小叶莲（Fructus Podophylli）为常用藏药，中国药典2005年版收载其基源为小檗科植物桃儿七Podophyllum hexandrum Royle的干燥成熟果实。具有活血调经功能，常用于治疗多种妇科疾病，同时也具有较好的抗肿瘤作用。但是目前关于小叶莲的研究却少见报道。为了更好的开发和利用藏药资源，探明其内在有效成分，完善小叶莲药材的质量标准，本文对小叶莲进行了化学成分、抗肿瘤活性和含量测定研究。

通过对小叶莲的化学成分进行研究，利用现代色谱技术和光谱方法从其乙醇提取物中分离并鉴定了10个化合物的结构。其中木脂素类化合物3个：鬼臼毒素（Se1）、去氧鬼臼毒素（Se2）、4′-去甲去氧鬼臼毒素（Se3）；黄酮类化合物5个：8-异戊烯基山柰酚（Se4）、槲皮素（Se5）、山柰酚（Se6）、8,2′-二异戊烯基槲皮素3-甲醚（Se7）、芦丁（Se8）；甾醇类化合物2个：β-谷甾醇（Se9）、胡萝卜苷（Se10）。其中，8,2′-二异戊烯基槲皮素3-甲醚（Se7）为新化合物，槲皮素、山柰酚和芦丁均为首次从小叶莲中分离得到。

对小叶莲乙醇提取物及其不同极性部分的抗肿瘤活性筛选实验表明：小叶莲乙醇提取物对白血病细胞株（HL-60）、人胃癌细胞株（BGC-823）、人肝癌细胞株（Bel-7402）和人鼻咽癌细胞株（KB）四种肿瘤细胞株均表现出一定的抑制作用，其对HL-60和BGC-823细胞株具有较强的抑制作用。石油醚部分高浓度（100μg/mL）对KB细胞株；乙酸乙酯部分高浓度（100μg/mL）对HL-60、Bel-7402和KB细胞株也表现出较强的抑制作用；正丁醇和水部分未表现出抑制作用。

小叶莲提取物对小鼠移植性乳腺癌的抑制作用实验显示，小叶莲组（500mg/kg，每天灌胃给药）和环磷酰胺组（20mg/kg，隔天腹腔注射）与阴性对照组（500mg/kg，每天灌胃给药）的平均瘤重相比，均具有显著性差异，提示肿瘤生长受抑，其抑制率分别为48%和46%。小叶莲组比CTX组的抑瘤率稍高，提示小叶莲乙醇提取物对小鼠移植性乳腺癌具有较强的抑瘤作用。

采用人乳腺癌细胞MDA-231和MCF-7进行了体外抑制作用实验，结果表明小叶莲乙醇提取物对两种细胞株均具抑制活性（48h的IC₅₀分别为14.0和84.3μg/mL）。在不同极性部分中，乙酸乙酯部分抑制作用最强（48h的IC₅₀分别为2.21和6.53μg/mL），其抑制作用强于阳性对照顺铂。在单体化合物中，三种鬼臼毒素类木脂素均表现出了较好的抗癌活性，其中4'-去甲去氧鬼臼毒素对MDA-231（48h的IC₅₀为0.889μg/mL）、去氧鬼臼毒素对MCF-7（48h的IC₅₀为2.52μg/mL）的抑制作用最为强烈；8-异戊烯基山柰酚高浓度对两种细胞均表现出一定的抑制作用。细胞周期实验中，小叶莲提取物及各化合物均将MDA-231细胞的周期阻滞在G₂/M期，影响肿瘤细胞的有丝分裂，从而阻滞癌细胞的生长。对MCF-7细胞周期的影响则表现不同，MCF-7细胞被阻滞在G₀/G₁期，进入S期的细胞数量减少。

建立了同时测定小叶莲中七种成分（3种木脂素类成分：鬼臼毒素、去氧鬼臼毒素、4′-去甲去氧鬼臼毒素和4种黄酮类成分：8-异戊烯基山柰酚、槲皮素、山柰酚、8,2′-二异戊烯基槲皮素3-甲醚）含量的HPLC方法，测定了6份样品，发现小叶莲中异戊烯基黄酮类成分的含量较高（8-异戊烯基山柰酚：0.6245~8.108 mg/g；8,2′-二异戊烯基槲皮素3-甲醚：5.046~53.02 mg/g），因此我们认为可以将黄酮类和鬼臼毒素类木脂素作为小叶莲质量控制的指标成分。

Fructus Podophylli (FP), a drug used commonly by Tibetan people in China, is dried mature fruit of Podophyllum hexandrum Royle. It is recorded by Chinese Pharmacopoeia and has been used to treat gynecological diseases for hundreds of years. Chemical studies revealed that podophyllum ligans and flavonoids had been isolated from FP. In the past few decades, the podophyllum ligans, such as podophyllotoxin, desoxypodophyllotoxin had drawn attention in pharmacological research because of the significant antineoplastic, antiviral and insecticidal activities.

However, up to now, limited research had been reported about FP. In this paper, in order to find bioactive constituents and make good use of FP, phytochemical research and pharmacological tests, including anti-tumor in vitro and in vivo, had been done. In addition, we developed a RP-HPLC method to simultaneously determine the three podophyllum ligans and four flavonoids for the quality control of FP.

In our phytochemical research of the ethanol extract of FP, 10 compounds had been isolated, and these compounds were identified and elucidated on the base of most kinds of chromatographic techniques and spectroscopic methods. They included 3 lignans (podophyllotoxin, desoxypodophyllotoxin and 4′-demethyldesoxypodophyllotoxin), 5 flavonoids (8-prenylkaempferol, quercetin, kaempferol and 8,2′-diprenylquercetin 3-methyl ether, rutin) and 2 others (β-sitosterol and daucosterol). Among them, 8,2′-diprenylquercetin 3-methyl ether was a new compound, quercetin, kaempferol and rutin were isolated from FP for the first time.

The in vitro anti-tumor activities were screened against the HL-60, BGC-823, Bel-7402 and KB cell lines. The ethanol extract gave activities in the 4 cells. Among them, HL-60 and BGC-823 cell lines were strongly inhibited. The extract of petroleum ether in high concentration (100μg/mL) showed inhibition against KB. And the extract of ethyl acetate in the same concentration showed inhibition against HL-60, Bel-7402 and KB. On the contrary the n-butanol and water extracts had no activities.

Inhibit activities against breast-tumor testing of ethanol extract and the compounds were determined in vivo and in vitro, respectively.

The in vivo anti-tumor activity was evaluated against human breast carcinoma model (MCF-7) in nude mice, cyclophosphamide was used as positive control. Extract of FP showed obvious activity (Inhibition rate = 48%)。

Ethanol extract showed strong activities in MDA-231 and MCF-7 cell lines(48h IC₅₀=14.0 and 84.3μg/mL, respectively). The ethyl acetate extract showed the strongest activities, which suggested that the low and medium polarity materials should be the effective section of anti-tumor activities. In the continuing screening tests of the compounds in vitro, the podophyllum lignans and 8-prenylkaempferol in high concentration exhibited the significant cytotoxic effects against the MCF-7 and MDA-231 cell lines. Among them, 4′-demethyldesoxypodophyllotoxin performed best against MDA-231, while against MCF-7, it is desoxypodophyllotoxin. The results suggested that the lignans and flavonoids may be the potential cytotoxic active components of this drug. Cell cycle was analyzed by flow cytometry (FCM). The extracts and compounds of FP blocked the cell cycle in G₂/M phase of MDA-231. Differently, G₀/G₁ phase was blocked in MCF-7.

Besides, this was the first reserach on the simultaneous quantification of three podophyllum ligans and four flavonoids in the samples of FP. (podophyllotoxin, desoxypodophyllotoxin, 4′- demethyldesoxypodophyllotoxin, 8-prenylkaempferol, quercetin, kaempferol and 8,2′-diprenylquercetin 3 methyl ether) A simple, accurate and rapid assay method was successfully established. The results suggested that this RP-LC method could be considered as a valid method for the quality control of FP. The method was applied in determinating 6 samples. It was found that the contents of prenylflavonoids were quite high in FP. (8-prenylkaempferol: 0.6245~8.108 mg/g; 8,2′-diprenylquercetin 3 methyl ether: 5.046~53.02 mg/g) Therefore the flavonoids and podophyllum ligans could be regarded as “ marker compounds” in FP.

前 言. 1

第一章 文献综述. 3

第一节 本草考证. 4

一、小叶莲的本草考证. 4

二、鬼臼的本草考证. 4

第二节 化学成分研究. 5

一、木脂素类（lignanoids）. 5

二、黄酮类（flavones）. 9

三、其它类. 10

第三节 质量评价研究. 10

一、含量测定和成分比较. 10

二、指纹图谱研究. 12

第四节 药理作用及临床应用. 13

一、药理作用. 13

二、毒性. 15

三、临床应用. 15

第五节 桃儿七资源的开发和利用. 16

第六节 小结和讨论. 17

一、质量控制标准有待建立. 17

二、指纹图谱研究有待深化和细化. 18

三、抗肿瘤药效学有待深入研究. 18

四、保护野生资源，合理开发利用. 18

第一章参考文献. 19

第二章 小叶莲的化学成分研究. 24

第一节 化合物的结构鉴定. 25

一、新化合物结构解析. 25

二、已知化合物结构鉴定. 28

第二节 实验部分. 32

一、仪器和试剂. 32

二、药材. 33

三、提取分离. 33

四、化合物的理化和波谱数据. 35

第二章参考文献. 38

第三章  抗肿瘤活性研究. 39

第一节 小叶莲乙醇提取物及各萃取部分对肿瘤细胞增殖的影响. 39

一、实验材料和仪器. 39

二、实验方法. 40

三、实验结果. 40

第二节 小叶莲提取物对小鼠移植性乳腺癌的抑制作用. 41

一、实验材料和仪器. 41

二、实验方法. 42

三、实验结果. 42

四、讨论. 43

第三节 小叶莲提取物及其主要成分对人乳腺癌细胞MDA-231和MCF-7的抑制作用  43

一、细胞增殖及增殖抑制实验. 44

二、细胞周期的检测. 50

三、小结. 56

第四节 小结与讨论. 57

一、小结. 57

二、讨论. 57

第三章参考文献. 59

第四章 小叶莲中木脂素类和黄酮类成分的含量测定. 60

第一节 小叶莲含量测定方法的建立. 60

一、仪器与试剂. 60

二、试药. 60

三、药材来源. 62

四、色谱条件. 62

五、提取方法考察. 63

六、方法学考察. 70

第二节 小叶莲含量测定结果. 75

一、含量测定方法. 75

二、样品测定结果. 76

第三节 结果分析与讨论. 76

第四章参考文献. 78

第五章 结论与讨论. 79

附图. 81

致 谢. 92

作者简历. 93