本文選題：民族醫(yī)藥 + 民族藥用植物��； 參考：《中央民族大學(xué)》2016年博士論文

【摘要】：由于缺乏抗腫瘤特效藥物,惡性腫瘤嚴(yán)重威脅著人類(lèi)尤其是少數(shù)民族地區(qū)人們的生命健康和社會(huì)的經(jīng)濟(jì)發(fā)展,成為阻礙我國(guó)全面建設(shè)小康社會(huì)的一大障礙。少數(shù)民族醫(yī)藥學(xué)是我國(guó)少數(shù)民族幾千年傳統(tǒng)文化知識(shí)的結(jié)晶,它們不但在歷史上為本民族的生存和繁衍昌盛做出過(guò)重大貢獻(xiàn),時(shí)至今日仍為本民族和其他民族防病治病和衛(wèi)生保健事業(yè)發(fā)揮重要作用。尋找抗腫瘤特效藥的希望就寄托在經(jīng)過(guò)長(zhǎng)期研究和臨床實(shí)踐及藥理驗(yàn)證,對(duì)治療腫瘤有著巨大優(yōu)勢(shì)和潛力的民族醫(yī)藥上。本研究是在對(duì)民族傳統(tǒng)醫(yī)藥知識(shí)文化的認(rèn)知和尊重的基礎(chǔ)上,以資源豐富、具有抗腫瘤潛力的民族藥物及民族藥用植物為研究對(duì)象,以DNA G-四鏈體為抗腫瘤民族藥物研發(fā)的新靶點(diǎn),運(yùn)用民族學(xué)、民族植物學(xué)、生物學(xué)、天然有機(jī)化學(xué)、儀器分析等多學(xué)科知識(shí),從民族藥物中尋找高效、低毒的抗腫瘤新型靶向制劑。本文開(kāi)展了傳統(tǒng)民族醫(yī)藥有效單體成分靶向結(jié)合G-四鏈體抗腫瘤活性研究,并基于上一部分實(shí)驗(yàn)材料和實(shí)驗(yàn)儀器適用范圍的摸索,建立了CD結(jié)合NMR高效、快速的篩選民族藥用植物中靶向G-四鏈體活性小分子化合物的方法。以畬藥紫玉蘭(Magnolia liliiflora Desr.)為民族植物學(xué)研究對(duì)象,CD結(jié)合NMR的方法靶向G-四鏈體追蹤紫玉蘭活性部位,進(jìn)而分離得到具有抗腫瘤潛力的小分子化合物。研究結(jié)果不但能為抗腫瘤特效藥物研發(fā)提供參考,而且能為傳統(tǒng)民族醫(yī)藥的現(xiàn)代開(kāi)發(fā)提供新思路,同時(shí)建立的活性篩選方法切實(shí)可行,能高效、快捷的篩選民族藥物復(fù)雜體系中結(jié)構(gòu)已知甚至是未知的活性化合物。研究思路和方法值得推廣應(yīng)用于其他民族醫(yī)藥的研究與開(kāi)發(fā)。具體研究如下：一、開(kāi)展了傳統(tǒng)民族醫(yī)藥有效單體成分靶向結(jié)合G-四鏈體抗腫瘤活性研究。通過(guò)查閱民族醫(yī)藥文獻(xiàn)發(fā)現(xiàn),藏藥掌葉大黃(Rheum palmatum L.)、維藥茜草(Rubia cordifolia L.)及新疆紫草[Arnebia euchroma (Royle) Johnst.]、傣藥木蓮(Manglietia fordiana Oliv.)均被當(dāng)?shù)厝擞脕?lái)清熱、解毒,其有效單體成分蘆薈大黃素、茜草素、紫草素、厚樸酚、和厚樸酚這些化合物在細(xì)胞水平上均具有一定抗腫瘤活性,但不明確是否作用于G-四鏈體達(dá)到抗腫瘤活性。小分子化合物空間上具有平面結(jié)構(gòu),理論上更容易靶向結(jié)合G-四鏈體。因此選擇具有平面結(jié)構(gòu)的上述民族醫(yī)藥有效單體成分為研究對(duì)象,研究靶向結(jié)合G-四鏈體活性,具體研究結(jié)果和結(jié)論如下：1、c-myc 2345序列形成了平行結(jié)構(gòu)構(gòu)型的G-四鏈體,可以用作民族藥用植物與G-四鏈體相互作用研究的分子靶點(diǎn)。藏藥掌葉大黃有效單體成分蘆薈大黃素通過(guò)π-π堆積結(jié)合模式作用于c-myc 2345 G-四鏈體平面,蘆薈大黃素與G-四鏈體以1：1形成絡(luò)合物。研究結(jié)果表明藏藥掌葉大黃有效單體成分蘆薈大黃素具有靶向G-四鏈體抗腫瘤作用的潛力。2、維藥茜草有效單體成分茜草素通過(guò)π-π堆積結(jié)合模式作用于c-myc 2345 G-四鏈體平面,茜草素與G-四鏈體以2：1形成絡(luò)合物；維藥新疆紫草有效單體成分紫草素能靶向結(jié)合c-myc 2345 G-四鏈體,形成2：1絡(luò)合物,并通過(guò)溝槽結(jié)合和3’端的π-π堆積結(jié)合模式作用于c-myc 2345 G-四鏈體平面。研究結(jié)果表明維藥茜草、新疆紫草有效單體成分茜草素、紫草素具有靶向G-四鏈體抗腫瘤作用的潛力。3、傣藥木蓮有效單體成分厚樸酚不能靶向結(jié)合c-myc 2345 G-四鏈體達(dá)到抗腫瘤作用,而其同分異構(gòu)體和厚樸酚能夠通過(guò)3’端π-π堆積和溝槽結(jié)合模式作用于c-myc 2345 G-四鏈體,和厚樸酚與G-四鏈體以2：1形成絡(luò)合物。研究結(jié)果表明傣藥木蓮有效單體成分厚樸酚無(wú)靶向G-四鏈體抗腫瘤活性,和厚樸酚具有靶向G-四鏈體抗腫瘤作用的潛力。4、紫外吸收光譜和熒光光譜法是研究小分子與生物大分子相互作用的一種簡(jiǎn)單、常用方法,但其對(duì)小分子化合物的紫外吸收光譜性質(zhì)要求較高,若小分子化合物與G-四鏈體的紫外吸收最大值波長(zhǎng)處重合,則無(wú)法應(yīng)用該方法進(jìn)行實(shí)驗(yàn)。同時(shí),紫外吸收光譜和熒光光譜法更善于發(fā)現(xiàn)π-π堆積結(jié)合模式的小分子化合物,NMR方法則能夠發(fā)現(xiàn)弱結(jié)合及多種結(jié)合模式(溝槽結(jié)合、堆積結(jié)合)的小分子化合物。二、基于上一部分實(shí)驗(yàn)材料和實(shí)驗(yàn)儀器適用范圍的摸索,建立了CD結(jié)合NMR高效、快速的篩選民族藥用植物中靶向G-四鏈體活性小分子化合物的方法。以畬藥紫玉蘭(Magnolia liliiflora Desr.)為民族植物學(xué)研究對(duì)象,CD結(jié)合NMR的方法靶向G-四鏈體追蹤紫玉蘭活性部位,進(jìn)而分離得到具有抗腫瘤潛力的小分子化合物,具體研究結(jié)果總結(jié)如下：1、CD變溫實(shí)驗(yàn)最終確定95%乙醇為最適宜提取溶劑,其乙酸乙酯萃取層為分離的重點(diǎn)極性部位,進(jìn)一步以石油醚-丙酮溶劑體系進(jìn)行梯度洗脫后得到5個(gè)餾分,NMR實(shí)驗(yàn)確定餾分Fr.2(5：1)和Fr.3(3：1)為重點(diǎn)分離部位。2、根據(jù)靶向G-四鏈體追蹤結(jié)果,利用常規(guī)硅膠柱層析(Silica gelCC),凝膠柱層析(Sephadex LH-20 CC)以及制備薄層層析(PTLC),共分離得到31個(gè)化合物。進(jìn)一步利用UV、R、EI-MS、1-NMR、 13C-NMR以及2D-NMR (1H-1H COSY、HMQC、HMBC)等現(xiàn)代波譜技術(shù),結(jié)合文獻(xiàn)數(shù)據(jù)對(duì)比確定了31個(gè)化合物的結(jié)構(gòu),其中4個(gè)為新的木脂素化合物：liliflorin B (1), liliflorin C (2), liliflorin D (3), liliflorin E(4)�；衔�5-22為已知的木脂素化合物,化合物23-27為已知的苯環(huán)化合物,化合物28-31為已知的倍半萜化合物。考慮到化合物的種類(lèi)、新穎程度以及化合物的質(zhì)量,選擇了8個(gè)化合物為研究對(duì)象,利用NMR開(kāi)展了其與G-四鏈體相互作用初步研究,發(fā)現(xiàn)化合物1,2,3,12,22具有靶向結(jié)合G-四鏈體的能力。3、化合物12(紫玉蘭素A)質(zhì)量足夠,活性好,因此選擇紫玉蘭素A為與G-四鏈體相互作用的深入研究對(duì)象。研究結(jié)果表明紫玉蘭素A(4.0μM)在K+緩沖液中可以將人端粒G-四鏈體的解鏈溫度提高至69.93℃(△Tm=3.22℃),且不與DNA其他二級(jí)結(jié)構(gòu)(雙鏈DNA ds26、DNA發(fā)卡結(jié)構(gòu)F10T、DNAG-四鏈體c-kit)相互作用,能夠特異性以溝槽模式結(jié)合人端粒G-四鏈體,形成摩爾比1：1絡(luò)合物。靶向G-四鏈體,從畬藥紫玉蘭中分離提取得到的紫玉蘭素A具有靶向G-四鏈體抗腫瘤作用的潛力。論文的研究結(jié)果為傳統(tǒng)民族醫(yī)藥靶向G-四鏈體抗腫瘤活性化合物的發(fā)現(xiàn)奠定物質(zhì)基礎(chǔ),為高效、快速篩選民族藥用植物靶向G-四鏈體的抗腫瘤活性小分子化合物提供了基礎(chǔ)科學(xué)數(shù)據(jù)和技術(shù)支撐。論文所建立的研究方法能夠應(yīng)用于民族傳統(tǒng)藥物的二次開(kāi)發(fā),促進(jìn)民族醫(yī)藥的現(xiàn)代利用,給少數(shù)民族人民帶來(lái)經(jīng)濟(jì)效益,更好的促進(jìn)我國(guó)民族地區(qū)經(jīng)濟(jì)和社會(huì)的發(fā)展。
[Abstract]:Due to the lack of antitumor drugs, malignant tumors seriously threaten people's life and health and social economic development, especially in ethnic minority areas. It has become a major obstacle to the overall construction of a well-off society in our country. Minority medical and pharmacy are the crystallization of traditional cultural knowledge of the ethnic minorities in China for thousands of years, and they are not only in history. It has made great contributions to the survival and prosperity of the nation, and today it still plays an important role in the prevention and treatment of diseases and health care for the nation and other nationalities. The hope of finding antitumor drugs is placed on the people who have a great advantage and potential in the treatment of cancer through long-term research, clinical practice and pharmacological validation. On the basis of the cognition and respect to the knowledge and culture of traditional medicine, this study is based on the rich resources, the national medicinal plants and ethnic medicinal plants with anti tumor potential, and the DNA G- four chain body is a new target for anti tumor national drug research and development, and it uses ethnology, ethnobotany, biology, and natural organic chemistry. In this paper, the anti-tumor activity of the effective single body component targeting G- four chain body was studied. Based on the exploration of the application range of the previous experimental materials and experimental instruments, the CD combined with NMR was established. A method for screening the active small molecules of G- four chain body in ethnic medicinal plants. Magnolia liliiflora Desr. was used as the object of Ethnological Study. CD combined with NMR was used to target G- four chain body to trace the active part of Magnolia, and then a small molecular compound with antitumor potential was obtained. It can not only provide reference for the research and development of antitumor drugs, but also provide new ideas for the modern development of traditional national medicine. At the same time, the active screening method is practical and effective. It can efficiently and quickly screen the known or unknown active compounds in the complex system of national drugs. The research and development of other ethnic medicine were studied as follows: first, the anti-tumor activity of the effective monomer targeting G- four chain body of traditional national medicine was studied. Through consulting the national medical literature, the Tibetan medicine Rheum palmatum L., the Rubia cordifolia L. and the euchroma (Royle) of Xinjiang purple grass [Arnebia (Royle) were found. Johnst.], Manglietia fordiana Oliv. is used by local people to remove heat and detoxify. The effective monomer components of aloe emodin, alizarin, herb, magnolol, and honokiol all have certain antitumor activity on the cell level, but it is not clear whether the G- four chain body can achieve the antitumor activity. Small molecule The compound has a plane structure in space, and it is more likely to target the binding of G- four chain body in theory. Therefore, to select the effective monomer components of the national medicine of the ethnic group as the research object, the target is combined with the activity of the G- four chain body. The specific results and conclusions are as follows: 1, c-myc 2345 sequence formed a parallel structure configuration of the G- four chain body, As a molecular target for the study of the interaction of ethnic medicinal plants with the G- four chain body. The effective monomeric aloe emodin of the palmatum palmatum of the palmatum palmatum of the Tibetan medicine is acted on the plane of the body of the c-myc 2345 G- four chain by the pion accumulation binding mode. The complex of the aloe emodin and the G- four chain body is formed by 1:1. The aloe emodin has the potential of anti tumor effect of the target G- four chain body.2, and the active monomer of the radix alizaris alizarin has the effect on the c-myc 2345 G- four chain body plane through the pion pion accumulation mode, and the alizarin and G- four chain body form the complex with 2:1, and the active monomer of the herb of Xinjiang purple herb can target c-myc 2345 G- four. The chain body forms the 2:1 complex and acts on the c-myc 2345 G- four chain body plane through the groove binding and the 3 'end pion accumulation binding mode. The results show that the Uygur herb, the active monomer of Alizarin Xinjiang, and the purple herb have the potential to target the anti-tumor potential of the G- four chain body, and the effective monomer magnolol of the Dai medicine Carpenter The antitumor effect was achieved by targeting the c-myc 2345 G- four chain body, and its isomers and honokiol can act on the c-myc 2345 G- four chain through the 3 'pion pion accumulation and the groove binding mode, and the complex of the magnolol and G- four chain body is formed by 2:1. The results show that the effective monomer magnolol of the Dai medicine is not targeted to the G- four chain body. The antitumor activity and the potential of magnolol to the anti-tumor effect of the target G- four chain body.4. The UV absorption spectrum and fluorescence spectrum are a simple and common method to study the interaction between small molecules and biological macromolecules, but the UV absorption spectra of small molecules are higher, if small molecules and the violet of the G- four chain body are purple. In addition, the method can not be used to experiment. At the same time, the UV absorption and fluorescence spectroscopy are better at finding small molecular compounds in the pion pion binding mode. The NMR method can find small molecular compounds with weak binding and a variety of binding modes (groove binding, heap binding). Two, based on the previous part The application range of experimental materials and experimental instruments was explored. A method of screening the active small molecular compounds targeting the G- four chain body in the ethnic medicinal plants with CD combined with NMR was established. The Magnolia liliiflora Desr. was used as the object of Ethnological Study. The CD junction NMR was targeted to the G- four chain to trace the activity of the Magnolia. The specific research results are summarized as follows: 1, the CD temperature experiment finally determines that 95% ethanol is the most suitable solvent, and the ethyl acetate extraction layer is the key polar part of the separation, and 5 fractions are obtained after the gradient elution of petroleum ether propanone solvent system, NMR The distillate Fr.2 (5:1) and Fr.3 (3:1) are the key separation sites.2. According to the tracking results of the target G- four chain bodies, 31 compounds are separated by conventional silica gel column chromatography (Silica gelCC), gel column chromatography (Sephadex LH-20 CC) and preparation of thin layer chromatography (PTLC). COSY, HMQC, HMBC) and other modern spectroscopy techniques, combined with literature and data comparison to determine the structure of 31 compounds, of which 4 are new lignan compounds: liliflorin B (1), liliflorin C (2), liliflorin D (3), liliflorin E (4). Compound 5-22 is known as lignan compound, compound 23-27 is known benzene ring compound, chemical combination, chemical combination, chemical compounds, compounds 23-27 Substance 28-31 is a known sesquiterpene compound. Considering the species, novelty and quality of the compound, 8 compounds are selected as the research object. A preliminary study on the interaction with the G- four chain body has been carried out by NMR, and it is found that compound 1,2,3,12,22 has the ability to target the binding of G- four chain bodies to.3, compound 12 (Magnolia A). It is sufficient and active, so the selection of Magnolia A is an in-depth study of the interaction with G- four chain body. The results show that the temperature of the A (4 M) in the K+ buffer can increase the solution chain temperature of the human telomere G- four chain body to 69.93 degrees C (delta Tm=3.22 C), and not with the other two structure of DNA (double stranded DNA ds26, DNA hairpin structure F10T). The interaction of four chain body c-kit) can form a mole ratio 1:1 complex by combining the trench pattern with the human telomere G- four chain body. The target G- four chain body and the extraction of the Magnolia A from the she medicine Magnolia Magnolia have the potential for the anti-tumor effect of the target G- four chain body. The research result of this paper is the traditional national medicine target to the G- four chain body. The discovery of tumor active compounds provides the material basis for the rapid screening of anti tumor active small molecule compounds targeting the G- four chain body by the national medicinal plant. The research method established in this paper can be applied to the two development of traditional national drugs and promote the modern utilization of national medicine. It will bring economic benefits to the minority people and better promote the economic and social development of our national regions.

【學(xué)位授予單位】：中央民族大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R29

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