【作者】 杨洁；

【导师】 李绪文；

【作者基本信息】 吉林大学 ， 分析化学， 2016， 博士

【摘要】 人参皂苷是五加科人参属植物人参、西洋参、三七等的主要功效成分,具有中枢神经调节、免疫调节、心脑血管保护、抗肿瘤、抗衰老等多种药理作用。人参皂苷分为原人参二醇(PPD)型、原人参三醇(PPT)型、奥克梯隆(Ocotillol)型、齐墩果烷(Oleanane)型及其他类型人参皂苷。随着研究的深入,人们发现常见人参皂苷降解产生的次级人参皂苷及苷元具有更强的药理作用,如人参皂苷Rg3、Rh2及其苷元PPD具有很强的抗癌活性,其中人参皂苷Rg3已作为抗癌药物上市销售,Rh2及PPD已进入临床研究阶段。近年来关于C-20位不连糖基的次级人参皂苷(人参皂苷Rg3、Rh2、Rg2和Rh1)及其苷元(PPD和PPT)体内、外的代谢研究表明C-20和C-24环氧的奥克梯隆型代谢产物可能是其体内发挥作用的真正有效成分。为了开展上述关于次级人参皂苷及其苷元代谢过程及代谢产物的研究,揭示奥克梯隆型人参皂苷的生物活性,寻找人参皂苷在体内发挥作用的真正有效成分,本文在前期已完成C-20位不连糖基的次级人参皂苷及其苷元制备方法的基础上,对12种上述人参皂苷及其苷元(20(S)-/20(R)-人参皂苷Rg3、20(S)-/20(R)-人参皂苷Rh2、20(S)-/20(R)-人参皂苷Rg2、20(S)-/20(R)-人参皂苷Rh1,20(S)-/20(R)-PPD、20(S)-/20(R)-PPT)进行氧化修饰,合成了相应的奥克梯隆型人参皂苷并对氧化产物进行了抗肿瘤、抗氧化生物活性的研究。首先本文以过氧化氢为氧化剂对12种C-20位不连糖基的次级人参皂苷及其苷元进行了氧化修饰,得到的氧化产物经硅胶、ODS柱层析及制备型液相色谱(PLC)等多种色谱分离手段进行纯化,共分得了40个单体化合物,利用MS、1D、2D-NMR等波谱学手段鉴定了其化学结构,其中24个为奥克梯隆型氧化产物,16个为非奥克梯隆型侧链环合的氧化产物。在上述化合物中,15个为新化合物,它们分别是20(S)-达玛-20,25-环氧-3β,12β,24α-三醇,20(R)-达玛-20,25-环氧-3β,12β,24α-三醇,(20R,24R)-3-O-甲酰-达玛-20,24-环氧-3β,12β,25-三醇,(20R,24S)-3-O-甲酰-达玛-20,24-环氧-3β,12β,25-三醇,20(R)-3-O-β-D-吡喃葡萄糖--20,25--3β,12β,24β-20(R)-3-O-β-D---20,25--3β,12β,24α-三醇,20(S)-3-O-[β-D-吡喃葡萄糖-(1→2)-β-D-吡喃葡萄糖]-达玛-20,25-环氧-3β,12β,24α-三醇,20(R)-3-O-[β-D-吡喃葡萄糖-(1→2)-β-D-吡喃葡萄糖]-达玛-12-酮-20,24-内酯-3β-醇,20(S)-达玛-20,24-内酯-3β,6α,12β-三醇,(20S,24R)-达玛-22-酮-20,24-环氧-3β,6α,12β,25-四醇,20(R)-达玛-20,24-内酯-3β,6α,12β-三醇,(20R,24R)-达玛-20,24-环氧-3β,6α,12β,25-四醇,(20R,24S)-达玛-20,24-环氧-3β,6α,12β,25-四醇,(20R,24R)-6-O-β-D-吡喃葡萄糖-达玛-20,24-环氧-3β,6α,12β,25-四醇,(20R,24S)-6-O-β-D-吡喃葡萄糖-达玛-20,24-环氧-3β,6α,12β,25-四醇,其中后4个化合物为新的奥克梯隆型人参皂苷。通过分析产物C-20及C-24位的立体构型,我们发现在过氧化氢为氧化剂的条件下,上述次级人参皂苷及其苷元的C-20位立体构型不发生改变,并且均以SN1的反应机理同时产生C-24(S)和C-24(R)两个立体异构的奥克梯隆型产物。13C NMR数据(δC,pyridine)显示,当奥克梯隆型氧化产物的C-20和C-24位立体构型不同时,二者的化学位移值存在如下规律:C-20为S构型,C-24为R构型时,核磁数据分别为δC86和δC85;C-20为R,C-24为S构型时,分别为δC86和δC87;C-20和C-24均为S构型时,分别为δC87和δC88;C-20和C-24均为R构型时,分别为δC86和δC86。以上碳谱数据规律为奥克梯隆型化合物立体结构的研究提供了波谱学依据。接着本文采用MTT法对上述一系列次级人参皂苷及其苷元的氧化产物进行了体外抗肿瘤活性研究,这些产物对He La和Hep G2肿瘤细胞均具有一定的抑制增殖作用,其中11个原人参二醇组皂苷及其苷元的氧化产物对He La和Hep G2细胞表现出更好的抑制作用(IC50<40μg/m L),而且拟人参皂苷元PDQ抑制He La肿瘤细胞的增殖作用最强(IC50=2.18μg/m L)。从实验结果可知,原人参二醇组人参皂苷及其苷元的氧化产物比原人参三醇组人参皂苷及其苷元的氧化产物抗肿瘤的活性更强,同时糖基数目和侧链C-20和C-24的立体构型对活性均有一定的影响。为了探究拟人参皂苷元PDQ的抗肿瘤作用机制,本文还进行了细胞凋亡形态学、Caspase酶活力及免疫印迹的研究。实验结果表明拟人参皂苷元PDQ具有诱导He La细胞凋亡的作用,并且激活Caspase-8依赖的细胞表面死亡受体介导的外源性凋亡通路和Caspase-9依赖的线粒体介导的内源性凋亡通路是引发He La细胞凋亡的原因。过量自由基能够引起DNA损伤,导致DNA携带的遗传信息错误复制;可进攻细胞膜上的不饱和脂肪酸,使细胞膜的结构和功能遭到破坏,从而影响信号的传导;还可损伤DNA的修复酶导致发生错误修复致使基因突变等引发癌症。为了考察这些奥克梯隆型氧化产物是否具有清除自由基的作用,本文选择其中12个具有较强抗肿瘤作用的氧化产物,采用3种化学模拟体系和2种化学模拟生物体系研究了它们的抗氧化能力。结果这些化合物在3种化学模拟体系中清除自由基的能力较弱,但在2种化学模拟生物体系中表现出了一定的抑制DNA损伤作用,而且对由羟自由基引发的DNA氧化损伤的抑制作用更加显著。羟自由基是生物体内存在的一种非常活泼的自由基,对由该自由基引起的DNA氧化损伤具有抑制效果,提示这些氧化产物可作为抗氧化剂应用于肿瘤的预防与治疗。鉴于拟人参皂苷PDQ及其24(R)-异构体在抑制肿瘤细胞增殖和抑制羟基自由基对DNA损伤中的良好活性,本文采用单因素分析法结合HPLC-ELSD检测手段,对拟人参皂苷PDQ及其异构体的合成条件进行了优化,确立了最佳反应条件。当原料起始浓度为4.6 mg/m L,氧化剂为间氯过氧苯甲酸(m-CPBA),氧化剂与原料的用量比(摩尔比)为9:1,反应温度为25℃,反应时间为1.0 h时,拟人参皂苷元PDQ及其异构体的产率最高。综上所述,本文在制备能够产生奥克梯隆型氧化产物的包括R/S异构体在内的12种C-20位不连糖基的次级人参皂苷的基础上,首次全面地合成了其对应的全部24种奥克梯隆型氧化产物,鉴定了其化学结构并探讨了其抗肿瘤及抗氧化生物活性,研究了部分抗肿瘤作用机制,并分析了活性与结构之间的关系。本文的研究结果为进一步开展人参皂苷的药代动力学及药效物质基础研究,揭示人参皂苷的代谢产物及有效成分,寻找新的具有更好生物医药用途的人参皂苷及其衍提供了科学依据。更多还原

【Abstract】 Ginsenosides are the mainly effective components in the plants of Araliaceae,Panax genus, Panax ginseng C. A. Meyer, Panax quinquefolium L. and Panax notoginseng(Burk.) F.H.Chen. It has been reported that ginsenosides have many kinds of pharmacological effects, such as central nerve regulation, immune regulation,cardiovascular protection, antitumor, antiaging, et al. Ginsenosides are classified into five types, i.e. protopanaxatriol type, protopanoxatriol type, ocotillol type, oleanane type and other types.With the research in-depth, it was found that the degraded secondary ginsenosides and its aglycone possessed better pharmacological effects, especially ginsenosides Rg3, Rh2 and PPD showed stronger antitumor activity. Ginsenoside Rg3 as anti-cancer drug has been on listed sale, and ginsenosides Rh2 and PPD have entered the stage of clinical research.In recent years, the metabolic researches of the secondary ginsenosides without glycosyl group at C-20(ginsenoside Rg3, Rh2, Rg2 and Rh1) and their aglycones(PPD and PPT) in vitro or vivo hint that their corresponding ocotillol type ginsenosides or aglycones maybe the active ingredients in vivo.In order to carry out the above research on metabolism process and metabolic products of secondary ginsenosides and their aglycones, reveal biological activities of ocotillol type ginsenosides, and explore the truly effective ingredients of ginsenosides in vivo, we oxidated twelve kinds of above-mentioned ginsenosides and aglycone(20(S)-/20(R)-ginsenoside Rg3, 20(S)-/20(R)-ginsenoside Rh2, 20(S)-/20(R)-ginsenoside Rg2, 20(S)-/20(R)-ginsenoside Rh1, 20(S)-/20(R)-PPD, 20(S)-/20(R)-PPT)which were provided by the preliminary preparations, synthesised the corresponding ocotillol type ginsenosides, and then evaluated the antitumor and antioxidant activities of these oxidative products.Firstly in this paper, we oxidated the twelve secondary ginsenosides and theirVI aglycones with hydrogen peroxide as oxidant. The oxidative products were isolated and purified by using the methods of column chromatography and preparative liquid chromatography. Forty compounds were isolated in total, and their chemical structure were identified by the means of MS, 1D and 2D-NMR spectroscopies. Among them,twenty-four compounds were confirmed as ocotillol type ginsenosids, and sixteen oxidative products of non-ocotillol type ginsenosids with the cyclized structure in the side chain. Fifteen new compounds were identified as20(S)-dammar-20,25-epoxy-3β,12β,24α-triol, 20(R)-dammar-20,25-epoxy-3β,12β,24α-triol,(20R,24R)-3-O-formyl-dammar-20,24-epoxy-3β,25-diol,(20R,24S)-3-Oformyl-dammar-20,24-epoxy-3β,25-diol, 20(R)-3-O-β-D-glucopyranosyl-dammar-20,25-epoxy-3β,12β,24β-triol, 20(R)-3-O-β-D-glucopyranosyl-dammar-20,25-epoxy-3β,12β,24α-triol, 20(S)-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-dammar-20,25-epoxy-3β,12β,24α-triol, 20(R)-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyr-anosyl]-dammar-12-one-20,24-lactone-3β-ol], 20(S)-dammar-20,24-lactone-3β,6α,12β-triol,(20S,24R)-dammar-22-one-20,24-epoxy-3β,6α,12β,25-tetraol,(20R,24R)-dam-mar-20,24-epoxy-3β,6α,12β,25-tetraol, 20(R)-dammar-20,24-lactone-3β,6α,12β- triol,(20R,24S)-dammar-20,24-epoxy-3β,6α,12β,25-tetraol,(20R,24R)-6-O-β-D-glucopyra-nosyl-dammar-20,24-epoxy-3β,6α,12β,25-tetraol,(20R,24S)-6-O-β-D-glucopyranosy-l-dammar-20,24-epoxy-3β,6α,12β,25-tetraol, respectively. Moreover, the last four products are new ocotillol type ginsenosides.Under the reaction condition, the configuration of each starting material at C-20 did not be changed. According to the SN1 reaction mechanism, they got a pair of ocotillol type isomers at C-24.By the analysis of 13 C NMR data in pyridine, the chemical shifts at C-20 and C-24 of all the four ocotillol type ginsenosides are as follows: 20 S,24R(δC86, δC85);20S,24S(δC87, δC88); 20 R,24R(δC86, δC86); 20 R,24S(δC86, δC87). The above carbon spectrum data can provide the spectroscopic evidence for the analysis of stereochemical structure of ocotillol type ginsenosides.This paper for the first time evaluated the antitumor activity of a series of oxidative products in vitro by MTT, and found all of them could inhibit cellsVII proliferation to a certain extent. Eleven compounds of them obtained from protopanaxatriol type ginsenosides and aglycones showed better inhibitory effects against He La and Hep G2 cells(IC50<40 μg/m L). The compound PDQ had the strongest inhibition on the proliferation of He La cells(IC50=2.18 μg/m L). The results showed the oxidative products from protopanaxadiol type ginsenosides have better effects than those from protopanaxatriol type ginsenosides. At the same time the number of glycosyl and the configuration at C-20 and C-24 also have certain influence on antitumor activity.To explore the antitumor mechanism of the compound PDQ, this paper investigated the cell morphology, Caspase activity and western blot analysis. The results suggested PDQ can induce He La cells apoptosis, and activated Caspase-8-dependent the extrinsic apoptotic pathways mediated by cell surface death receptor and Caspase-9-dependent intrinsic apoptosis pathway mediated by mitochondria.As we known, excess free radicals can cause cancer by various pathways including causing DNA damage to lead gene replicated by error, attacking unsaturated fatty acid on the cell membrane to destroy its structure and function and further affect signal transduction, and damaging DNA repair enzymes to gene mutations. In this paper, we took antioxidant model in vitro including three kinds of free radicals clearance and two kinds of inhibiting DNA damage experiments to evaluate antioxidant capacity of twelve oxidative products with better antitumor activities. The results showed that the tested compounds had weak abilities in terms of clearing free radicals in vitro, but possessed the abilities of inhibiting hydroxyl radical in DNA damage experiments. Hydroxyl radical is a kind of high reactive free radicals in vivo,thus these oxidative products are worthy of further research and application to the prevention and treatment for tumor as antioxidant reagent.Given the compound PDQ and its isomer great activities on antitumor and antioxidation, we studied its synthetic conditions by univariate analysis method combined with HPLC-ELSD detection, and established the optimum reaction conditions. When starting material initial concentration is 4.6 mg/m L, m-CPBA isused as oxidant, molar proportion of oxidant and starting material is 9:1, reaction temperature is set at 25℃, reaction time is 1.0 h, the yield of PDQ and its isomer is highest.On the basis of the preparations of twelve kinds of 20(S/R)-secondary ginsenosides without glycosyl at C-20 and aglycones, this article firstly comprehensively synthesized their corresponding ocotillol type ginsenosides,identified their chemical structure, discussed a portion of antitumor mechanism, and analyzed the relationships between the activities and structures. The results of this paper provide new scientific basis for the research of pharmacokinetics and efficacy material base of ginsenosides, reveal the metabolite and active ingredients of ginsenosides, and develop new ginsenosides and their derivatives with better medicine applications.更多还原