九节龙中新的三萜皂苷成分及其对人恶性胶质瘤的细胞毒性和诱导凋亡作用.doc

【精品文档】如有侵权，请联系网站删除，仅供学习与交流九节龙中新的三萜皂苷成分及其对人恶性胶质瘤的细胞毒性和诱导凋亡作用.精品文档.九节龙中新的三萜皂苷成分及其对人恶性胶质瘤的细胞毒性和诱导凋亡作用汤海峰1* 汤海峰(1971-), 第四军医大学西京医院药剂科副主任, 副教授、副主任药师, 硕士生导师, 博士后。Tel: 029-84775471, E-mail: natprod, 田冶1,2, 王晓娟3, 邱峰2, 程光4, 文爱东1 (1. 第四军医大学西京医院药剂科，陕西 西安 710032; 2. 沈阳药科大学中药学院天然药物教研室，辽宁 沈阳 110015；3. 第四军医大学口腔医学院药剂科，陕西 西安 710032；4. 第四军医大学西京医院脑科医院，陕西 西安 710032)摘要：目的 研究中药九节龙中具有抗恶性胶质瘤活性的三萜皂苷成分。方法 应用多种色谱手段和波谱方法分离和鉴定化合物。采用MTT法测定肿瘤细胞毒性，流式细胞术、透射电镜、Hoechst 33342荧光检测、DNA Ladder、Western blot等技术研究凋亡及其信号途径。结果 分离鉴定了10种三萜皂苷类化合物，包括6个新化合物，分别命名为ardipusillosides III (1), IV (2)和ardipusillosides AD (36)，4个已知化合物为ardisiacrispin B (7), ardisiacrispin A (8), ardipusilloside I (9)和ardipusilloside II (10)，其中7和8首次从本种得到。除皂苷6无活性、皂苷8具有中等强度活性外，其余8种皂苷均对人恶性胶质瘤显示显著细胞毒性，并均不影响人原代星形胶质细胞的生长。1可诱导人恶性胶质瘤U251MG细胞凋亡，机制研究表明涉及到BAD介导的细胞内凋亡信号途径以及caspase-8和caspase-3介导的细胞外凋亡信号途径。结论 皂苷3的分子中苷元通过1个丙三醇基与糖醛酸连接，是三萜皂苷中非常罕见的结构特征；皂苷5和6的苷元以及皂苷4的30位羧基连接阿拉伯糖的结构特征，均为首次在本属(紫金牛属)植物中发现；皂苷1的苷元和皂苷2的六糖链结构在本属中也比较少见。这些皂苷显著的肿瘤细胞毒性及对其具体作用机制的研究结果为进一步开发抗恶性胶质瘤新药提供了有价值的先导化合物和科学依据。关键词：九节龙；三萜皂苷；恶性胶质瘤；细胞毒性；凋亡；BAD；caspaseNew Triterpenoid Saponins from Ardisia pusilla, Their Cytotoxicity and Ability to Induce Apoptosis in Human Glioblastoma U251MG CellsTANG Hai-feng1, TIAN Ye1,2, WANG Xiao-juan3, QIU Feng2, CHENG Guang4, WEN Ai-dong1 (1. Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xian 710032; 2. Department of Phytochemistry, Shenyang Pharmaceutical University, Shenyang 110015; 3. Department of Pharmacy, School of Stomatology, Fourth Military Medical University, Xian 710032; 4. Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xian 710032)ABSTRACT: OBJECTIVE To study the anti-glioblastoma triterpenoid saponins from Ardisia pusilla. METHODS A combination of multi-chromatography as well as spectral analysis were employed for the separation and determination of the compounds from the plant. The cytotoxicities against human glioblastoma U251MG cells were determined using the MTT assays. The ability of ardipusilloside III to induce apoptosis in U251MG cells, as well as the involvement of apoptotic signaling pathways were also studied. RESULTS Ten triterpenoid saponins (1-10) were obtained and elucidated, among which six are new compounds and named ardipusillosides III (1), IV (2) and ardipusillosides A-D (3-6). Nine saponins showed in vitro cytotoxicity against U251MG cells. Both the intrinsic pathway of apoptosis, mediated by BAD dephosphorylation and cleavage, and the extrinisic pathway of apoptosis, mediated by caspase-8 and caspase-3 activation, were involved in ardipusilloside III-induced apoptosis. CONCLUSION The structural features of new saponins 1-6 are relative rare among triterpenoid glycosides or saponins from genus Ardisia. Our studies focused on cytotoxic saponins of A. pusilla have provided valuable leads for pursuing new anti-glioblastoma drugs, and established a homebase for further development of the title plant with abundant resources in Southern China.KEY WORDS: Ardisia pusilla; triterpenoid saponin; glioblastoma; cytotoxicity; apoptosis; BAD (Bcl-xL/Bcl-2- associated death promoter homolog); caspase1. 前言人恶性胶质细胞瘤是颅内肿瘤中最常见的一种，新型安全高效的化疗药物的开发是治疗该病的迫切要求，我们的前期研究表明中药九节龙的总皂苷可抑制人恶性胶质瘤U251MG细胞生长。九节龙Ardisia pusilla A. DC为紫金牛科紫金牛属植物，广泛分布于我国南部地区，具有清热解毒的功效，药用全草。本属植物主要含三萜皂苷，且被证明为主要有效成分。文献报道已从九节龙中分离鉴定了3种原生的三萜皂苷，即ardipusillosides I, II和一个未命名的皂苷，均对多种肿瘤显示较强的抑制作用。鉴于对其化学成分研究尚不充分，我们以发现新的抗胶质瘤活性成分为目的，对其中的三萜皂苷进行了系统分离。除获得已知的2种皂苷ardipusillosides I (9)和II (10)外，还得到了6种新的三萜皂苷ardipusillosides III (1)、IV (2)、ardipusillosides AD (36)以及2种在本属其他植物中曾分离到的皂苷ardisiacrispins B (7)和A (8)。应用现代波谱技术(1HNMR, 13CNMR, DEPT, 1H-1H COSY, TOCSY, HSQC, HMBC, NOESY, ESI-MS, MS/MS, HR-ESI-MS等)和化学手段鉴定了它们的结构(见Fig. 1)。MTT法筛选表明：除皂苷6无活性、皂苷8具有中等强度活性外，其余8种皂苷均对人恶性胶质瘤U251MG细胞显示显著细胞毒性，并均不影响人原代星形胶质细胞的生长。对皂苷1的具体作用机制研究表明：1可诱导人恶性胶质瘤U251MG细胞凋亡，且涉及到BAD介导的细胞内凋亡信号途径以及caspase-8和caspase-3介导的细胞外凋亡信号途径。本文阐述了这些皂苷的分离鉴定，以及对人恶性脑胶质瘤U251MG细胞的抑制作用和作用机制研究结果。2. 实验部分2.1 材料和仪器九节龙全草于2006年9月采自四川夹江县，由第四军医大学口腔医院药剂科王晓娟教授鉴定。标本存于第四军医大学口腔医院药剂科。仪器和试剂略。2.2 提取和分离药材粗粉(3 kg) 用95% EtOH回流提取，提取物分散于水中，石油醚萃取脱脂，正丁醇萃取得到含总皂苷的部位，之后经过多种色谱手段(正相和反相硅胶柱色谱、Sephadex LH-20 凝胶过滤、高效液相制备等)分离获得10个皂苷成分：110。采用波谱和化学手段鉴定了它们的结构。2.3 结构鉴定皂苷1：无色无定形粉末，mp 240241, = 3.6 (c 0.15, MeOH)，Liebermann-Burchard和Molish反应阳性。ESI-MS (positive ion mode) m/z: 1099 M+Na+; ESI-MS (negative ion mode) m/z: 1075 MH-; ESI-MS/MS (negative ion mode, m/z 1075) m/z: 943 1075Xyl-, 913 1075Glc-, 781 943Glc-, 619 781Glc-, 487 619Ara- (aglyconeH-), 471; HR-ESI-MS (positive ion mode) m/z: 1099.5310 M+Na+ (calc. for C52H84O23Na: 1099.5301). 1H NMR和13C NMR数据见Table 1。Fig. 1 Elucidated structures of triterpenoid saponins isolated from Ardisia pusilla1的酸水解和糖衍生物的分析：采用2 mol/L三氟乙酸进行酸水解并制备糖的三甲基硅醚化衍生物，以L-Chirasil-Val 柱(25 m ´ 0.32 mm i.d.)进行GC分析，标准糖制备的相应衍生物作对照。标准糖衍生物的色谱保留时间分别为8.68, 9.60 min (D-阿拉伯糖), 8.76, 9.64 min (L-阿拉伯糖), 10.84, 11.86 min (D-木糖), 10.87, 11.91 min (L-木糖), 14.56 min (D-葡萄糖), 14.50 min (L-葡萄糖)。可以看到，采用上述条件能够分辨不同绝对构型的糖。分析表明：1的寡糖链由3种糖基组成，即L-阿拉伯糖, D-木糖和D-葡萄糖，组成比约为1:1:2。其余化合物的波谱数据和化学反应略。2.4 抗肿瘤作用筛选和作用机制研究 具体实验方法略。Table 1 1H (600MHz) and 13C NMR (150MHz) data of ardipusilloside III (1) in C5D5N ( in ppm, J in Hz).PositionHCPositionHC11.60 brd (13.2), 0.79 m39.1Ara22.00 m, 1.81 m26.514.80 d (5.4)104.333.13 dd (11.4, 4.2)89.024.51 m79.9439.534.25 m73.550.64 d (11.4) 55.644.20 m78.861.39 m17.953.64 brd (10.8), 4.60 brd (12.0)63.671.50 m, 1.18 m34.2Glc I842.515.48 d (7.8)104.791.21 d (12.6)50.424.04 m76.21036.734.21 m78.2111.66 m, 1.41 m19.144.16 m71.9122.58 m, 2.04 m31.153.97 m78.01387.264.50 m, 4.34 dd (11.4, 4.2)62.81444.9Glc II152.18 dd (13.8, 4.8), 1.48 m36.915.05 d (7.8)104.1164.20 m76.123.92 t (9.0)85.21742.434.17 m77.4181.86 m49.144.14 m71.0192.15 m34.453.75 m78.32048.564.38 dd (11.4, 3.6), 4.24 m62.2212.57 m30.8Xyl221.99 m, 1.84 m28.114.94 d (7.8)107.5231.18 s28.023.96 m75.9241.00 s16.633.99 m77.6250.81 s16.344.08 m70.6261.29 s18.554.53 m, 3.71 m67.2271.55 s19.7285.12 s99.2291.06 s24.3309.70 s207.93. 结果与讨论皂苷710通过波谱解析和化学手段，并与文献对照确定为已知皂苷ardisiacrispin B、ardisiacrispin A、ardipusilloside I和ardipusilloside II，其中7和8首次从本种植物中得到。皂苷1为无定形粉末，mp 240241, = 3.6 (c 0.15, MeOH)，Liebermann-Burchard和Molish反应阳性。HR-ESI-MS分析确定分子式为C52H84O23。准分子离子峰M+Na+和MH均比皂苷8的相应离子峰多16个质量单位。负离子模式的ESI-MS/MS (m/z 1075)中可观察到一系列失去糖基的碎片，如m/z 943 1075Xyl-, 913 1075Glc-, 781 943Glc-, 619 781Glc-, 487 619Ara- (aglyconeH-)等。1的13C NMR谱显示52个碳信号，其中30个可归属为苷元部分。1的13C NMR数据与cyclamiritin A非常相似，同样具有30 位醛基和16-OH，3位连接有寡糖链。3位寡糖基的相对构型通过NOESY谱中Hax-3和Me-23以及H-5的相关峰确认为，由C-16的化学位移值可推断16-OH为构型(相似三萜苷元的文献值：16-OH者约为 77.0，16-OH者约为 64.0)。在已知皂苷8的苷元中13,28-环氧桥的特征碳信号显示为 86.2 (C-13, C)和 77.7 (C-28, CH2)，但是1的13C NMR谱未显示 77.7左右的CH2信号，相反观察到1个 99.2的CH信号。结合分子式，推测C-28连接有1个OH。C-17, C-18和C-22信号发生了高场位移，而C-15, C-19, C-21等信号发生了小的低场位移，HMBC谱中可观察到H-28与C-13, C-16以及C-18的远程相关峰，这些都进一步确证了上述推测。16b位的氢与H-28存在NOE相关，表明28-OH为a构型。基于上述分析，推定1的苷元为3b,16a,28a-trihydroxy-13b,28-epoxy-oleanan-30-al。1的NMR谱中显示4个糖基的端基氢信号及相对应的端基碳信号，提示其寡糖链由4个糖基组成。1经三氟乙酸水解后对糖的三甲硅醚化衍生物采用L-Chirasil-Val柱进行GC分析表明其连接有L-阿拉伯糖, D-木糖和D-葡萄糖(1:1:2)。糖端基的相对构型通过相应端基氢的多重性、偶合常数以及NOESY分析分别推断为, , 和。糖基的氢和碳信号经由2D NMR的仔细分析得到归属 (Table 1)，通过HMBC和NOESY谱对糖残基的连接顺序和连接位置作了确认 (Fig. 2)。综上所述，鉴定1的结构为3b-O-b-D-xylopyranosyl- (12)-b-D-glucopyranosyl-(14)-b-D-glucopyranosyl-(12)-a-L-arabinopyranosyl-16a,28a-dihydroxy-13b,28-epoxy-oleanan-30-al，命名为ardipusilloside III。Fig. 2 Key HMBC and NOESY correlations of saponin 1新皂苷26亦根据相似的方式分别进行了结构鉴定，并分别命名为为ardipusilloside IV和ardipusillosides AD。对这些新皂苷的结构特征进行分析，表明：皂苷3的分子中苷元通过1个丙三醇基与糖基连接，是三萜皂苷中非常罕见的结构特征，这是自然界发现的第三个该类型皂苷；皂苷5和6的苷元以及皂苷4的30位羧基连接阿拉伯糖的结构特征，均为首次在本属植物中发现；皂苷2的寡糖基由6个糖组成，以及皂苷1的苷元结构，在本属发现的三萜皂苷中也比较少见。MTT法筛选表明：除皂苷6无活性、皂苷8具有中等强度活性外，其余8种皂苷均对人恶性胶质瘤U251MG细胞显示显著细胞毒性，并均不影响人原代星形胶质细胞的生长 (Table 2)。Table 2 Cytotoxic activity of saponins 1-10 against human glioblastoma U251MG cells and primary cultured astrocytes in vitro (IC50, mM).Cell line123456U251MG3.95 ± 0.37a2.57 ± 0.211.55±0.212.20±0.354.93 ± 0.25> 100astrocytes>100> 100>100>100> 100> 100Cell line78910ACNUbU251MG3.33 ± 0.29a12.20 ± 1.143.48 ± 0.164.51 ± 1.320.98 ± 0.06astrocytes> 100> 100> 100> 100> 100a The data represent the mean plus SD of three independent experiments in which each compound concentration was tested in three replicate wells.b Nimustine hydrochloride (ACNU) as positive control.对1的具体抗肿瘤作用进行了深入研究，发现其可诱导胶质瘤U251细胞凋亡，并深入探讨了作用机制。采用Annexin V-FITC/PI染色、DNA电泳、Hoechst 33342染色和透射电镜检测U251细胞凋亡；Western Blot分析U251细胞BAD蛋白表达和磷酸化水平(Fig. 3, Fig. 4, Fig. 5)。发现1以剂量和时间依赖方式抑制U251细胞活性，并诱导细胞染色质浓聚边集、DNA断裂等典型凋亡改变。Western blot结果表明1诱导的凋亡细胞中BAD的总体表达水平上升而磷酸化BAD的表达水平呈时间依赖性迅速下降。研究还观察到随着凋亡时间的延长，tBADS的表达水平从无到有稳步上升。既往分子生物学研究认为tBADS各磷酸化位点很少被磷酸化，从而具有更强的促凋亡能力，并且tBADS具有高效的线粒体外膜定位能力，它与caspase蛋白相互激活的正反馈放大了这种能力。这表明BAD去磷酸化和酶解凋亡信号途径参与到1诱导的恶性胶质瘤细胞凋亡进程，是其重要的内在分子机制。此外，鉴于1还促使caspase-8和caspase-3激活，因此其诱导凋亡的分子机制既涉及到BAD介导的细胞内凋亡信号途径，也涉及到caspase-8和caspase-3介导的细胞外凋亡信号途径。本文对九节龙中三萜皂苷的分离鉴定，肿瘤细胞毒性筛选及其具体作用机制的系统研究结果为进一步开发抗恶性胶质瘤新药提供了有价值的先导化合物和科学依据。该研究也为九节龙这一在我国南部地区资源丰富的植物的开发利用打下了基础。Fig. 3 Effect of ardipusilloside III on cell cycle progression, intracellular structures, phosphatidylserine externalization and DNA fragmentation of human glioblastoma U251MG cells: (A) Flow cytometric analysis of cell cycle phase distribution of U251MG cells after treatment with different concentrations of ardipusilloside III for the indicated time periods; (B) Electron microscopy showed the organization of subcellular organelles of U251MG cells, including the presence of vacuoles containing cytoplasm and nucleolus fragments budding off the plasma membrane (center, magnification 915,000) and marginal chromatin condensation (right, magnification 97,500); (C) Representative dot plots showing flow cytometric analysis of glioblastoma U251MG cells treated with different concentrations of ardipusilloside III for the indicated time periods then stained with FITC- conjugated Annexin V and propidium iodide; (D) The characteristic DNA ladder suggesting apoptotic DNA damage in the ardipusilloside III-treated U251MG cells is shown in Lane 3. Cells were treated with 8.2 g/ml ardipusilloside III for 24 h.Fig. 4 Morphological alterations of U251MG cells treated with different concentrations of ardipusilloside III for 24 h. Treated cells shrank, aggregated, and detached from the bottom of culture flask as shown in the upper panels (magnification 9600). Nuclear morphology was detected using Hoechst 33342, and visualized by fluorescence microscopy as shown in the lower panels (magnification 9600): (A) Cell condensation and fracturing of marginal chromatin; (B) The lobulated gemmules of the cell nucleus; (C) Apoptotic body.Fig. 5 Activation of BAD and caspases involved in ardipusilloside III-induced apoptosis of human glioblastoma U251MG cells: (A) BAD was dephosphorylated at serine136 and cleaved to generate tBADS (15 kDa), as indicated by the arrow. Phospho-BAD expression decreased, while tBADS expression increased, during the indicated time course. The expression of total BAD was slightly decreased after ardipusilloside III treatment. Rapid and substantial caspase-8 and -3 cleavage was detected after treatment with 8.2 lg/ml ardipusilloside III for the indicated time periods; (B) Statistical analysis of the Western blots. Bars, ±SE; *P<0.05; *P<0.01.参考文献略。