【作者】 孙晓敏；

【导师】 贾贝西；

【作者基本信息】 郑州大学 ， 药学（专业学位）， 2020， 硕士

【摘要】 厚叶五味子,为五味子科(Schisandraceae)南五味子属(Kadsura)植物厚叶五味子Kadsuracoccinea(Lem.)A.C.Smith的根及茎藤,具有行气止痛,散癖通络等功效。厚叶五味子在我国南方各省广泛分布,常做民族药用于风湿痹痛,妇科痛症以及急慢性胃肠道疾病等,疗效显著,但缺乏深入研究。近三十多年来,国内外学者对厚叶五味子进行了化学成分及药理活性研究,发现木脂素和三萜类为其主要活性成分,具有抗氧化、抗病毒、抗肿瘤、抗炎、肝脏保护等作用。我们课题组前期的研究已经证实厚叶五味子95%醇提取物对大鼠佐剂性关节炎(AA)具有良好的治疗作用,但其抗炎活性组分及作用机制尚不明确。本文在文献综述的基础上,利用亲和超滤-液质联用技术,对厚叶五味子提取物中治疗炎症的COX-2潜在抑制剂进行了研究。(1)选取与炎症直接相关的作用靶点环氧合酶-2(COX-2),建立亲和超滤结合高效液相色谱和四级杆飞行时间质谱(AUF-HPLC-Q-TOF/MS)的分析方法,用于快速筛选和表征厚叶五味子中的COX-2配体。同时,设置空白组和灭活组以排除实验方法的非特异性干扰。通过该方法共推断了 21个化学成分,通过计算相对结合度,将大于25%的8个化合物作为COX-2特异性配体,分别是KadsulignanN(2)、KadsulignanL(4)、Neokadsuranin(5)、Kadsutherin(6)、Kadsuralignan F(8)、(+)-anwulignan(10)、Kadsuracoccinic acid A(14)、3,4-seco(24Z)-cychmrt-4(28),24-diene-3,26-dioic acid 3-methyl ester(19),其中化合物2、4、5和10不仅含量较大而且与COX-2有较好的结合亲和力。(2)通过AutoDock 4.2软件对8个COX-2特异性配体进行分子对接评价,来确定具有较好活性的潜在抑制剂,并通过结合力和结合模式了解可能的作用机理。将选择性抑制剂SC-558做对接,证明参数设置的合理性。从相互作用的结合能和抑制常数可以看出,KadsulignanN(2)、Kadsulignan L(4)、Neokadsuranin(5)和(+)-anwulignan(10)的抑制活性相对较好,且与COX-2活性位点的氨基酸残基相互作用,确定了这4个配体为潜在的抑制剂。(3)将厚叶五味子醇提物用石油醚萃取后得到石油醚部位,对其进行化学成分研究。实验采用硅胶柱色谱、凝胶Sephadex LH-20柱层析及重结晶等分离纯化方法,结合核磁碳谱(13C-NMR)和氢谱(1H-NMR)等波谱学技术,对分离得到的单体化合物进行结构鉴定,共确定了6个化合物的结构,其中木脂素类化合物有 2 个:(+)-anwulignan(Kc-1)和 Kadsulignan N(Kc-6);三萜类化合物有 3 个 Kadsuracoccinic acid B(Kc-2)、Nigranoic acid(Kc-4)和 Manwuweizic acid(Kc-5);甾体类化合物有 1 个:β-sitosterol(Kc-3)。化合物 Kc-1 和 Kc-6为实验确定的COX-2潜在抑制剂,又通过体外COX-2抑制剂筛选实验,来验证其抑制活性。研究发现,Kc-1和Kc-6的IC50为3.80和1808.13μg/mL(相当于11.52和4204.95μM)。该试剂盒中的阳性对照塞来昔布的IC50为0.45μM。更多还原

【Abstract】 Kadsura coccinea(Lem.)A.C.Smith is an evergreen liana belings to the Kadsura of the Schisandraceae family,which roots and stems are widely used for medicinal purposes,with the functions of promoting Qi,relieving pain,dispersing blood stasis,and dredging collaterals.K.coccinea is widely distributed in the southern provinces of China,which often used as the national medicine for rheumatism and pain,gynecological pain,and acute and chronic gastrointestinal diseases.Its efficacy is significant,but lack of in-depth research.In the past three decades,scholars at home and abroad have studied the chemical constituents and pharmacological activities of K.coccinea,and found that lignans and triterpenes are the main active ingredients,which have antioxidant,antiviral,antitumor,anti-inflammatory,liver protection effects.Our previous research had confirmed that 95%alcohol extract of K.coccinea had good therapeutic effect on the adjuvant arthritis(AA)of rats.However,the effective ingredients and mechanism of action of the plant for treating inflammation have not yet been clarified.Based on the literature review,this paper investigated the potential inhibitors of COX-2 for treating inflammation from K.coccinea extract by affinity ultrafiltration coupled with high-performance liquid chromatography and quadruple time-of-flight mass spectrometry(AUF-HPLC-Q-TOF/MS).(1)The target COX-2 was selected directly related to the inflammation and an analytical method based on AUF-HPLC-Q-TOF/MS was established for rapidly screening and characterizing COX-2 ligands from K.coccinea.Meanwhile,the blank group and the inactivated group were set up to exclude non-specific interference of the experimental method.As a result,a total of 21 chemical compositions were inferred,and 8 compounds were considered as COX-2 specific ligands,which relative binding degree more than 25%,namely Kadsulignan N(2),Kadsulignan L(4),N eokadsurani n(5),Kadsutherin(6),Kadsuralignan F(8),(+)-anwulignan(10),Kadsuracoccinic acid A(14),3,4-seco(24Z)-cychmrt-4(28),24-diene-3,26-dioic acid 3-methyl ester(19),among which compounds 2,4,5 and 10 not only have a large content but also have good binding affinity with COX-2.(2)The molecular docking evaluation of 8 COX-2 specific ligands was performed by AutoDock 4.2 software to identify potential inhibitors with good activities,and to understand the possible mechanism of action through binding force and binding mode.The selective inhibitor SC-558 was docked to prove the reasonableness of the parameter setting.The results of binding energy and inhibition constant shown that,Kadsulignan N(2),Kadsulignan L(4),Neokadsuranin(5),and(+)-anwulignan(10)have relatively better inhibitory activity,and interact with amino acid residues at the active site of COX-2,which identified as potential inhibitors.(3)After extracting the alcohol extract of K.coccinea with petroleum ether,the petroleum ether fraction was obtained,which chemical composition was studied.In the experiment,separation and purification methods such as silica gel column chromatography,Sephadex LH-20,and recrystallization,combining nuclear magnetic carbon spectrum(13C-NMR)and hydrogen spectrum(1H-NMR)spectroscopy techniques,the structures of 6 compounds were isolated and identified,of which were two lignans:(+)-anwulignan(Kc-1)and Kadsulignan N(Kc-6),three terpenoids:Kadsuracoccinic acid B(Kc-2),Nigranoic acid(Kc-4)and Manwuweizic acid(Kc-5),one steroidal compound:β-sitosterol(Kc-3).Compounds Kc-1 and Kc-6 were experimentally identified COX-2 potential inhibitors,and their inhibitory activity were verified by COX-2 inhibitor screening experiments in vitro,which shown their IC50 were 3.80 and 1808.13μg/mL(equivalent to 11.52 and 4204.95μM).The IC50 of the positive control celecoxib in the kit is 0.45μM.更多还原