CN116041411A - 一种海参烷型新化合物及其制备方法和用途 - Google Patents
一种海参烷型新化合物及其制备方法和用途 Download PDFInfo
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- CN116041411A CN116041411A CN202211421869.4A CN202211421869A CN116041411A CN 116041411 A CN116041411 A CN 116041411A CN 202211421869 A CN202211421869 A CN 202211421869A CN 116041411 A CN116041411 A CN 116041411A
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- holotoxin
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- sea cucumber
- glucopyranose
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Abstract
本发明公开了两种新的海参烷型皂苷类化合物及其制备方法和用途。以海参为原料，通过有机溶剂提取，硅胶和/或反相柱层析分离和/或制备型高效液相色谱分离纯化可以获得这两种化合物。这两种化合物中前者对三阴性乳腺癌MDA‑MB‑231细胞、肝癌HepG2细胞具有细胞毒作用，后者对MDA‑MB‑231细胞具有细胞毒作用。
Description
技术领域
本发明属于天然药物化学研究领域。
背景技术
仿刺参(Apostichopus japonicus Selenka)属棘皮动物门(Echinopermata)、海参纲(Holothuroidea)、楯手目(Aspidochirotida)、刺参科(Stichopodidae)、仿刺参属(Apostichopus)海洋生物，主要分布于中国、日本、朝鲜、俄罗斯等地。仿刺参是我国传统的食材与中药材，是我国北方主要的食用海参品种，是最为名贵的海参品种，被誉为“参中之冠”。传统医学认为，仿刺参具有滋补功效，可以治疗经血亏损、虚弱劳祛、阳痿梦遗、小便频数、肠燥便艰等多种症状。现代医药学研究表明，仿刺参富含氨基酸、维生素、脂肪酸、常量和微量元素等人体必需的营养物质，可以调节人体的生长和代谢，增强人体免疫力，促进人脑发育。仿刺参还含有皂苷、脑苷脂、神经节苷脂、酸性粘多糖、凝集素等结构新颖的次生代谢产物，这些物质具有抗肿瘤、抗菌、抗病毒、抗凝血、抗辐射等多种生物活性。
海参皂苷是仿刺参中重要的活性成分，由苷元和糖链通过β-糖苷键结合组成的，大多属于羊毛甾醇型三萜类寡糖苷。仿刺参中皂苷的糖链一般由2-6个单糖组成，单糖一般为：木糖(Xyl)、葡萄糖(Glc)、喹诺糖(Qui)、3-甲基葡萄糖(MeGlc)等，连接苷元的第一个单糖常为木糖。仿刺参中皂苷的苷元常为四环三萜和五环三萜，按照苷元的结构可以将其分为两种：海参烷型(Holostane-type)和非海参烷型(Non-holostane-type)。海参烷型苷元一般含有18(20)-内酯环，而非海参烷型苷元有18(16)-内酯环或无内酯环结构。仿刺参中还有一种甾族皂苷(Steroid glycosides)，其苷元部分一般包含胆甾(cholest)的结构。目前仿刺参中报道的海参皂苷共有49种，其中，海参烷型皂苷比较多，非海参烷型皂苷比较少。仿刺参中的海参皂苷具有抗肿瘤活性、抗氧化活性、抑菌活性、抗凝血活性等多种生物活性。
发明内容
本发明提供了从仿刺参中分得的两种具有抗癌活性的新的海参烷型皂苷类化合物，分别是Holotoxin E1，即3β-O-{[3-O-甲基-β-D-吡喃葡萄糖-(1→3)-β-D-吡喃木糖-(1→4)-β-D-吡喃喹诺糖-(1→2)]-[β-D-吡喃葡萄糖-(1→4)]-β-D-吡喃木糖}-海参烷-7(8),25(26)-二烯-16-酮和Holotoxin E2，即3β-O-{2-O-[3-O-甲基-β-D-吡喃葡萄糖-(1→3)-β-D-吡喃木糖-(1→4)-β-D-吡喃葡萄糖]-4-O-[β-D-吡喃葡萄糖-(1→3)-β-D-吡喃葡萄糖]-β-D-吡喃木糖}-海参烷-7(8),25(26)-二烯-16-酮，同时提供了其制备的方法和用途。本发明的具体内容如下。
以鲜海参为原料，有机溶剂提取，滤过，回收溶剂，获得海参粗提物；将海参粗提物加水超声溶解，获得海参粗提物混悬液；有机溶剂萃取海参粗提物混悬液，有机相减压回收溶剂，获得含有两种海参烷皂苷类新化合物的提取物(简称提取物)；将提取物反复进行硅胶柱层析，收集含有两种海参烷皂苷类新化合物的组分后再进行反向柱层析分离纯化和/或制备型高效液相色谱分离纯化，得到Holotoxin E1和Holotoxin E2单体。
所述的提取方法为取鲜海参用匀浆机匀浆后用有机溶剂水溶液浸泡或回流提取，合并提取液，回收溶剂，获得海参粗提物。提取用有机溶剂水溶液为乙醇水溶液或甲醇水溶液，优选比例为75％-95％。将海参粗提物加水超声溶解，获得海参粗提物混悬液，依次用有机溶剂萃取海参粗提物混悬液，取正丁醇相减压回收溶剂，获得提取物。萃取用有机溶剂依次为石油醚、乙酸乙酯、正丁醇。
取正丁醇萃取物，选用体积比为10:1:0.1～10:7:0.1的乙酸乙酯-乙醇-水混合溶液为流动相进行硅胶柱层析梯度洗脱，TLC检验，合并相同组分，回收溶剂，得到1-1～1-7共7个部分。取含有Holotoxin E1的1-5组分用体积比为13:2.5:0.3的二氯甲烷-甲醇-水混合溶液为流动相，再次进行硅胶柱层析梯度洗脱，获得5-1～5-9共9个部分。取含有HolotoxinE1的5-9组分采用制备型高效液相色谱分离纯化，获得Holotoxin E1单体化合物，流动相选用乙腈-水混合溶液，优选体积比为38:62。取含有Holotoxin E2的1-6组分，选用甲醇-水混合溶液为流动相，优选体积比为10:1，进行ODS反相柱层析分离纯化，获得6-1～6-4共4个组分。取含有Holotoxin E2的6-3组分再次进行ODS反相柱层析分离纯化，收集含有HolotoxinE2的组分，减压回收溶剂，得到Holotoxin E2单体。或进一步采用制备型高效液相色谱法分离纯化，选用乙腈-水混合溶液为流动相，优选体积比为36:64，获得纯度更高的HolotoxinE2单体化合物。本发明制备方法得到的Holotoxin E1和Holotoxin E2单体纯度可以达到99％以上。
本发明获得的Holotoxin E1和Holotoxin E2单体可用于仿刺参及其相关产品的质量控制以及制备抗癌药物组合物及其他产品。
本发明利用MS和NMR等手段鉴定了分得的Holotoxin E1和Holotoxin E2的化学结构，Holotoxin E1和Holotoxin E2的化学结构以及结构鉴定过程及相关数据如下：
Holotoxin E1和Holotoxin E2的结构式
化合物1为白色粉末，高分辨液相色谱-质谱(HR-ESI-MS)分析得到m/z1239.7435[M+Na]+，提示化合物1的分子量为1216.7545，推测其分子式为C59H92O26。化合物1的13C NMR(C5D5N,150MHz)谱共给出59个碳信号，其中包括30个母核碳信号和29个糖基碳信号。结合DEPT 90°谱和DEPT 135°谱可知，低场区出现的δC 213.10和δC 178.79均为季碳信号，推测应为羰基碳信号。δC145.74，δC 144.20，δC 122.04，δC 110.68应为母核上双键的碳信号，其中δC 122.04应为母核环上双键中CH的碳信号，而δC 110.68应为母核链上双键中CH2的碳信号。δC 105.80，δC 105.65，δC 105.46，δC 105.37和δC 103.59推测为糖的端基碳信号。谱中共给出8个伯碳信号，分别为δC 60.96，δC 32.14，δC 28.88，δC 26.38，δC 24.29，δC 22.44，δC 18.20，δC 17.52，其中δC 60.69应为-OCH3的碳信号，而δC 18.20的存在预示糖基中可能含有奎诺糖。化合物1的1H NMR(C5D5N,600MHz)谱中，给出8个甲基氢信号，结合HSQC分析，δH1.13(s,H-19)，δH 1.32(s,H-21)，δH 1.58(s,H-27)，δH 1.04(s,H-28)，δH 1.19(s,H-29)和δH 1.07(s,H-30)这6个信号是海参烷骨架结构的特征甲基氢信号，而δH 1.62(d,J＝6.0Hz)和δH 3.75(s)应归属为糖链上的甲基氢信号。低场区δH 4.64(d,J＝7.2Hz)，δH 5.04(d,J＝7.2Hz)，δH4.74(d,J＝7.8Hz)，δH 5.21(d,J＝7.8Hz)和δH 4.92(d,J＝7.8Hz)可归属为糖的端基质子信号，这进一步证明化合物1中存在五分子糖，由于它们的J值均大于7.2Hz，可知糖的构型均为β-D构型。在HMBC谱中，δH 1.07(s,H-30)与δC 144.20(C-8)远程相关，这表明存在7(8)位的双键，受双键影响，30位的CH3信号向低场位移。一个木糖的端基质子信号δH4.64(d,J＝7.2Hz)与δC 89.18(C-3)相关，可以判断母核的C-3位连接一分子木糖。喹诺糖的端基质子信号δH 5.04(d,J＝7.2Hz)与连接母核的木糖的2位碳信号δC 83.76存在C-H远程相关，说明喹诺糖与木糖2位相连。另一个木糖的端基质子信号δH 4.74(d,J＝7.8Hz)与喹诺糖的4位碳信号δC86.07存在C-H远程相关，说明该木糖与喹诺糖4位相连。3-甲基葡萄糖的端基质子信号δH 5.21(d,J＝7.8Hz)与木糖的3位碳信号δC 87.58存在C-H远程相关，说明3-甲基葡萄糖连于木糖的3位。此外，葡萄糖的端基质子信号δH 4.92(d,J＝7.8Hz)与连接母核木糖的4位碳信号δC 77.44存在C-H远程相关，说明连接母核的木糖的4位连有一分子葡萄糖。结合1H-1H COSY，HSQC，HMBC和文献相互佐证，可以确定化合物1为3β-O-{(3-O-甲基-β-D-吡喃葡萄糖-(1→3)-β-D-吡喃木糖-(1→4)-β-D-吡喃喹诺糖-(1→2)-[β-D-吡喃葡萄糖-(1→4)]-β-D-吡喃木糖}-海参烷-7(8),25(26)-二烯-16-酮。经文献检索，未见该化合物的文献报道，确认其为新化合物，命名为Holotoxin E1。Holotoxin E1的13C NMR和1HNMR化学位移值见表1。
化合物2为白色粉末，高分辨液相色谱-质谱(HR-ESI-MS)分析得到m/z1418.0654[M+Na]+，提示化合物2的分子量为1395.0746，推测其分子式为C65H102O32。化合物2的13C NMR(C5D5N,150MHz)谱共给出65个碳信号，其中包括30个母核碳信号和35个糖基碳信号。结合DEPT 90°谱和DEPT 135°谱可知，低场区出现的δC 213.09和δC 178.79均为季碳信号，推测应为羰基碳信号。δC 145.75，δC 144.17，δC 122.00，δC 110.68应为母核上双键的碳信号，其中δC 122.00应为母核环上双键中CH的碳信号，而δC 110.68应为母核链上双键中CH2的碳信号。δC 106.04，δC 105.94，δC 105.65，δC 105.48，δC 105.10和δC 103.03为糖的端基碳信号。谱中共给出7个伯碳信号，分别为δC 60.97，δC 32.12，δC 28.96，δC 26.38，δC 24.24，δC22.45，δC 17.56，其中δC 60.69应为OCH3的碳信号。与化合物1的13C NMR(C5D5N,150MHz)谱对比，可以发现两种化合物的母核部分相同。化合物2的1H NMR(C5D5N,600MHz)谱中，给出7个甲基氢信号，结合HSQC分析，δH 1.09(s,H-19)，δH 1.31(s,H-21)，δH 1.57(s,H-27)，δH1.00(s,H-28)，δH 1.12(s,H-29)和δH 1.04(s,H-30)这6个信号是海参烷骨架结构的特征甲基氢信号，而δH 3.75(s)是糖链上的甲基氢信号。低场区δH 4.64(d,J＝7.2Hz)，δH 5.14(d,J＝7.8Hz)，δH 4.99(d,J＝7.8Hz)，δH 5.20(d,J＝7.8Hz)，δH 4.88(d,J＝7.8Hz)和δH 5.20(d,J＝7.8Hz)可归属为糖的端基质子信号，这进一步证明化合物2中存在六分子糖，由它们的J值均大于7.2Hz，可知糖的构型均为β-D构型。在HMBC谱中，有一个木糖的端基质子信号δH 4.64(d,J＝7.2Hz)与δC 89.18(C-3)相关，可以判断母核的C-3位连接一分子木糖。一分子葡萄糖的端基质子信号δH 5.14(d,J＝7.8Hz)与连接母核的木糖的2位碳信号δC 83.50存在C-H远程相关，说明该葡萄糖与木糖2位相连。另一个木糖的端基质子信号δH 4.99(d,J＝7.8Hz)与上述葡萄糖的4位碳信号δC 80.44存在C-H远程相关，说明该木糖与该葡萄糖4位相连。3-甲基葡萄糖的端基质子信号δH 5.20(d,J＝7.8Hz)与木糖的3位碳信号δC 87.69存在C-H远程相关，说明3-甲基葡萄糖连于木糖的3位。此外，一分子葡萄糖的端基质子信号δH4.88(d,J＝7.8Hz)与连接母核木糖的4位碳信号δC 77.73存在C-H远程相关，说明连接母核的木糖的4位连有一分子葡萄糖。一分子葡萄糖的端基质子信号δH 5.20(d,J＝7.8Hz)与上述葡萄糖的3位碳信号δC 88.23存在C-H远程相关，说明该葡萄糖连于上述葡萄糖的3位。结合1H-1H COSY，HSQC，HMBC和文献相互佐证，可以确定化合物2为3β-O-{2-O-[3-O-甲基-β-D-吡喃葡萄糖-(1→3)-β-D-吡喃木糖-(1→4)-β-D-吡喃葡萄糖]-4-O-[β-D-吡喃葡萄糖-(1→3)-β-D-吡喃葡萄糖]-β-D-吡喃木糖}-海参烷-7(8),25(26)-二烯-16-酮。经文献检索，未见该化合物的文献报道，确认其为新化合物，命名为Holotoxin E2。Holotoxin E2的13CNMR和1H NMR化学位移值见表1。
表1Holotoxin E1和Holotoxin E2的13C NMR和1H NMR数据(C5D5N,150/600MHz)
分析Holotoxin E1和Holotoxin E2两种化合物纯度的HPLC-ELSD色谱条件如下：
色谱柱-Agilent Eclipse XDB-C18(5μm,4.6×250mm)；流动相-甲醇-水(64:36)；流速-1mL/min；柱温-37.5℃；进样量-20μL；
ELSD检测器漂移管温度95℃；撞击状态-关闭；载气压力-0.45Mpa；雾化器流速-2.0L/min；放大倍数-1.0倍。
另外，本发明采用CCK-8法测定了Holotoxin E1和Holotoxin E2对两种癌细胞MDA-MB-231和HepG2的抑制生长活性。其中，Holotoxin E1对MDA-MB-231细胞和HepG2细胞均有显著的抑制活性；Holotoxin E2对MDA-MB-231细胞有显著的抑制活性。
附图说明
图1.Holotoxin E1的质谱图
图2.Holotoxin E1的1H-NMR图
图3.Holotoxin E1的13C-NMR图
图4.Holotoxin E1的DEPT90°,135°图
图5.Holotoxin E1的1H-1H COSY图
图6.Holotoxin E1的HMBC图
图7.Holotoxin E1的HMQC图
图8.Holotoxin E2的质谱图
图9.Holotoxin E2的1H-NMR图
图10.Holotoxin E2的13C-NMR图
图11.Holotoxin E2的DEPT90°,135°图
图12.Holotoxin E2的1H-1H COSY图
图13.Holotoxin E2的HMBC图
图14.Holotoxin E2的HMQC图
图15.Holotoxin E2浓度与MDA-MB-231细胞存活率关系曲线
图16. Holotoxin E1浓度与HepG2细胞存活率关系曲线
图17. Holotoxin E1浓度与MDA-MB-231细胞存活率关系曲线
具体实施方式
以下结合本发明的优选实施例进一步阐明本发明。应当理解，此处所描述的优选实施例仅用于说明和解释本发明，而不是用于限定本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的优选实施方法与材料仅作示范之用。
实施例1
取30kg鲜海参匀浆，加入95％乙醇回流提取3次，液料比均为10:1(W/V)，回流时间均为6h，滤去残渣，合并提取液，减压回收乙醇，然后加水1L，超声溶解，获得海参粗提物混悬液。分5次加入正丁醇萃取海参粗提物混悬液，正丁醇与混悬液体积比为2:1，合并正丁醇层，回收正丁醇，获得提取物Z(134g)。
取提取物Z，用乙酸乙酯-乙醇-水(体积比为10:1:0.1～10:7:0.1)进行硅胶柱层析梯度洗脱，TLC检验，合并相同组分，回收溶剂，得到Z1～Z7共7个部分。取含有HolotoxinE1的Z5组分用二氯甲烷-甲醇-水(体积比为13:2.5:0.3)再次进行硅胶柱层析梯度洗脱，TLC检验，合并相同组分，回收溶剂，获得Z5-1～Z5-9共9个部分。取含有Holotoxin E1的Z5-9组分，以乙腈-水为(体积比为38:62)流动相，采用制备型高效液相色谱法分离纯化，获得Holotoxin E1单体5.2mg。经HPLC-ELSD检测其纯度为99.4％。
取含有Holotoxin E2的Z6组分再次进行硅胶柱层析分离纯化，用二氯甲烷-甲醇-水(体积比为13:3.3:0.3)为流动相洗脱洗脱，获得Z6-1～Z6-4共4个组分。取含有HolotoxinE2的Z6-3组分以乙腈-水(体积比为36:64)为流动相，采用制备型高效液相色谱进行分离纯化，获得Holotoxin E2单体5.6mg。经HPLC-ELSD检测其纯度为99.5％。
实施例2
取20kg鲜海参匀浆，加入85％甲醇回流提取3次，液料比均为10:1(W/V)，回流时间均为6h，滤去残渣，合并提取液，减压回收甲醇，然后加水1L，超声溶解，获得海参粗提物混悬液。用正丁醇萃取海参粗提物混悬液共5次，正丁醇与混悬液体积比为2:1，合并正丁醇层，回收正丁醇，获得提取物Z(120g)。
取提取物Z，用乙酸乙酯-乙醇-水(体积比为10:1:0.1～10:7:0.1)进行硅胶柱层析梯度洗脱，TLC检验，合并相同组分，回收溶剂，得到Z1～Z7共7个部分。取含有HolotoxinE1的Z5组分，以甲醇-水(体积比为8:1)为流动相进行ODS反相柱层析分离纯化，获得Z5-1-Z5-7共7个部分。取含有Holotoxin E1的Z5-7组分，采用制备型高效液相色谱分离纯化，获得Holotoxin E1单体4.5mg。使用的流动相为乙腈-水(体积比为38:62)。经HPLC-ELSD检测其纯度为98.3％。
取含有Holotoxin E2的Z6组分，用丙酮-水(体积比为10:1)为流动相进行ODS反相柱层析分离纯化，获得Z6-1～Z6-5共5个部分。
取含有Holotoxin E2的Z6-4组分，采用制备型高效液相色谱分离纯化，获得Holotoxin E2单体3.6mg。使用的流动相为乙腈-水(体积比为36:64)。经HPLC-ELSD检测其纯度为98.6％。
实验实施例1
三阴性乳腺癌MDA-MB-231细胞用DMEM高糖培养基(其中含有10％胎牛血清(FBS)和1％青霉素-链霉素双抗溶液)于37℃、5％ CO2培养箱中培养。实验时使用对数生长期的细胞。将贴壁生长的肿瘤细胞用0.25％胰酶-EDTA溶液消化1min左右，然后用含血清培养基终止消化。离心，弃去上清液后用培养基重悬细胞制成细胞悬液，通过细胞计数控制细胞悬液终浓度为8×104个/mL。在96孔板中每孔接种100μL细胞悬液，于37℃、5％ CO2条件下培养24h，然后按不同浓度梯度分别加入Holotoxin E2的样品溶液(每个实验组设置3个复孔，以PBS缓冲溶液作为阴性对照组)，于37℃、5％ CO2培养箱中孵育24h，样品溶液浓度梯度为：0μM、5μM、10μM、15μM、20μM。
检测时弃去原培养基，向每孔中加入100μL含有10％ CCK-8检测试剂的培养基，在细胞培养箱中反应约1h。最后用酶标仪检测各孔在450nm处的光密度值(OD450nm)，计算细胞存活率公式如下：
结果表明，Holotoxin E2能够显著抑制MDA-MB-231细胞的活性，IC50值为10.35μmol/L。
实验实施例2
肝癌HepG2细胞用DMEM高糖培养基(其中含有10％胎牛血清(FBS)和1％青霉素-链霉素双抗溶液)于37℃、5％ CO2培养箱中培养。实验时使用对数生长期的细胞。将贴壁生长的肿瘤细胞用0.25％胰酶-EDTA溶液消化1min左右，然后用含血清培养基终止消化。离心，弃去上清液后用培养基重悬细胞制成细胞悬液，通过细胞计数控制细胞悬液终浓度为8×104个/mL。在96孔板中每孔接种100μL细胞悬液，于37℃、5％ CO2条件下培养24h，然后按不同浓度梯度分别加入Holotoxin E1的样品溶液(每个实验组设置3个复孔，以PBS缓冲溶液作为阴性对照组)，于37℃、5％ CO2培养箱中孵育24h，样品溶液浓度梯度为：0μM、1μM、2μM、4μM、6μM、15μM、20μM、25μM。
检测时弃去原培养基，向每孔中加入100μL含有10％ CCK-8检测试剂的培养基，在细胞培养箱中反应约1h。最后用酶标仪检测各孔在450nm处的光密度值(OD450nm)，计算细胞存活率公式如下：
结果表明，Holotoxin E1能够显著抑制HepG2细胞的活性，IC50值为14.26μmol/L。
实验实施例3
三阴性乳腺癌MDA-MB-231细胞用DMEM高糖培养基(其中含有10％胎牛血清(FBS)和1％青霉素-链霉素双抗溶液)于37℃、5％ CO2培养箱中培养。实验时使用对数生长期的细胞。将贴壁生长的肿瘤细胞用0.25％胰酶-EDTA溶液消化1min左右，然后用含血清培养基终止消化。离心，弃去上清液后用培养基重悬细胞制成细胞悬液，通过细胞计数控制细胞悬液终浓度为8×104个/mL。在96孔板中每孔接种100μL细胞悬液，于37℃、5％ CO2条件下培养24h，然后按不同浓度梯度分别加入Holotoxin E1的样品溶液(每个实验组设置3个复孔，以PBS缓冲溶液作为阴性对照组)，于37℃、5％ CO2培养箱中孵育24h，样品溶液浓度梯度为：0μM、1μM、2μM、4μM、6μM、8μM、10μM。
检测时弃去原培养基，向每孔中加入100μL含有10％ CCK-8检测试剂的培养基，在细胞培养箱中反应约1h。最后用酶标仪检测各孔在450nm处的光密度值(OD450nm)，计算细胞存活率公式如下：
结果表明，Holotoxin E1能够显著抑制MDA-MB-231细胞的活性，IC50值为2.386μmol/L。
上述实施例仅为本发明的优选实施例而已，并不能用于限制本发明的实质技术内容范围，本发明的实质技术内容是广义地定义于申请的权利要求范围中，任何相关技术人员完成的技术实体或方法，若是与申请的权利要求范围所定义的完全相同，也或是一种等效的修改、等同替换、改进等，均应包含于该权利要求范围之中。
Claims (10)
1.一种海参烷型新化合物，其特征在于所述海参烷型新
化合物分别为Holotoxin E1即3β-O-{[3-O-甲基-β-D-吡喃葡萄糖-(1→3)-β-D-吡喃木糖-(1→4)-β-D-吡喃喹诺糖-(1→2)]-[β-D-吡喃葡萄糖-(1→4)]-β-D-吡喃木糖}-海参烷-7(8),25(26)-二烯-16-酮和Holotoxin E2，即3β-O-{2-O-[3-O-甲基-β-D-吡喃葡萄糖-(1→3)-β-D-吡喃木糖-(1→4)-β-D-吡喃葡萄糖]-4-O-[β-D-吡喃葡萄糖-(1→3)-β-D-吡喃葡萄糖]-β-D-吡喃木糖}-海参烷-7(8),25(26)-二烯-16-酮。
2.权利要求1中所述的Holotoxin E1和Holotoxin E2的制备方法，其特征在于通过如下步骤获得这两种海参烷型皂苷类化合物：(1)将鲜海参用有机溶剂提取，滤过，回收溶剂，获得海参粗提物；(2)将提取物进行硅胶柱层析分离；(3)将硅胶柱层析分离得到的含有Holotoxin E1和Holotoxin E2的组分分别进行反相柱层析分离和/或制备型高效液相色谱分离纯化，得到Holotoxin E1和Holotoxin E2单体。
3.权利要求2所述的制备方法，其特征在于所述的提取用有机溶剂为乙醇水溶液或甲醇水溶液。
4.权利要求2所述的制备方法，其特征在于所述的硅胶柱层析洗脱剂为石油醚-乙酸乙酯-乙醇混合溶剂，梯度洗脱体积比为10:1:0.1～10:7:0.1或二氯甲烷-甲醇-水混合溶剂，梯度洗脱体积比为13:2.5:0.3～13:3.3:0.3。
5.权利要求2所述的制备方法，其特征在于所述的反相柱层析固定性为ODS，洗脱剂为甲醇-水混合溶剂，梯度洗脱体积比为10:1～4:1或丙酮-水混合溶剂，梯度洗脱体积比为12:1～2:1。
6.权利要求2所述的制备方法。其特征在于所述的制备型高效液相色谱流动相为乙腈-水混合溶剂，优选体积比为38:62～36:64。
7.权利要求1所述的Holotoxin E1和Holotoxin E2在制备抗癌药物中的应用。
8.权利要求1所述的Holotoxin E1和Holotoxin E2与医学上可接受的药用辅料组成的药物。
9.权利要求1所述的Holotoxin E1和Holotoxin E2在制备具有抗癌功能的保健品中的应用。
10.权利要求1所述的Holotoxin E1和Holotoxin E2与其他辅料组成的保健品。
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