小花鬼针草抗前列腺增生活性成分的研究
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摘要
前列腺增生是老年男性的多发病。在我国,随着人口年龄的增长,前列腺增生的发病率急剧上升,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。
本课题组以体外抑制前列腺癌细胞(LNCaP cells)分泌前列腺特异性抗原(PSA)作为抗前列腺增生活性筛选体系,在对100多种中药提取物进行抗前列腺增生活性筛选过程中,发现小花鬼针草60%乙醇提取物具有较好的抑制LNCaP细胞分泌PSA活性。
本文对小花鬼针草60%乙醇提取物进行了活性部位的确认,并采取反复硅胶柱色谱、Sephadex LH-20柱色谱、ODS柱色谱和制备高效液相等手段,对活性部位的化学成分进行了追踪分离。从小花鬼针草的活性部位即大孔吸附树脂柱30%和60%乙醇洗脱部分,共分离得到34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33个化合物的结构,分别为苄基β-D-吡喃葡萄糖昔(1);4-(2-羟基-2,6,6-三甲基环己-4-O-β-D-吡喃葡萄糖)-3-丁烯-2-酮(2);3-(3,4-二羟基)苯基丙酸甲酯(3);色原酮7-O-β-D-吡喃葡萄糖苷(4);(1S,2S)-1-(3-甲氧基-4-羟基)苯基丙三醇(5);(3R)8(E)-癸烯-4,6-二炔-1,3,10-三醇(6);5-羟基色原酮7-O-β-D-吡喃葡萄糖苷(7);8(E)-癸烯-4,6-二炔-3,10-二醇1-O-β-D-吡喃葡萄糖苷(8);3,5-二甲氧基苯丙烯醇4-O-β-D-吡喃葡萄糖苷(9);没食子酸(10);原儿茶酸(11);6-甲氧基香豆素-7-O-β-D-吡喃葡萄糖苷(12);槲皮素(13);木犀草素(14);山奈酚(15);2-羟基-3,4,4',6'-四甲氧基查尔酮(16);3,4,2',4',6'-五甲氧基二氢查尔酮(17);3,4,2',4'-四羟基查尔酮(18);金合欢素7-O-(α-D-芹菜呋喃糖)(1→6)-β-D-吡喃葡萄糖苷(19);硫磺菊素(20);海生菊苷(21);硫磺菊苷(22);金丝桃苷(23);木犀草素7-O-β-D-葡萄糖醛酸苷(24);山奈酚3-O-β-D-芸香糖苷(25);芦丁(27);槲皮苷(28);山奈酚3-O-(α-L-阿拉伯呋喃糖)(1→2)-β-D-吡喃葡萄糖苷(29);3,4,2'-三羟基查尔酮4'-O-β-D-葡萄糖苷(30);5,3',4'-三羟基二氢黄酮7-O-β-D-葡萄糖苷(31);木犀草素7-O-β-D-葡萄糖苷(32);(2S,3S)二氢槲皮素(33);二氢木犀草素(34)。34个化合物中,主要为黄酮类化合物,共22个,其中化合物6为新化合物,23个化合物为首次从该植物中分离得到。
分离得到的34个化合物中,26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:黄酮类、苯丙素类和酚酸类。黄酮类化合物的活性强于苯丙素类和酚酸类,当测试浓度为15μM时,前者抑制分泌PSA的相对量在8.7%～55.4%,后两者抑制分泌PSA的相对量在9.0%～32.5%。在黄酮类化合物中,又以黄酮醇和黄酮的活性最好,查尔酮、二氢黄酮、橙酮的活性次之,黄酮苷类化合物的活性较弱。
本文首次以体外细胞活性和总黄酮含量为评价指标,考察了小花鬼针草药材的最佳提取方法和工艺,结果表明提取物的活性强弱和总黄酮含量具有同比性,在水提醇沉、碱提酸沉、醇提水沉、醇提溶剂萃取和醇提大孔树脂分离的提取方法中,以60%乙醇提取大孔树脂分离的活性和总黄酮含量最高,洗脱部分活性富集作用好。而提取工艺则是以加20倍量60%乙醇提取三次,每次0.5 h的工艺最好,得到浸膏收率和总黄酮的含量最高。
本文首次对小花鬼针草药材的质量控制进行了初步研究,对28批不同产地的小花鬼针草药材进行了高效液相指纹图谱研究,使用“中药色谱指纹图谱相似度评价系统(中国药典2004A版)”和SPSS软件对这些样品进行了相似度分析和聚类分析,得到小花鬼针草的对照色谱图。也研究了用反相高效液相色谱同时测定小花鬼针草中四种成分的含量的方法,为小花鬼针草药材的质量控制提供了一定的理论依据。
综上所述,本文在活性筛选的基础上对小花鬼针草药材抗前列腺增生活性成分进行了研究,并阐明了其主要活性物质为黄酮类、酚酸类和苯丙素类。又在活性测试的基础上考察了小花鬼针草提取方法和提取工艺,确定了小花鬼针草作为抗前列腺增生药物开发时的最佳提取方法和工艺。本文也进行了小花鬼针草指纹图谱和含量测定方法的研究工作,为小花鬼针草药材的开发利用提供了药材质量控制的理论依据。
Benign prostate hyperplasia(BPH) is an universal disease among old men.With the trends in aging,the incidence of BPH is stably increasing in China.How to prevent and treat this disease 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 our previous study,we tested over 100 tradition Chinese medicines' extracts by evaluating their inhibition effect on secreted prostate specific antigen(PSA) in LNCaP cells with ELISA method,and found the EtOH extract of Bidens parviflora Willd.showed strong activity.
In this thesis,the EtOH extract of Bidens parviflora Wind.was applied to a macroporous adsorptive resins column,and found that the 60%and 30%EtOH eluate are the active fractions of the whole plant,further study on the active constitutes of the two fractions were carried out by bioassay-guided isolation.34 compounds were isolated from the 60%and 30%EtOH eluate of Bidens parviflora Willd by repeatedly applied to silica gel columns,Sephadex LH 20 columns, ODS columns and preparative HPLC.33 compounds' structures were elucidated on the basis of physicochemical property and spectroscopic analysis.The 33 compounds were Benzylβ-D-gluco-pyranoside (1),4-(2-hydroxy-2,6,6-trimethylcyclohexane 4-O-β-D-glucosyl)-3-butylene-2-one(2), 3-(3,4-dihydroxy) phenylpropanoic acid(3),Chromone 7-O-β-D-glucopyranoside(4),(1S,2S)-1-(3-Methoxy-4-hydroxy) phenylglycerol(5),(3R) 8(E)-Decene-4,6-diyne-1,3,10-triol(6*), 5-Hydroxychromone 7-O-β-D-glucopyranoside(7),8(E)-Decene-4,6-diyne-3,10-diol 1-O-β-D-glucopyranoside (8),3,5-Dimethoxyphenylpropylenol 4-O-β-D-glucopyranoside(9),Gallic acid (10),Protocatechuic acid(11),6-Methoxycoumarin 7-O-β-D-glucopyranoside(12),Quercetin(13), Luteolin(14),Kaempferol(15),2'-Hydroxy- 3,4,4',6'-tetramethoxychalcone(16),3,4,2',4', 6'-Pentamethoxydihydrochalcone(17),3,4,2',4'-Tetrahydroxychalcone(18),Acacetin 7-O-(α-D-apiofuranosyl) (1→6)-β-D-glucopyranoside(19),Sulfuretin(20),Maritimein(21),Sulfurein(22), Hyperoside(23),Luteolin 7- O-β-D-glucuronide(24),Kaempferol 3-O-β-D-rutinoside(25),Rutin (27),Quceritrin(28),Kaempferol 3-O-(α-D-arabifuranosyl)(1→2)-β-D-glucopyranoside(29),3,4, 2'-Trihydroxychalcone 4'-O-β-D-glucopyranoside(30),5,3',4'-trihydroxyflavanone 7-O-β-D-gluco-pyranoside (31),Luteolin 7-O-β-D-glucopyranoside(32),((2S,3S) Dihydroquercetin(33), Dihydrolueotin(34) respectively.Compounds 6 was a new compound,twenty-third compounds were isolated for the first time from the Bidens parviflora Willd.
Among the 34 compounds,26 compounds showed inhibition effect on PSA secretion in LNCaP cells at different levels.These 26 active compounds belong to three structural types,which were flavanoids,phenyl propanoids and phenolic acids.Flavanoids were more effective than phenyl propanoids and phenolic acids in general.At 15μM test concentration,flavanoids' inhibition rate of PSA secretion in LNCaP cells was about 8.7%to 55.4%,while phenyl propanoids and phenolic acids was about 9.0%to 32.5%.Among these flavonoid compounds,flavone and flavonol showed stronger activity than flavonoid glucoside,while chalcone,flavonone and aurone showed an activity weaker than that of flavone but stronger than that of flavonoid glucoside.
In this thesis,with bioassay results in vitro and total contents of flavones as evaluate indexes, the optimal extract method and extract procedure of Bidens parviflora Willd.were firstly studied. The results showed that bioassay results in vitro are parallel with total contents of flavones,the optimal extract method with the best bioactivity and the highest total contents of flavones is to extract by EtOH and then apply to macroporous adsorption resin As an extract procedure,the optimal one is plant material of Bidens parviflora Willd.being extracted by 20 times volume of 60%EtOH under reflux for 3 times,each time lasting 0.5 hour.
In this thesis,we first studied on the quality control methods for Bidens parviflora Willd..28 batches of plant materials were collected from different place in domestic,and being used for HPLC fingerprint chromatograph research."Tradition Chinese medicine fingerprint chromatograph similarity evaluation system(China Pharmacopeia 2004A)" and "SPSS" software were used to analyze these chromatographs,which lead to a standard HPLC fingerprint chromatograph.We also studied on the method for simultaneous determination of the contents of 4 active constituents in Bidens parviflora Willd.by Rp-HPLC.Both researches provide quality control method for Bidens parviflora Willd..
In conclusion,based on the bioassay evaluation,this thesis studied active constitutes of the raw material plant ofBidens parviflora Willd.,and clarified the main active components of B.P.Willd. for treatment of BPH were flavanoids,phenyl propanoids and phenolic acids.We also studied and established the optimal extract method and procedure of Bidens parviflora Willd.on the basis of bioassay evaluation and content determination of the total flavone.This thesis also studied the HPLC fingerprint chromatograph and determination method of Bidens parviflora Willd.which provide a theory support for the quality control of the material plant when being developed and utilized as a anti-benign prostate hyperplasia medicine.
本课题组以体外抑制前列腺癌细胞(LNCaP cells)分泌前列腺特异性抗原(PSA)作为抗前列腺增生活性筛选体系,在对100多种中药提取物进行抗前列腺增生活性筛选过程中,发现小花鬼针草60%乙醇提取物具有较好的抑制LNCaP细胞分泌PSA活性。
本文对小花鬼针草60%乙醇提取物进行了活性部位的确认,并采取反复硅胶柱色谱、Sephadex LH-20柱色谱、ODS柱色谱和制备高效液相等手段,对活性部位的化学成分进行了追踪分离。从小花鬼针草的活性部位即大孔吸附树脂柱30%和60%乙醇洗脱部分,共分离得到34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33个化合物的结构,分别为苄基β-D-吡喃葡萄糖昔(1);4-(2-羟基-2,6,6-三甲基环己-4-O-β-D-吡喃葡萄糖)-3-丁烯-2-酮(2);3-(3,4-二羟基)苯基丙酸甲酯(3);色原酮7-O-β-D-吡喃葡萄糖苷(4);(1S,2S)-1-(3-甲氧基-4-羟基)苯基丙三醇(5);(3R)8(E)-癸烯-4,6-二炔-1,3,10-三醇(6);5-羟基色原酮7-O-β-D-吡喃葡萄糖苷(7);8(E)-癸烯-4,6-二炔-3,10-二醇1-O-β-D-吡喃葡萄糖苷(8);3,5-二甲氧基苯丙烯醇4-O-β-D-吡喃葡萄糖苷(9);没食子酸(10);原儿茶酸(11);6-甲氧基香豆素-7-O-β-D-吡喃葡萄糖苷(12);槲皮素(13);木犀草素(14);山奈酚(15);2-羟基-3,4,4',6'-四甲氧基查尔酮(16);3,4,2',4',6'-五甲氧基二氢查尔酮(17);3,4,2',4'-四羟基查尔酮(18);金合欢素7-O-(α-D-芹菜呋喃糖)(1→6)-β-D-吡喃葡萄糖苷(19);硫磺菊素(20);海生菊苷(21);硫磺菊苷(22);金丝桃苷(23);木犀草素7-O-β-D-葡萄糖醛酸苷(24);山奈酚3-O-β-D-芸香糖苷(25);芦丁(27);槲皮苷(28);山奈酚3-O-(α-L-阿拉伯呋喃糖)(1→2)-β-D-吡喃葡萄糖苷(29);3,4,2'-三羟基查尔酮4'-O-β-D-葡萄糖苷(30);5,3',4'-三羟基二氢黄酮7-O-β-D-葡萄糖苷(31);木犀草素7-O-β-D-葡萄糖苷(32);(2S,3S)二氢槲皮素(33);二氢木犀草素(34)。34个化合物中,主要为黄酮类化合物,共22个,其中化合物6为新化合物,23个化合物为首次从该植物中分离得到。
分离得到的34个化合物中,26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:黄酮类、苯丙素类和酚酸类。黄酮类化合物的活性强于苯丙素类和酚酸类,当测试浓度为15μM时,前者抑制分泌PSA的相对量在8.7%～55.4%,后两者抑制分泌PSA的相对量在9.0%～32.5%。在黄酮类化合物中,又以黄酮醇和黄酮的活性最好,查尔酮、二氢黄酮、橙酮的活性次之,黄酮苷类化合物的活性较弱。
本文首次以体外细胞活性和总黄酮含量为评价指标,考察了小花鬼针草药材的最佳提取方法和工艺,结果表明提取物的活性强弱和总黄酮含量具有同比性,在水提醇沉、碱提酸沉、醇提水沉、醇提溶剂萃取和醇提大孔树脂分离的提取方法中,以60%乙醇提取大孔树脂分离的活性和总黄酮含量最高,洗脱部分活性富集作用好。而提取工艺则是以加20倍量60%乙醇提取三次,每次0.5 h的工艺最好,得到浸膏收率和总黄酮的含量最高。
本文首次对小花鬼针草药材的质量控制进行了初步研究,对28批不同产地的小花鬼针草药材进行了高效液相指纹图谱研究,使用“中药色谱指纹图谱相似度评价系统(中国药典2004A版)”和SPSS软件对这些样品进行了相似度分析和聚类分析,得到小花鬼针草的对照色谱图。也研究了用反相高效液相色谱同时测定小花鬼针草中四种成分的含量的方法,为小花鬼针草药材的质量控制提供了一定的理论依据。
综上所述,本文在活性筛选的基础上对小花鬼针草药材抗前列腺增生活性成分进行了研究,并阐明了其主要活性物质为黄酮类、酚酸类和苯丙素类。又在活性测试的基础上考察了小花鬼针草提取方法和提取工艺,确定了小花鬼针草作为抗前列腺增生药物开发时的最佳提取方法和工艺。本文也进行了小花鬼针草指纹图谱和含量测定方法的研究工作,为小花鬼针草药材的开发利用提供了药材质量控制的理论依据。
Benign prostate hyperplasia(BPH) is an universal disease among old men.With the trends in aging,the incidence of BPH is stably increasing in China.How to prevent and treat this disease 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 our previous study,we tested over 100 tradition Chinese medicines' extracts by evaluating their inhibition effect on secreted prostate specific antigen(PSA) in LNCaP cells with ELISA method,and found the EtOH extract of Bidens parviflora Willd.showed strong activity.
In this thesis,the EtOH extract of Bidens parviflora Wind.was applied to a macroporous adsorptive resins column,and found that the 60%and 30%EtOH eluate are the active fractions of the whole plant,further study on the active constitutes of the two fractions were carried out by bioassay-guided isolation.34 compounds were isolated from the 60%and 30%EtOH eluate of Bidens parviflora Willd by repeatedly applied to silica gel columns,Sephadex LH 20 columns, ODS columns and preparative HPLC.33 compounds' structures were elucidated on the basis of physicochemical property and spectroscopic analysis.The 33 compounds were Benzylβ-D-gluco-pyranoside (1),4-(2-hydroxy-2,6,6-trimethylcyclohexane 4-O-β-D-glucosyl)-3-butylene-2-one(2), 3-(3,4-dihydroxy) phenylpropanoic acid(3),Chromone 7-O-β-D-glucopyranoside(4),(1S,2S)-1-(3-Methoxy-4-hydroxy) phenylglycerol(5),(3R) 8(E)-Decene-4,6-diyne-1,3,10-triol(6*), 5-Hydroxychromone 7-O-β-D-glucopyranoside(7),8(E)-Decene-4,6-diyne-3,10-diol 1-O-β-D-glucopyranoside (8),3,5-Dimethoxyphenylpropylenol 4-O-β-D-glucopyranoside(9),Gallic acid (10),Protocatechuic acid(11),6-Methoxycoumarin 7-O-β-D-glucopyranoside(12),Quercetin(13), Luteolin(14),Kaempferol(15),2'-Hydroxy- 3,4,4',6'-tetramethoxychalcone(16),3,4,2',4', 6'-Pentamethoxydihydrochalcone(17),3,4,2',4'-Tetrahydroxychalcone(18),Acacetin 7-O-(α-D-apiofuranosyl) (1→6)-β-D-glucopyranoside(19),Sulfuretin(20),Maritimein(21),Sulfurein(22), Hyperoside(23),Luteolin 7- O-β-D-glucuronide(24),Kaempferol 3-O-β-D-rutinoside(25),Rutin (27),Quceritrin(28),Kaempferol 3-O-(α-D-arabifuranosyl)(1→2)-β-D-glucopyranoside(29),3,4, 2'-Trihydroxychalcone 4'-O-β-D-glucopyranoside(30),5,3',4'-trihydroxyflavanone 7-O-β-D-gluco-pyranoside (31),Luteolin 7-O-β-D-glucopyranoside(32),((2S,3S) Dihydroquercetin(33), Dihydrolueotin(34) respectively.Compounds 6 was a new compound,twenty-third compounds were isolated for the first time from the Bidens parviflora Willd.
Among the 34 compounds,26 compounds showed inhibition effect on PSA secretion in LNCaP cells at different levels.These 26 active compounds belong to three structural types,which were flavanoids,phenyl propanoids and phenolic acids.Flavanoids were more effective than phenyl propanoids and phenolic acids in general.At 15μM test concentration,flavanoids' inhibition rate of PSA secretion in LNCaP cells was about 8.7%to 55.4%,while phenyl propanoids and phenolic acids was about 9.0%to 32.5%.Among these flavonoid compounds,flavone and flavonol showed stronger activity than flavonoid glucoside,while chalcone,flavonone and aurone showed an activity weaker than that of flavone but stronger than that of flavonoid glucoside.
In this thesis,with bioassay results in vitro and total contents of flavones as evaluate indexes, the optimal extract method and extract procedure of Bidens parviflora Willd.were firstly studied. The results showed that bioassay results in vitro are parallel with total contents of flavones,the optimal extract method with the best bioactivity and the highest total contents of flavones is to extract by EtOH and then apply to macroporous adsorption resin As an extract procedure,the optimal one is plant material of Bidens parviflora Willd.being extracted by 20 times volume of 60%EtOH under reflux for 3 times,each time lasting 0.5 hour.
In this thesis,we first studied on the quality control methods for Bidens parviflora Willd..28 batches of plant materials were collected from different place in domestic,and being used for HPLC fingerprint chromatograph research."Tradition Chinese medicine fingerprint chromatograph similarity evaluation system(China Pharmacopeia 2004A)" and "SPSS" software were used to analyze these chromatographs,which lead to a standard HPLC fingerprint chromatograph.We also studied on the method for simultaneous determination of the contents of 4 active constituents in Bidens parviflora Willd.by Rp-HPLC.Both researches provide quality control method for Bidens parviflora Willd..
In conclusion,based on the bioassay evaluation,this thesis studied active constitutes of the raw material plant ofBidens parviflora Willd.,and clarified the main active components of B.P.Willd. for treatment of BPH were flavanoids,phenyl propanoids and phenolic acids.We also studied and established the optimal extract method and procedure of Bidens parviflora Willd.on the basis of bioassay evaluation and content determination of the total flavone.This thesis also studied the HPLC fingerprint chromatograph and determination method of Bidens parviflora Willd.which provide a theory support for the quality control of the material plant when being developed and utilized as a anti-benign prostate hyperplasia medicine.
引文
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[5]于普林,郑宏,苏鸿学等.中国六城市老年人前列腺增生的患病率及相关因素.中华流行病学杂志,2000,21(4):276-279.
[6]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.
[7]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.
[8]Michael Marberger.Drug Insight:5α-reductase inhibitors for the treatment of benign prostatic hyperplasia.Nature clinical practice urology,2006,3(9):495-503.
[9]Carbone DJ,Hodges S.Medical therapy for benign prostatic hyperplasia:sexual dysfunction and impact on quality of life.International Journal of Impotence Research,2003,15:299-306.
[10]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.
[11]Dvorkin L,Song KY.Herbs for benign prostatic hyperplasia.The Annals of Pharmacotherapy,2002,36:1443-1452.
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[13]Timothy J Wilt,Areef Ishani,Indulis Rutks,et al.Phytotherapy for benign prostatic hyperplasia,Public Health Nutrition,2000,3(4A):459-472.
[14]朱宏建,南勋义,党建功.中药苦参和南瓜子对前列腺增生影响的实验研究.中国中西医结合外科杂志,1999,5(4):258-261.
[15]吉田恭子.以睾酮5a2还原酶的抑制作用为指标筛选对前列肥在有效的生药.国外医学·中医药中分 册,1998,20(3):57.
[16]朱宏建,南勋义,党建功.鸦油乳对前列腺增生动物模型的影响及机理研究.中华泌尿外科杂志,1999,20(11):680-682.
[17]吴国欣,林跃金,欧敏锐.白芥子提取物抑制前列腺增生的实验研究(Ⅰ).中国中药杂志,2002,27(10):766-768.
[18]杜旭,窦秋莲,李春艳等.黑龙江葵花粉治疗前列腺增生的药效学研究.中国中医药科技,2002,9(2):94-95.
[19]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.
[20]William D.Steers.5α-reductase activity in the prostate.Urology,2001,58(Supplement 6A):17-24.
[21]周瑞海编译.5α-还原酶抑制剂非那甾胺.国外医药(合成药、生化药、制剂分册),1994,15(1):23.
[22]张军编译.α-受体阻制剂治疗前列腺增生的评价.国外医药(合成药、生化药、制剂分册),1996,17(3):166.
[23]吴佳清.雌激素及其受体在良性前列腺增生中的作用.中华男科学,2002,8(3):212.
[24]Royuela M,de Miguel MP,Bethencourt FR.Estrogen receptors alpha and beta in the normal,hyperplastic and carcinomatous human prostate.J Endocrinol,2001,168(3):447-454.
[25]Walsh PC,Retik AB,Vaughan ED,etc.Eds.Campbell's Urology.Philadelphia,Saunders.1998,2(7):1381-1418.
[26]Fiorelli G,De Bellis A,Longo A,etc.Growth factors in the human prostate.J Steroid Biochem Mol Biol.,1991,40(1-3):199-205.
[27]Claus S,Berges R,Senge T,etc.Cell kinetic in epithelium and stroma of benign prostatic hyperplasia.J Urol.,1997,158:217-221.
[28]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.
[29]Lilja HA.kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein.J Clin.Invest.,1985,76:1899-1903.
[30]Gittes RF.Carcinoma of the prostate,N Engl.J Med.,1991,324:236-245.
[31]Leonard S.Marks,Claus G.Roehrbom,Gerald L.Andriole,Prevention of Benign Prostatic Hyperplasia Disease,The journal of urology,2006,176:1299-1306.
[32]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》泌尿系统分册,1999,19(2):49-50.
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[34]Rcehrborn CG,boyle P,Gould Al,etc.,Serum prostate specific antigen as a predictor of prostate volume in men with benign prostatic hyperplasia.Urology,1999,53(3):581-589.
[35]Chiu K.Y.,Yong C.R.Effects of finasteride on prostate volume and prostate-specific antigen.J Chin Med Assoc,2004,67:571-574.
[36]吴宇芳,关晓东.ELISA法测定前列腺特异性抗原的临床意义.广东医学院学报.1999,17(4):361.
[37]高中伟,张寒,王明珠等.前列腺增生患者前列腺特异性抗原测定.郑州大学学报(医学版).2003,38(1):72.
[38]Xia,G.;Zhang,J.;Chen,X.;Ma,L.The resource utilization of Bidens parviflora Willd.in Baoding district.Chin.Trad.Herb.Drugs 1985,16:37-40.
[39]Jiangsu New Medical College.Dictionary of Chinese Crude Drugs;Shanghai Technological Publisher:Shanghai,P.R.China,1986;pp.1413,1694,2239.
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