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KIB OpenIR  > 昆明植物所硕博研究生毕业学位论文  > 学位论文
题名: 四类天然产物的生物活性研究
作者: 杨为民
学位类别: 博士
答辩日期: 2004
授予单位: 中国科学院昆明植物研究所
授予地点: 中国科学院昆明植物研究所
导师: 刘吉开
关键词: 地花菌素 ; 豚鼠 ; 气管平滑肌 ; 收缩 ; 脱敏 ; 抗氧化 ; 抗伤害 ; 小鼠 ; 干巴菌 ; 对联三苯化合物 ; 石栎木 ; 二氢查耳酮葡萄糖苷化合物 ; 高脂血症 ; 岩白菜素 ; 止咳 ; 祛痰
学位专业: 植物学
中文摘要: 本论文主要对四类天然产物进行了生物活性研究,包括:1.地花菌(Albatrellus confiuens)中的地花菌素;2.干巴菌(Thelerhoraganbajun)中三种对联三苯类化合物;3.石栎木(L ithocarPus pachyphyllus)中三种二氢查耳酮葡萄糖苷类化合物;4.岩白菜素及其衍生物。另外对高脂血症及动脉粥样硬化动物模型及治疗药物的研究进展进行了综述。论文选题主要围绕研究组其它成员的植物化学研究成果,选择其中四类天然产物进行有关体内外生物活性及机制研究,为今后进一步的研究开发奠定了重要基础。对重要的天然产物生物活性进行研究也是近年来植物化学研究的发展趋势,对天然药物研究和开发具有重要意义。1.地花菌素的生物活性研究地花菌素(albaconol)是本研究组成员从云南产地花菌(A lbatrellus confluens)中提取分离得到的一个新骨架类型化合物,结构部分己发表。本论文对其生物活性进行了如下研究:1.1地花菌素诱导豚鼠气管平滑肌收缩及脱敏作用实验研究地花菌素对离体豚鼠气管平滑肌的收缩及脱敏作用,以及受体vanilloid recePtor(VR)激动剂辣椒素、拮抗剂。apsazePine及细胞外c扩十对其作用的影响,探讨地花菌素的这种作用是否通过VR受体而起作用。结果证明,虽然地花菌素及辣椒素都引起气管平滑肌收缩,但地花菌素效价不及辣椒素,它们的-log(M)EC50值分别为4.23±0.18(n=10)及7.33±2.1(n=10)。2.5μM capsazepine(vR拮抗剂)竞争性地拮抗地花菌素及辣椒素的收缩作用。地花菌素增强低浓度辣椒(10-10-10-9M)的收缩反应,却非竞争性地拮抗高浓度辣椒素(10-8-10-3M)的作用。地花菌素(1或100 mM)及辣椒素(3或10μM)都能降低离体豚鼠气管平滑肌第二次接触地花菌素的反应,即引起脱敏反应。capsazePine(5.0μM)明显抑制这种脱敏反应。细胞外Ca2+对于地花菌素的收缩反应是关键的,但并不影响上述脱敏反应。总之,上述结果提示,地花菌素为VR部分激动剂,诱导离体豚鼠气管的收缩和脱敏反应。1.2地花菌素的体外杭氧化活性体外评价地花菌素抗氧化活性,测定其对大鼠肝匀浆脂质过氧化、超氧化物歧化酶(SOD)、1,1-二苯基-2-苦基肼(D PPH)自由基的影响。结果证明,地花菌素抑制大鼠肝匀浆脂质过氧化水平,IC50为104.2μg/mL,阳性对照叔丁基甲氧基苯酚(B HA)及维生素E(VE)IC50分别是40.4和127.2μg/mL。地花菌素提高SOD活性,EC50为106.3μg/mL,而BHA EC50为76.4μg/mL。另外,地花菌素具有一定的清除DPPH自由基作用,EC50为51.9μg/mL,BHA EC50为19.9μ/mL。上述结果提示地花菌素通过提高抗氧化酶SOD活性及质子供体作用,体外抑制脂质过氧化,具有体外抗氧化活性。1.3地花菌素的抗伤害作用采用一定强度温热刺激及化学刺激,引起疼痛反应,观察地花菌素的抗伤害作用。地花菌素(6、12μM/kg,iv)显著延长热板法小鼠舔足反应潜伏期及热水缩尾法小鼠缩尾潜伏期。地花菌素(3、6、12μM/kg,iv)明显抑制醋酸(0.6%,0.1mL/10g,in)诱导的小鼠扭体反应次数。地花菌素(6μM/kg,iv)的抗伤害作用可被VR激动剂辣椒素(0.125μM/kg,iv)所增强,而被VR拮抗剂。capsazepine(20μM/kg,iv)所抑制。另外,地花菌素的刺激性弱于辣椒素。结果提示地花菌素可能通过激动VR,具有体内抗伤害作用。2.干巴菌中对联三苯类化合物的抗氧化活性本研究组成员从云南产干巴菌(ThelePhora ganbajun)中分离鉴定了一系列对联三苯类化合物(干巴菌素A-G),结构鉴定部分已经发表。本论文选择其中含量较大的化合物(1,2,3)进行了抗氧化活性研究。我们对化合物1、2、3进行了对大鼠肝匀浆的脂质过氧化活性、SOD及DPPH自由基清除方面的实验研究。实验证明化合物1、2、3抑制大鼠肝匀浆脂质过氧化水平,ICS。分别为40以48、54μM,VE及BHA IC50为295、222μM。化合物1、2、3提高大鼠肝匀浆SOD活性,EC50分别为182、74、204μM,BHA EC50为424μM。另外,化合物1、2、3也具有清除DPPH活性,EC5o分别为49、1233、55μM,BHA EC50为110μM。结果提示化合物1、2、3具有体外抗氧化活性,其活性不仅取决于其提高抗氧化酶SOD活性,也由于其对DPPH自由基的清除作用。3.石栋木二氢查耳酮葡萄糖苷化合物的生物活性研究3.1体外抗氧化活性三种甜味二氢查耳酮葡萄糖苷类化合物,即trilobatin2″-acetate(1)、Ph1orin(2)和triI0batin(3),是从石栎木Lithocarpus pachyphyllus叶中提取分离得到的。其中化合物3在叶中含量高达1%。为探讨其体外抗氧化活性,本研究实验测定了其对于大鼠肝匀浆中脂质过氧化水平、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)及DPPH自由基的影响。结果显示,化合物1、2、3均抑制大鼠肝匀浆脂质过氧化水平,ICS。分别为 124.6、12.0、38.2μg/ML,阳性对照BHA及VE ICS。分别为40.4、127.2μg/mL.化合物1、2、3不仅增强soD活性(Ecso分别为275.1、72.2、55.8μg/mL,BHA为76.4μg/mL),而且提高GSH-Px活性(EC50分别为342.8、151.4、56.2μg/mL,VE为112.6μg/mL)。化合物1、2、3在清除DPPH自由基方面活性较弱(EC50分别为247.9、950.8、188.6μg/mL,BHA为19.9g/mL)。提示化合物1、2、3主要通过提高抗氧化酶活性,抑制脂质过氧化水平,从而具有较强的抗氧化活性,这种活性强弱与它的分子结构及取代基位置有关。3.2对高脂血症小鼠的调节血脂及抗氧化作用为进一步研究三个二氢查耳酮葡萄糖苷化合物在动物体内生物活性,实验建立高脂血症模型小鼠(灌胃给予高脂乳剂,连续7天),将受试物灌胃给予小鼠(连续7天),测定血清或肝组织中总胆固醇(TC)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、脂质过氧化物(LPO)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平。与正常对照组比较,高脂乳剂诱导的高脂血症模型组的TC、LDL-C、LPO水平明显上升,而HDL-C、SOD、GSH-Px水平明显下降。与模型组比较,62.5,250 mg/kg化合物2、3明显抑制高脂乳剂造成的TC、LDL-C上升,同时化合物2(高、低剂量)及化合物3(高剂量组)显著升高HDL-C;另外,化合物1(250 mg/kg)明显降低LDL-C,而升高HDL-C。化合物1、2、3(62.5,250mg/kg)都明显抑制高脂乳剂造成的血清和肝组织LPO水平上升;与模型组比较,除了化合物3(低剂量)外,其余各剂量(62.5,250mg/kg)的化合物均显著升高血清或肝组织的SOD活性及GSH-Px活性(约为模型组的1—3倍);并且三个化合物升高GSH-Px活性比VE强。另外,实验还证明具有降血脂作用的阳性药物非诺贝特(250mg/kg,ig)具有抗氧化活性,而VE(250mg/kg,ig)也具有一定降脂活性。结果提示化合物1、2、3对高脂血症小鼠具有降脂及抗氧化作用。4.岩白菜素及其衍生物的生物活性初步研究4.1岩白莱素及其衍生物的止咳、祛痰活性初步筛选本课题组成员通过修饰结构,运用成熟的有机反应合成了系列岩白菜素衍生物,以期改变其油/水分配系数,增加疗效,延长半衰期,筛选出部分镇咳作用更强的衍生物。本论文初步筛选研究了岩白菜素(1)及其衍生物(2,3,4)在动物体内的止咳、祛痰活性。通过构橼酸溶液喷雾,反射性地引起豚鼠咳嗽,观察岩白菜素(1)及其衍生物2、3、4(iP)对化学性刺激引起咳嗽的影响。另外,毛细管法测定岩白菜素及其衍生物(iP)对大鼠痰液量分泌的影响。结果表明,岩白菜素及其衍生物2、4(62.5 mg/kg)明显延长豚鼠喷雾拘橼酸咳嗽潜伏期,岩白菜素及衍生物2明显减少豚鼠拘橼酸喷雾咳嗽次数,其中岩白菜素衍生物2对豚鼠喷雾构橼酸引咳的止咳活性较其岩白菜素、衍生物4效果更显著。毛细管法测定大鼠痰液量实验表明,岩白菜素及其衍生物2(62.5、250mg/kg)剂量依赖性地明显增加大鼠痰液量分泌,但衍生物3、4高低剂量没有明显增加痰液量分泌。提示岩白菜素及其衍生物2具有增加大鼠痰液量的作用,从而具有祛痰效应,并且衍生物2作用较岩白菜素强。4.2岩白菜素对高脂血症小鼠的调节血脂及抗氧化活性为了进一步研究岩白菜素对高脂血症模型小鼠脂代谢及抗氧化作用的影响。将高脂乳剂灌胃(ig)给予ICR小鼠,连续7天,同时将受试物19给予动物,连续7天。测定血清及肝组织中TC、LD-C、HDL-C、LPO、SOD、GSH-Px水平的变化。与正常对照组比较,高脂血症模型组的TC、LDL-C、LPO水平显著升高,而HDL-C、SOD、GSH-Px却明显下降。与模型组比较,62.5、250 mg/kg岩白菜素显著降低血清TC、LD-C水平,同时明显升高HDL-C水平。抗氧化活性检测发现,62.5、250mg/kg岩白菜素明显提高高脂血症小鼠血清及肝组织中SOD、GSH-Px活性,使其恢复近正常值,明显抑制LPO上升。具有提高SOD、GSH-Px活性的作用,活性高于同等剂量的VE(250mg/kg),结果提示岩白菜素对高脂血症小鼠具有调节脂代谢作用及抗氧化活性。5.高脂血症及动脉粥样硬化动物模型及治疗药物研究进展对近年来高脂血症及动脉粥样硬化动物模型研究进展进行了综述,从病因上主要包括饮食型及转基因动物模型,而从动物品系上包括了灵长类、兔、鸟类、啮齿类的大鼠、小鼠等。另外,对高脂血症及动脉粥样硬化治疗药物进行了综述,包括现有临床药物及新的研究方向等。
英文摘要: In the thesis, the bioactivities of four kinds of natural products were investigated. It includes: albaconol isolated from mushroom Albatrellus confluens; three p-terphenyls from mushroom Thelephora ganbajun; three dihydrochalcone-glucosides from plant Lithocarpus pachyphyllus; bergenin and its three derivatives. In addition, the recent development of animal models and drugs for hyperlipidemia and atherosclerosis were reviewed. 1. Bioactivities of Abaconol from the Mushroom Albatrellus confluens 1.1 Albaconol Induced Contraction and Desensitization in Guinea Pig Trachea Albaconol was isolated from the basidiomycetes Albatrellus confluens. The contraction of guinea pig trachea muscle and desensitization induced by albaconol and the influences of capsazepine, capsaicin and extracellular Ca2+ were examined to investigate whether the actions were mediated via a specific VR in guinea pig trachea spiral strips in vitro. Both albaconol and capsaicin were contractors of tracheal smooth muscle, but albaconol was not so potent as capsaicin, with -log (M) EC50 values of 4.23±0.18 (n=10) and 7.33±0.21 (n=10) respectively. 2.5 and 5.0 uM capsazepine competitively antagonized the contractile response to albaconol, with-log (M) pKB values of 6.60±0.39 (n=10) and 7.36±0.45 (n=10) respectively. Albaconol increased the contraction induced by a low dose of capsaicin (10-10-10-9 M), but non-competitively antagonized the contraction induced by a high dose of capsaicin (10-8-10-3 M). Either albaconol (1, 100 mM) or capsaicin (3.0, 10 μM) was able to desensitize the isolated guinea pig bronchi to subsequent addition of albaconol. Capsazepine (5.0 μM) significantly prevented the desensitization induced by either albaconol (1, lOOmM) or capsaicin (3, 10 μM). Extracellular Ca2+ was essential for albaconol induced excitation, but it unaffected albaconol- or capsaicin-induced desensitization. In summary, the results from the present study suggested that albaconol induce contraction and desensitization of guinea pig trachea in vitro as a partial agonist for VR. 1.2 The Antioxidative Effect of Albaconol In order to evaluate the antioxidative activity of albaconol in vitro, the rat liver homogenate lipid peroxidation inhibitory activity, superoxide dismutase (SOD) activity, and l,l-dipheny-2-picrylhydrazyl (DPPH) free radical scavenging activity were investigated. Albaconol inhibited lipid peroxidation in rat liver homogenate with IC50 values of 104.2 μg/mL compared with butylated hydroxyanisole (BHA, IC50 40.4 μg/mL) and vitamine E (VE, IC50127.2 μg/mL). Albaconol increased SOD activity with EC50 values of 106.3 μg/mL, and BHA (EC5076.4 μg/mL) as control. It scavenged DPPH radical scavenging with EC50 values of 51.9 μg/mL, compared with BHA (EC50 19.9 μg/mL). The results suggested the albaconol possessed antioxidative activity due to its effect on the antioxidant enzyme SOD and proton-donating action. 1.3 Antinociceptive Activities of Albaconol in vivo Albaconol (6 and 12 μmol/kg, iv) had significantly increased the latency to foot lick on the hot plate and the tail flick withdrawal latency in mice; albaconol (3, 6 and 12 μmol/kg, iv) significantly inhibited the writhing rate induced by acetic acid (0.6%, 0.1 ml/10, ip). The antinociceptive action of albaconol (6 μmol/kg, iv) was increased by capsaicin (0.125 μmol/kg, iv), but prevented by capsazepine (20 μmol/kg, iv). The irritancy of albaconol was slighter than capsaicin. The results suggested that albaconol possessed antinociceptive activity in vivo via VR. 2.Antioxidant Properties of Natural p-Terphenyl Derivatives from the Mushroom Thelephora ganbajun The antioxidant activity in vitro of three poly (phenylacetyloxy)-substituted l,1':4',l"-terphenyl compounds from the edible mushroom Thelephora ganbajun were investigated. The IC50 values of compounds 1 - 3 for lipid peroxidation in rat liver homogenate were 400, 48, 54 μM, respectively. Compounds 1 - 3 increased superoxide dismutase (SOD) activity with EC50 values of 182, 74, 204 μM. They were also assessed on the DPPH (l,l-diphenyl-2-picrylhydrazyl) radical scavenging activity with EC5o values of 49,1233, 55 μM. 3.Bioactivities of Three Dihydrochalcone-glucosides from Plant Lithocarpus pachyphyllus 3.1 Antioxidative Activity Three sweet dihydrochalcone-glucoside compounds, a new trilobatin 2"-acetate (1) and the known phlorizin (2) and trilobatin (3), were isolated from the leaves of Lithocarpus pachyphyllus. For the purpose of evaluating the antioxidative activity of three compounds in vitro, the rat liver homogenate lipid peroxidation inhibitory activity, superoxide dismutase (SOD) activity, gluthathione peroxidase (GSH-Px) activity and l,l-dipheny-2-picrylhydrazyl (DPPH) free radical scavenging activity were investigated. Compound 1,2,3 inhibited lipid peroxidation in rat liver homogenate with IC50 values of 124.6, 12.0, 38.2 μg/mL, respectively, and butylated hydroxyanisole (BHA, ICso4O.4 μg/mL) and vitamine E (VE, IC50 127.2 μg/mL) as control. Compound 1,2,3 not only increased SOD activity with EC50 values of 275.1, 72.7, 55.8 μg/mL, respectively, and BHA. (EC5076.4 μg/mL) as control, but also elevated GSH-Px activity with EC50 values of 342.8, 151.4, 56.2 μg/mL, respectively, and compared with VE (EC50112.6 μg/mL). Although compound 1, 2, 3 possessed potent lipid peroxidation inhibitory activity, SOD and GSH-Px activity in rat liver homogenate, they exhibited weaker DPPH radical scavenging activity with EC50 values of 247.9, 950.0, 188.6 μg/mL, respectively, than control BHA (EC5019.9 μg/mL). The results suggested the compound 1,2,3 possessed potent antioxidative activity mainly ascribed to their effects on the antioxidant enzymes but not to proton-donating action, and their activities are related to their substitution and polymerization patterns. 3.2 Hypolipidemic and Antioxidative Activity in Hyperlipidemic Mice Three sweet dihydrochalcone-glucoside compounds, a new trilobatin 2"-acetate (1) and the known phlorizin (2) and trilobatin (3), were isolated from the leaves of Lithocarpus pachyphyllus. For the purpose of investigating the hypolipidemic and antioxidative activity of three compounds in vivo, hyperlipidemic mice was induced by high cholesterol emulsion administered intragastrically once daily for 7 days, while the drugs also administered intragastrically for 7 days. The levels of total cholesterol (TC), low density lipoprotein cholesteral (LDL-C), high density lipoprotein cholesteral (HDL-C), lipid peroxidation (LPO), superoxide dismutase (SOD), ghithathione peroxidase (GSH-Px) in blood serum and liver or brain tissues were measured. 62.5, 250 mg/kg of compound 2 and 3 significantly decreased serum TC, LDL-C, but increased serum HDL-C dose-dependently, while compound 1 (250 mg/kg) only reduced serum LDL-C and elevated serum HDL-C. In antioxidative test, compound 1, 2 and 3 at a dose of 62.5, 250 mg/kg significantly lowered LPO of both in liver and brain tissues, but just compound 3 (62.5, 250 mg/kg) and compound 1, 2 (250 mg/kg) showed significantly decreased LPO serum levels. SOD levels were significantly elevated by compound 1 (62.5, 250 mg/kg) in serum and liver tissues, by compound 2 (62.5,250 mg/kg) in liver, and by compound 2 (250 mg/kg) did in serum, and by compound 3 (250 mg/kg) in liver. GSH-Px levels were significantly increased by compound 1 (62.5, 250 mg/kg) in serum and liver tissues, and by compound 2 and 3 (62.5,250 mg/kg) in serum, and by compound 2 (250 mg/kg) in liver. The results suggested compound 1,2,3 possessed hypolipidemic and antioxidative activity in hyperlipidemic mice, and their activities are related to their substitution and polymerization patterns. 4.Bioactivities of Bergenin and its Three Derivatives 4.1 Relieving Cough and Expectorant Activities The effect of bergenin and its three derivatives on cough and expectoration in guinea pigs and rats were investigated. The test in cough model guinea pigs and in rats suggested that bergenin and its derivative 2 had potent relieving cough and expectorant activities, and derivative 2 was more potent than begenin. 4.2 Hypolipidemic and Antioxidative Activity of Bergenin For the purpose of investigating the hypolipidemic and antioxidative activity of bergenin in vivo, hyperlipidemic mice was produced by high cholesterol emulsion administered intragastrically once daily for 7 days, while the drug also administered intragastrically for 7 days. The levels of total cholesterol (TC), low density lipoprotein cholesteral (LDL-C), high density lipoprotein cholesteral (HDL-C), lipid peroxidation (LPO), superoxide dismutase (SOD), gluthathione peroxidase (GSH-Px) in blood serum or liver tissues were measured. Compared with normal control animals, TC, LDL-C and LPO levels were significantly elevated in emulsion control animals, while HDL-C, SOD and GSH-Px levels were significantly reduced. Compared with emulsion control animals, 62.5, 250.0 mg/kg of bergenin significantly lowered serum TC and LDL-C levels, while recovered the reduction of serum HDL-C level towards normalization. In antioxidative test, 62.5, 250.0 mg/kg of bergenin significantly decreased liver LPO level in a dose-dependent manner. In addition, bergenin significantly recovered the reduction of serum or liver SOD and GSH-Px activity towards normalization. Its effects of elevating SOD activity and especially GSH-Px activity was more potent than VE at the dose of 62.5,. 250.0 mg/kg. The results suggested bergenin possessed hypolipidemic and antioxidative activity in Iryperlipidemic mice. 5. Review: Animal Models and Drugs for Hyperlipidemia and Atherosclerosis The recent development of animal models for hyperlipidemia and atherosclerosis and the drugs were reviewed.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/706
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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四类天然产物的生物活性研究.杨为民[d].中国科学院昆明植物研究所,2004.20-25
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