), 1272
[149] Wang,X., Martindale, J.L., Holbrook, N.J. Requirement for ERK activation in cisplatin-induced apoptosis, J. Biol. Chem. 2000, 275, 39435-39443.
[150] Schweyer S., Soruri A., Meschter O., et al. Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation, Br. J. Cancer, 2004,91, 589-598.
[151] Andre Tanel, Diana A. Averill-Bates. P38 and ERK mitogen-activated protein kinases mediate acrolein-induced apoptosis in Chinese hamster ovary cells. Cell Signal. 2007, 19(5), 968-77
[152] Sandeep Singh, Ankur Kumar Upadhyay, Amrendra Kumar Ajay, et al. p53 regulates ERK activation in carboplatin induced apoptosis in cervical carcinoma: A novel target of p53 in apoptosis, FEBS Lett, 2007, 581(2), 289-95.
[2] Roehrborn CG, McConnell JD. Etiology, pathophysiology, epidemiology and natural history of benign prostatic hyperplasia. In: Walsh PC, Retik AB, Vaughn EB Jr, et al, editors. Campbell's urology. 8th edition. Philadelphia: WB Saunders Co. 2002, p38, 1297-1330.
[3] Nakajima H. In: Denis L, Griffiths K, Khoury S, et al, editors. 4th International Consultation on Benign Prostatic Hyperplasia, Pads, July 2-5, 1997. Plymoth, UK: Plymbridge Distributors Ltd., 1998, p20.
[4] Gopal GARG, Deependra SINGH, Swamlata SARAF, et al. Management of Benign Prostate Hyperplasia: An Overview of a-Adrenergic Antagonist, Biol. Pharm. Bull, 2006, 29(8), 1554—1558.
[5] Sarma AV et al. A population based study of incidence and treatment of benign prostatic hyperplasia among residents of Olmsted County, Minnesota: 1987 to 1997. J Urol, 2005, 173, 2048-2053.
[6] William D. Steers. 5α-reductase activity in the prostate, Urology), 2001, 58(Supplement 6A), 17-24.
[7] Michael Marberger. Drug Insight: 5α-reductase inhibitors for the treatment of benign prostatic hyperplasia Nature clinical practice urology, 2006, 3(9), 495-503.
[8] DJ Carbone Jr, S Hodges. Medical therapy for benign prostatic hyperplasia: sexual dysfunction and impact on quality of life, International Journal of Impotence Research, 2003, 15, 299-306.
[9] Roehrborn CG, Schwinn DA. Alphal-adrenergic receptors and their inhibitors in lower urinary tract symptoms and benign prostate hyperplasia. J Urol, 2004, 171(3), 1029-1035.
[10] Dvorkin, L., Song, K. Y. Herbs for benign prostatic hyperplasia, The Annals of Pharmacotherapy, 2002, 36, 1443-1452.
[11] V. Steenkamp, M. C. Gouwsa, M. Gulumian, et al. Studies on antibacterial, anti-inflammatory and antioxidant activity of herbal remedies used in the treatment of benign prostatic hyperplasia and prostatitis, Journal of Ethnopharmacology, 2006, 103, 71-75.
[12] Timothy J Wilt, Areef Ishani, Indulis Rutks, et al. Phytotherapy for benign prostatic hyperplasia, Public Health Nutrition, 2000, 3(4A), 459-472.
[13] Yan Kit Fong, Sibylle Marihart, Mike Harik, et al. Preventing progression in men with mild symptoms of benign prostatic hyperplasia: A Potential Role for Phytotherapy, Reviews in urology, 2004, 6(4), 187-191.
[14] 薛蔚．良性前列腺增生的治疗与预后，中华老年医学杂志，2006，25(9)，716-718
[15] 阳诺，王志华(综述)，胡志全，叶章群(审校)．激素难治性前列腺癌的化疗进展，国际泌尿系统杂志 2006, 26(5), 625-629.
[16] Willian R, Berry, The evolving role of chemotherapy in Androgen-Independent(Homrmone—Refractory) prostate cancer, Urology, 2005, 65, 2-7.
[17] 吴晁(综述)，陈同钰(审校)．前列腺癌相关分子标志物研究进展，国际泌尿系统杂志，2006，26(5)，641-644．
[18] S. H. Landis, T. Murray, S. Bolden, P. A. Wingo. Cancer statistics, Ca Cancer J. Clin, 1999, 49, 8-31.
[19] M. R. Smith, J. B. Nelson. Future therapies in hormone-refractory prostate cancer, Urology, 2005, 65(5 Suppl.), 9-16.
[20] A. S. Kibel, An interdisciplinary approach to treating prostate cancer, Urology, 2005, 65(6 Suppl.), 13-18.
[21] 赵洪，张琚，冯剑利等．血清PSA水平与前列腺癌的检测，Chin J Urol，1998，19(10)，638-640．
[22] Lilja HA. kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein. J Clin Invest, 1985, 76, 1899-1903.
[23] Gittes RF. Carcinoma of the prostate, N Engl J Med, 1991, 324, 236-245.
[24] Kelly WK, Scher HI, Mazumdar M, et al. Prostate specific antigen as a measure of disease outcome in hormone-refractory prostatic cancer. J Clin Oncol, 1993 11, 607-615.
[25] Leonard S. Marks, Claus G. Roehrborn, Gerald L. Andriole, Prevention of Benign Prostatic Hyperplasia Disease, The journal of urology, 2006, 176, 1299-1306.
[26] 彭明强，张钊，鲍镇美(审校)．前列腺特异抗原临床研究进展，《国外医学》泌尿系统分，1999，19(2)，49-51．
[27] Horoszewicz, J. S., Leong, S. S., Kawinski, E., et al. LNCaP model of human prostatic carcinoma. Cancer research, 1983, 43, 1809-1818.
[28] Chiu K. Y., Yong C. R. Effects of finasteride on prostate volume and prostate-specific antigen. J Chin Med Assoc, 2004, 67, 571-574.
[29] Bandana Chatterjee. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer Molecular and Cellular Biochemistry, 2003, 253, 89-101.
[30] Wu CP, Gu FL. The prostate in eunuchs, Prog Clin Biol Res 1991, 370, 249-255.
[31] Culig Z, Klocker H, Bartsch G, et al. Androgen receptors in prostate cancer endocrine-related, Cancer, 2002, 9, 155-170.
[32] Grossmann ME, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer, J Nat Cancer Inst, 2001, 93, 1687-1697.
[33] 韩慧英前列康及其原料油菜花粉(Brassica napus L．pollen)活性成分的研究，沈阳药科大学硕士学位论文，2001．
[34] 专利申请：云南白药实业股份有限公司，一种治疗前列腺疾病的药物，1996，授权号：96121261 公告号：1183291．
[35] 江苏中医学院，中药大辞典，下册，上海人民出版社．
[36] 傅沛云．中国杭子稍属植物研究，植物研究，1987，7(4)，11-55．
[37] 中华人民共和国药典，1977年版，一部，民卫生出版社．
[38] 毛友昌．消乳癖丸及制备方法，专利申请，申请号CN03124461.0 公开号CN1548118
[39] 泰利，陈新民，陈维新等．大红袍中单宁化学成分的研究，天然产物研究与开发1991，3(4)，31-34
[40] 鲁学照，徐文豪，沈家祥等．大红袍化学成分的研究，中国中药杂志，1997，22(11)，681-682
[41] L. Denisa M. S, Mortonb K. Griffithsb. Diet and Its Preventive Role in Prostatic Disease. Eur Urol 1999, 35, 377-387.
[42] Colm Mordssey, R. William G. Watson. Phytoestrogens and Prostate Cancer, Current Drug Targets, 2003, 4, 231-241.
[43] Pamela J. Magee, Ian R. Rowland. Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer, British Journal of Nutrition, 2004, 91, 513-531.
[44] Adlercreutz H, Bannwart C, Wahala K, et al. Inhibitionof human aromatase by mammalian lignans and isoflavonoid phytoestrogens, J Steroid Biochem Mol Biol, 1993a, 44, 147-153.
[45] Morton MS, Chan PS, Cheng C et al. Lignans and isoflavonoids in plasma and prostatic fluid in men: samples from Portugal Hong Kong and the United Kingdom, Prostate, 1997, 32, 122-128.
[46] Evans BA, Griffiths K, Morton MS. Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids, J Endocrinol, 1995, 147(2), 295-302.
[47] Geller J, Sionit L, Partido C, et al. Genistein inhibits the growth of human-patient BPH and prostate cancer in histoculture, Prostate, 1998, 34, 75-79.
[48] Kyle E, Neckers L, Takimoto C, et al. Genistein-induced apoptosis of prostate cancer cells is preceded by a specific decrease in focal adhesion kinase activity, Mol Pharmaco, 1997, 151, 193-200.
[49] Xing N, Chen Y, Mitchell SH, et al. Quercetin inhibits the expression and function of the androgen receptor in LNCaP prostate cancer cells, Carcinogenesis, 2001, 22(3), 409-414.
[50] Thelen P, Seseke F, Ringert RH, et al. Pharmacological potential of phytoestrogens in the treatment of prostate cancer, Urologe A, 2006, 45(2), 195-201.
[51] Joanne N. Davis, Omer Kucuk, Fazlul H. Sarkar. Expression of prostate-specific antigen is transcriptionally regulated by genistein in Prostate Cancer Cells. Molecular carcinogenesis, 2002, 34, 91-101.
[52] 王明东，杨松松．锦灯笼化学成分及药理作用综述，2005，7(4)，341-342．
[53] Kawai M, Yamamoto T, Makino B, et al. The structure of physalin T from Physalis alkekengi var. franchetti, J Asian Nat Prod Res, 2001, 3(3), 199-205.
[54] Antoun MD, Abramson D, Tyson RL, et aL Potential antitumor agents. ⅩⅦ. physalin B and 25, 26-epidihydrophysalin C from Witheringia coccoloboides, JNat Prod, 1981, 44(5), 579-85.
[55] Chiang HC, Jaw SM, Chen CF, et al. Antitumor agent, physalin F from Physalis angulata L, Anticancer Res, 1992, 12(3), 837-43.
[56] Chiang HC, Jaw SM, Chen PM. Inhibitory effects ofphysalin B and physalin F on various human leukemia cells in vitro, Anticancer Res, 1992, 12(4), 1155-62.
[57] Chai HB, Castillo JJ, Soejarto DD, et al. Cytotoxic constituents of Brachistus stramoniifolius Fang L, Phytother Res. 2003, 17(5), 520-3.
[58] Lee CC, Houghton P. Cytotoxicity of plants from Malaysia and Thailand used traditionally to treat cancer, J Ethnopharmacol. 2005, 100(3), 237-43.
[59] Magalhaes HI, Veras ML, Torres MR, et al. In-vitro and in-vivo antitumour activity of physalins B and D from Physalis angulata. J Pharm Pharmacol. 2006, 58(2), 235-41.
[60] Grorge N. Thalmann, Ploutarchos E. Anezinis, Shi-Ming Chang, et al. Androgen-independent cancer progression and bone metastasis in the LNCaP model of Human prostate cancer. Cancer research, 1994, 54, 2577-2581.
[61] Sramkoski, R. M., Pretlow, T. G., Giaconia, J. M., Pretlow, T. P., et al. A new human prostate carcinoma cell line, 22Rvl. In Vitro Cell. Dev. Biol. Anim., 35, 403-409, 1999.
[62] Clifford G. Tepper, David L. Boucher, Philip E. Ryan, et al. Characterization of a Novel Androgen Receptor Mutation in a Relapsed CWR22 Prostate Cancer Xenograft and Cell Line, Cancer research, 2002, 62, 6606-6614.
[63] Horoszewicz, J. S., Leong, S. S., Kawinski, E., et al. LNCaP model of human prostatic carcinoma. Cancer research, 1983, 43, 1809-1818.
[64] Kaighn, M. E., Narayan, K. S., Ohnuki, Y., et al. Establishment and characterization of a human prostatic carcinoma cell line(PC-3). Investig. Urol, 1979, 17, 16-23.
[65] Stone, K. R., Mickey, D. D., Wunderli, H., et al. Isolation of a human prostate carcinoma cell line(DU 145). Int. J. Cancer, 1978, 21, 274-281.
[66] 娥登高．细胞调亡与谷光甘肽-s-转移酶关系的研究．解剖学报，1995，26(4)，32．
[67] Thompson CB. Apoptosis in the pathogenesis and treatment of disease, Science, 1995, 267, 1456.
[68] Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKs, TIBS, 1995, 20, 117
[69] Minden A, Lin A, Claret FX, et al. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTP ases Rac and Cdc42Hs. Cell, 1995, 81, 1147.
[70] Toumier C, Whitmarsh AJ, Cavanagh J, et al. Mitogen-aetivated protein kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc Natl Acad Sci USA, 1997, 94(14), 7337
[71] Minden A, Lin A, Mcmahon M et al. Differential activation of ERK and JNK mitogen-activated protein kinase by Raf-1 and MEKK, Science, 1994, 266, 1719
[72] Minden A, Lin A, Smeal T, et aL c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinase, Mol Cell Biol, 1994, 14, 6683.
[73] Raingeaud J, Whitmarsh AJ, Barrett T, et al. MKK3 and MKK6 regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transducion pathway, Mol Cell Biol, 1996, 16(3), 1247.
[74] Pages G, Lenormand P, L Allemain G et al. Mitogen-activated proein kinase p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA, 1993, 90: 8319
[75] Xia Z, Dickens M, Raingeand J, et al. Opposing effects of ERK and JNK-p38 MAP kinase on apoptosis, Science, 1995, 270, 13326.
[76] Chen YR; Wan XP, Templeton D, et al. The role of c-Jun terminal kinase(JNK) in apoptosis induced by ltraviolet C and γ radiation, J Biol Chem, 1996, 271(50), 31929.
[77] Chen YR, Meyer CF, Tan TH. Persistent activation of c-Jun N-terminal kinasel(JNK1) in γ radiation-induced apoptosis, J Biol Chem, 1996, 271, 631.
[78] Cohen GM. Caspase: the executioners ofapoptosis. Biochem J. 1997, 326, 1-16.
[79] Villa P, Kaufmann SH, Eamshaw WC. Caspase and caspase inhibitors. Trend Biochem Sci. 1997, 22, 388-393.
[80] Stennicke HR, Salvesen S. Properties of the caspases. Biochimica et Biophysica Acta. 1998, 1387, 17-31.
[81] Salvesen GS, Dixit VM. Caspase: intracellular signaling by proteolysis. Cell. 1997, 91, 443-446.
[82] Takahashi A, Eamshaw WC. ICE-related proteases in apoptosis. Curt Opin Gene Dev. 1996, 6, 50-55.
[83] Enari M, Sakahira H, Yokoyama H, et al. A caspase-activated Dnase that degrades DNA during apoptois, and inhibitor ICAD. Nature. 1998, 391, 43-50.
[84] Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med. 1997, 3, 614-619.
[85] Hengartner MO, Horvitz HR. C. elegans cell survival gene ted-9 encodes a functional homolog of the mammalian proto-oncogenebcl-2. Cell, 1994, 76, 665-676.
[86] Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998, 281, 1309-1311.
[87] Grooss A, McDonnell JM, Korsmeyer SJ. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev, 1999, 13, 1899-1911.
[88] Joza N. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 2001, 410, 549-554.
[89] Harris MH, Thompson CB. The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability, Cell Death Differ, 2000, 7, 1182-1191.
[90] Shi Y. A structural view of mitochondria-mediated apoptosis. Nat Struct Biol, 2001, 8, 394-401.
[91] Korsmeyer SJ. Bcl-2 gene family and the regulation of programmed cell death. Cancer Res. 1999, 59, 1693-1700.
[92] Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998, 281, 1312-1316.
[93] Haldar S, Basu A, Croce CM. Bcl-2 is the guardian of microtubule integrity. Cancer Res. 1997, 57, 229-233.
[94] Chan Zeng Wang, De Quan Yu. Lignan and acetylenic glycosides from Aster auriculatus, Phytochemistry, 1998, 48(4), 711-717.
[95] 原忠，李铣．8-O-4′型异木脂素立体构型的测定方法，波谱学杂志，2003，20(3)，307-314．
[96] Barbara vermes, Otto seligmann, Hildebert Wagner. Synthesis of biologically active terahydro-furofuranlignan-(syrining, pinoresinol)-mono-and bis-glucosides, Phytochemistry, 1991, 30(9), 3087-3089.
[97] Malada M. A., Rahman, Paul M., Dewick, David E., et al. Lignans of Forsythia intermedia, Phytochemistry, 1990, 29(6), 1971-1980.
[98] S. Matsuda, S. Kadota, T. Tai, T. Kikuchi. Isolation and structures ofhedyotisol-A, -B, and -C novel diligans from Hedyotis Lawsoniae, Chem. Pharm. Bull., 1984, 32, 5066-5069.
[99] Kaori Kijima, Hideaki Otsuka, Toshinori Ide, Choei Ogimi, et al. Sesquiterpene glycosides and sesquilignans glycosides from stems of Alangium premnifolium, Phytochemistry, 1998, 48(4), 669-676.
[100] Barrero, Alejandro F., Haidour, Ali, Munoz Dorado, M. Sesquipinsapols A and B: two sesquilignans from Abies pinsapo, Natural Product Letters, 1993), 2(4), 255-62.
[101] HANS ACHENBACH, MARKUS STOCKER, MANUEL A. Flavonoid and other constituents of Bauhinia Manca, Phytochemistry, 1988, 27(6), 1835-1841.
[102] Sebastiao F. Fonseca, Lawrence T. Nielsen, Ecmundo A. Ruveda. Lignans ofAraucaria angustifolia and ~(13)C NMR analysis of some phenyltetralin lignans, Phytochemirrry, 1979, 18, 1703-1708.
[103] 张梅，董小萍，邓赞等．藏药波棱瓜子中一个新的倍半降木脂素，药学学报，2006，41(7)，659-661．
[104] Osama Salama, Ratan K. Chaudhuri, Otto sticher. A lignan glucoside from Euphrasia rostkoviana, Phytochemistry, 1978, 20(11), 2603-2604.
[105] Shogo Inoshiri, Manami Sasaki, Hiroshi Kohda, et al. Aromatic glycosides from Berchemia racemosa, Phytochemistry, 1987, 26(10), 2811-2814.
[106] Tsutomu Hatano, Yasuhiro Aga, Yasushi Shintani, et al. Minor favonoids from licorice, Phytochemistry, 2000, 55, 959-963.
[107] Kimiye Baba, Youko Matsuyama, Mitsugi Koazwa. Studies on coumarins from the root of Angelica pubescens maxim. Ⅳ. structures of angelol-type prenylcoumadns, Chem. Phare. Bull., 1982, 30(6), 2025-2035.
[108] Jiang-Hua Liu, Sui-Xu Xu, Xin-sheng Yao, et al. Angelol-type coumarins from Angelica pubescence F. Biserrata and their inhibitory effect on platelet aggregation, Phytochemistry, 1995, 39(5), 1099-1101.
[109] 刘元艳，张村，李丽，肖永庆．白云花根的化学成分研究Ⅰ．中国中药杂志，2006，31(4)，309-311．
[110] 孙视，刘斌，孔令义，张涵庆，贺善安．福参化学成分研究，中国药科大学学报，2002，33(3)，181-183．
[111] 孙友福，肖永庆，刘晓宏，羌活化学成分的研究Ⅲ．中国中药杂志，1994，19(2)，99-100．
[112] E. Wong, G. C. M. Latch. Leguminosae Coumestans in diseased white clover, Phytochemistry, 1971, 10, 466-468.
[113] LIU Yi, MA Xiao xia, CHEN Hu biao. Chemical Constituents of Hedysamm gmelinii, Journal. of Chinese Pharmaceutical Sciences, 2005, 14(2), 75-78.
[114] Oldˇrich Lapˇc'lk, Jan Stursa, Tereza Kleinova, et al. Synthesis of hapten and conjugates ofcoumestrol and development of immunoassay, Steroids, 2003, 68, 1147-1155.
[115] 张雯洁，刘玉清，李兴从等．云南“生态茶”的化学成分，云南植物研究，1995，17(2)，204-208．
[116] 秦力，陈新民，陈维新等．大红袍中单宁化学成分的研究，天然产物研究与开发，1991，3(4)，31-34．
[117] 贾志胜，周波，杨立等．绿茶中茶多酚的2D NMR研究，波谱学杂志，1998，15(1)，23-30．
[118] Chien-Chang Shen, Yuan-Shiun Chang, Li-Kang Ho. Nuclera magnetic resonance studies of 5, 7-dihydroxyflavonoids, Phytochemistry, 1993, 34(3), 843-845.
[119] 徐德平，江汉湖，庞自洁．丹贝异黄酮的提取分离与结构鉴定，食品与发酵工业，27(3)，1-4．
[120] MA Chunhui, Li Bogang, XU Qing, et al. Chemical study on the stem ofLonicera landeolata, Chin J Appl Environ Biol, 2006, 12(4), 487-495.
[121] Beate Baderschneider, Peter Winterhalter. Isolation and Characterization of Novel Benzoates, Cinnamates, Flavonoids, and Lignans from Riesling Wine and Screening for Antioxidant Activity. J. Agric. Food Chem, 2001, 49, 2788-2798.
[122] 李霄，石任兵，刘斌等．清脑宣窍方有效部位的化学成分研究(Ⅱ)，北京中医药大学学报，2006，29(8)，545-550．
[123] 魏玉辉，武新安，张承忠等．光茎大黄化学成分研究(Ⅱ)，中国药学杂志，2006，41(4)，253-254．
[124] Ramarathnam, N., Osawa, T., Namiki, M., et al. Chemical studies on novel rice hull antioxidants. 2. Identification of isovitexin, a C-glycosyl flavonoid. Journal of the Agricultural and Food Chemistry, 1989, 37, 316-319.
[125] 李玉林，丁晨旭，刘健全等．红直獐牙菜的苷类成分，中草药，2002，33(2)，104-106．
[126] Yinrong Lu, L. Yeap Foo. The polyphenol constituents of grape pomace, Food Chemistry, 1999, 65, 1-8.
[127] 王景华，王亚琳，楼凤昌．槐树种子的化学成分研究，2001，32(6)，471-473．
[128] Salvatore De Rosa, Alfonso De Giulio, Giuseppina Tommonaro. Aliphatie and aromatic glycosides from the cell cultures of Lycopersicon esculentum, Phytochemistry, 1996, 42(40), 1031-1034.
[129] 王建忠，王峰鹏．川党参的化学成分研究，天然产物研究与开发，1996，8(2)，8-12．
[130] Leonard G. Gomella. Contemporary use of hormonal therapy in prostate cancer: managing complications and addressing quality-of-life issues, BJU international, 2007, 99, Supplement 1, 25-29.
[131] Richard A. Dixon, M. S. Srinivasa Reddy. Biosynthesis of monolignols. Genomic and reverse genetic approaches, Phytochernistry Reviews 2, 2003, 289-306.
[132] Brenda Winkel-Shirley. Flavonoid Biosynthesis. A Colorful Model for Genetics, Biochemistry, Cell Biology, and Biotechnology, Plant Physiology, 2001, 126, 485-493.
[133] P. M. Dewick, W. Barz, H. Grisebach. Biosynthesis of coumestrol in Phaseolus aureus, Phytochemistry, 1970, 9, 775-783.
[134] David R Gang, Michael A Costa, Masayuki Fuijita et al. Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis, chemistry and Biology, 1999, 6(3), 143-151.
[135] Sylvain Tranchimand, Thierry Tron, Christian Gaudin, et al. Synthesis of bis-lactone lignans through laccase catalysis, Journal of Molecular Catalysis B: Enzymatic, 2006, 42, 27-31.
[136] Joshua D. Ford, Kai-sheng Huang, Huai-Bin Wang, et al. Biosynthetic Pathway to the Cancer Chemopreventive Secoisolariciresinol Diglucoside-Hydroxymethyl Glutaryl Ester-Linked Lignan Oligomers in Flax (Linum usitatissimum) Seed, J. Nat. Prod, 2001, 64, 1388-1397.
[137] F. Bourgaud, A. Hehn, R. Larbat, et al. Biosynthesis of coumafins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes, Phytochem Rev, 2006, 5, 293-308.
[138] Setchell KDR, Adlercrentz H. Mammalian lignans and phytoestrogens. Recent studies on their formation metabolism and biological role in health and disease. In: Rowland I(ed) Role of the gut flora in toxicity and cancer. Academic Press, London, UK, 1998, 315-345
[139] Evans BA, Griffiths K, Morton MS. Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids. J Endocrinol. 1995,147(2), 295-302.
[140] Bylund A, Zhang JX, Sergh A, et al, Rye bran and soy protein delay growth and increase apoptosis of human LNCaP prostate adenocarcinoma in nude mice, Prostate, 2000,42,304-314.
[141]Dalu A, Haskel JF, Coward L, et al. Genistein, a component of soy. inhibits the expression of the EGF and ErbB2/Neu receptors in the rat dorsolated prostate. Prostate, 1998, 37(1): 36—43.
[142] Fotsis F, Pepper M, Adlercreutz H, et al. Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis. J Nutr, 1995,125(Suppl), 790-797.
[143] Davis J, Muqim N, Bhuiyan M, et al. Inhibition of prostate specific antigen expression by genistein in prostate cancer cells. Int J Oncol, 2000, 16, 1091-1097.
[144] Rosenberg Zand RS, Jenkins DJ, et al. Flavonoids can block PSA production by breast and prostate cancer cell lines, Clin Chim Acta, 2002,317, 17-26.
[145] Pages G, Lenormand P, L Allemain G, et al. Mitogen-activated proein kinase p42mapk and p44mapk are required for fibroblast proliferation. Proc NatlAcadSci USA, 1993, 90, 8319.
[146] Xia Z, Dickens M, Raingeand J, et al. Opposing effects of ERK and JNK-p38 MAP kinase on apoptosis, Science, 1995,270, 13326
[147] Chen YR, Wan XP, Templeton D, et al. The role of c-Jun terminal kinase (JNK) in apoptosis induced by ultraviolet C and γ radiation, J Biol Chem, 1996,271(50), 31929
[148] Li W, Whaley CD, Mondino A. Blocked signal transduction to the ERK and JNK protein kinase in anergrc CD4T cells, Science, 1996,271(5253), 1272
[149] Wang,X., Martindale, J.L., Holbrook, N.J. Requirement for ERK activation in cisplatin-induced apoptosis, J. Biol. Chem. 2000, 275, 39435-39443.
[150] Schweyer S., Soruri A., Meschter O., et al. Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation, Br. J. Cancer, 2004,91, 589-598.
[151] Andre Tanel, Diana A. Averill-Bates. P38 and ERK mitogen-activated protein kinases mediate acrolein-induced apoptosis in Chinese hamster ovary cells. Cell Signal. 2007, 19(5), 968-77
[152] Sandeep Singh, Ankur Kumar Upadhyay, Amrendra Kumar Ajay, et al. p53 regulates ERK activation in carboplatin induced apoptosis in cervical carcinoma: A novel target of p53 in apoptosis, FEBS Lett, 2007, 581(2), 289-95.