【作者】 楼燕；

【导师】 邱峰；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2009， 博士

【摘要】 温郁金为姜科姜黄属（Curcuma）多年生草本植物温郁金Curcuma wenyijin的干燥根茎,分布于我国浙江省。在中国及日本的传统用药中,温郁金常用于治疗血液循环综合症,即由血液循环障碍引起的炎症,如关节痛,痛经等。其挥发油作为抗病毒,抗肿瘤药物收载于2005年《中华人民共和国药典》。前期的研究结果表明,温郁金中主要含有倍半萜类和二苯庚烷类化学成分,其中部分成分具有显著的舒张血管作用和保肝作用。为进一步明确其生物活性成分,了解其发挥药效的物质基础,本论文对温郁金的干燥根茎进行了较系统的化学研究,利用现代色谱分离手段,从温郁金中分离得到43个化合物,通过各种谱学数据（UV、NMR、MS）分析鉴定了40个化合物,包括三十三个倍半萜类化合物:其中十六个愈创木烷型化合物zedoalactone D（3）、zedoalactone E（4）、zedoalactone F（5）、zedoalactone H（6）、（1S,4S,5S,10R）-zedoarondiol（7）、procurcumenol（14）、zedoarondiol（15）、isozedoarondiol（16）、zedoalactone A（17）、zedoalactone B（18）、zedoalactone C（19）、aerugidiol（20）、zedoarolide B（21）、curcumenol（22）、curcumol（23）、alismoxide（24）;八个吉玛烷型化合物:curdionolide A（9）、curdionolide B（10）、curdionolideC（11）、curdione（25）、（4S,5S）-（+）-germacrone-4,5-epoxide（26）、（1S,10S）,（4S,5S）-（+）-germacrone-1（10）,4-diepoxide（27）、aeruginolactone（28）、（1E,4Z）-8-hydroxy-6-oxogermacra-1（10）,4,7（11）-trieno-12,8-lactone（29）;四个桉烷型化合物:curcodione（1）、curcolide（2）、1β,4α-dihydroxy-5α,8β（H）-eudesm-7（11）Z-en-12,8-olide（32）、curcolonol（33）;二个卡拉布烷型化合物:curcumenone（30）、4S-dihydrocurcumeno-ne（31）;一个榄烷型化合物:hydroxyisogermafurenolide（34）;一个苍耳烷型化合物:curcumadionol（12）;一个裂环吉玛烷型化合物:6R-dehydroxylsipanolinolide（8）;二个二苯庚烷类化合物:dicurcuminol（13）、1,5-epoxy-3α-hydroxyl-1-（3,4-dihydroxy-5-metho-xyphenyl）-7-（4-hydro-xy-3-methoxyphenyl）heptane（35）;二个酚酸类衍生物femlic acid（36）、p-hydroxy cinnamicacid（37）;一个黄酮类化合物5,7,4’-trihydroxy-flavanone（38）;二个甾醇及其苷类daucosterol（39）、β-sitosterol（40）。新化合物13个（化合物1-13）,其中化合物13是具有六元吡喃环结构的二苯庚烷二倍体,具有该骨架的二苯庚烷型化合物至今未见有文献报道,化合物11是首次发现的吉玛烷内酰胺类化合物;首次从本属植物中分离得到的化合物4个（化合物29、32、35、38）。首次从该种植物中分离得到的化合物6个（化合物14、24、28、33、36、37）。上述研究结果丰富了温郁金化学成分的结构类型,同时为进一步的生物活性研究奠定了物质基础。一氧化氮（NO）是在生物体内由L-精氨酸（L-arginine,L-Arg）在一氧化氮合酶（NOS）的作用下生成的一种无机小分子自由基,是一种生物体内重要的信使分子和神经递质,参与调节机体的许多生理或病理过程,如血管舒张,非特异性宿主防御反应,局部缺血再灌损伤,慢性或急性炎症反应等。研究证实在一些类型的细胞如巨噬细胞,上皮细胞,和平滑肌细胞中通过应答一些前炎症因子如白介素-1β（IL-1β）,肿瘤坏死因子（TNF-α）和脂多糖（LPS）等的刺激可以刺激其中诱导型一氧化氮合酶（iNOS）表达而导致过量产生NO。论文采用Griess法考察了其中38个化合物抑制脂多糖诱导的大鼠RAW264.7巨噬细胞NO释放活性,结果表明有6个倍半萜类化合物（化合物22,26,28,31,34,35）表现出很强的活性。另外,化合物25表现出与阳性对照药接近的抑制活性,化合物1,5,29,32较弱的抑制活性。其他化合物的IC₅₀值均大于100,没有表现出抑制活性。这些化合物对NO的抑制作用初步阐明了倍半萜类,二苯庚烷类化合物是治疗关节痛,痛经等血液循环阻碍引起的炎症的主要活性物质。2005版药典规定莪术醇为临床抗病毒、抗肿瘤用药莪术油质量控制的指标成分。近年研究发现莪术醇具有与莪术油一致的药理活性,因此,莪术醇是莪术油的主要药效成分之一。有关莪术醇的体内代谢研究截至目前仅有一篇文献报道,但代谢产物并未明确。本室已毕业的张卉博士曾经对莪术醇大鼠体内的代谢进行了阶段性研究,分离鉴定了10种代谢产物。为了全面了解莪术醇体内代谢信息,本论文继续对莪术醇的体内代谢进行了研究。利用现代色谱分离手段,从大鼠灌胃莪术醇的尿液中共分离得到了17个莪术醇的代谢产物,通过波谱学等手段确定其中14个代谢产物的结构。分别为:10α,14,15-trihydroxy-（1αH,7βH）-guai-4-en-3,8-dione（M-1）,10α-hydroxy-（1αH,7βH,11βH）-guai-8（12）,8（14）-diepoxy-4-en-3-one（M-2）,10α-hydroxy-（1αH,7βH）-guai-4-en-3,8-dioxo-12-oic acid（M-3）,（1αH,7βH）-guai-4,10（14）-dien-3,8-dioxo-12-oic acid（M-4）,5p,10β-dihydroxy-（1αH,7βH,11αH）-guai-8（12）,8（14）-diepoxide（M-5）,5α,10β-dihydroxy-（1αH,7βH,11βH）-guai-8（12）,8（14）-diepoxide（M-6）,8β-hydroxy-（1αH,7βH,11βH）-guai-8（12）-epoxy-4,10（14）-dien-3-one（M-7）,10β,14-dihy-droxy curcumol（M-8）,5β,10β,14-trihydroxy-（1αH,7βH）-guai-8-one（M-9）,10β-hydroxy-（1αH,7βH,11αH）-guai-8（12）,8（14）-diepoxy-4-en-3-one（M-10）,10β-hydroxy-（1αH,7βH,11βH）-guai-8（12）,8（14）-diepoxy-4-en-3-one（M-11）,10α-hydroxy-（1αH,7βH,11αH）-guai-8（12）,8（14）-diepoxy-4-en-3-one（M-12）,10β,14-dihydroxy-（1αH,7βH）-guai-4-en-3,8-dione（M-13）,10α,14-dihydroxy-（1αH,7βH）-guai-4-en-3,8-dione（M-14）。所有的代谢产物都是一相代谢产物,其中9个代谢产物为新化合物（M-1～M-9）,并且M-2、M-10、M-11、M-12;M-13、M-14;M-5、M-6是三对立体异构体。根据分离得到的代谢产物,我们发现莪术醇在大鼠体内主要发生羟基化、环氧化、醇氧化、脱水、水合反应、缩酮化、环氧环开环反应。本论文通过对莪术醇在哺乳动物体内代谢产物进行研究,明确了其在体内的代谢途径和转化过程,为其进一步研究提供了重要的体内代谢信息。更多还原

【Abstract】 Curcuma wenyujin,a perennial herbaceous plant of Zingiberaceae,is distributed in the south of China,especially in Zhejiang Province.In traditional Chinese and Japanese medicine, the rhizomes of Curcuma wenyujin are generally used to treat the Oketsu syndromes(various syndromes caused by the obstruction of blood circulation such as arthralgia and dysmenorrhea).The essential oil of Curcuma wenyujin is currently embodied in the Pharmacopoeia of the P.R.China(2005),as an anti-cancer and anti-virus remedy.Previous chemical investigations on the Curcuma wenyujin have led to the isolation of sesquiterpenoids and diarylheptanoids,some of these compounds possess significant vasorelaxant and hepatoprotective activities.In order to clarify the bioactive constituents of Curcuma wenyujin, the systematic investigation on the chemical constituents was carried out.As a result,43 compounds were isolated from the rhizomes of Curcuma wenyujin.On the basis of chemical evidences and spectral analysis,the structures of 40 compounds were elucidated as follows: sixteen guaiane-type sesquiterpenes:zedoalactone D(3),zedoalactone E(4),zedoalactone F(5),zedoalactone H(6),(1S,4S,5S,10R)-zedoarondiol(7),procurcumenol(14),zedoaron -diol(15),isozedoarondiol(16),zedoalactone A(17),zedoalactone B(18),zedoalactone C (19),aerugidiol(20),zedoarolide B(21),curcumenol(22),curcumol(23),and alismoxide (24);eight germacrane-type sesquiterpenes:curdionolide A(9),curdionolide B(10), curdionolide C(11),curdione(25),(4S,5S)-(+)-germacrone-4,5-epoxide(26), (1S,10S),(4S,5S)-(+)-germacrone- 1(10)-4-diepoxide(27),aeruginolactone(28),and(1E,4Z) -8-hydroxy-6-oxogermacra- 1(10),4,7(11)-trieno- 12,8-lactone(29);four eudesmane-type sesquiterpenes:curcodione(1),curcolide(2),1β,4α-dihydroxy-5α,8β(H)-eudesm-7(11) Z-en -12,8-olide(32),and curcolonol(33);two carabrane-type sesquiterpenes:curcumenone(30) and 4S-dihydrocurcumenone(31);one elemane-type sesquiterpene:hydroxyiso germafurenolide (34);one xanthane-type sesquiterpene:curcumadionol(12);one seco-germacrane -type sesquiterpene:6R-dehydroxylsipanolinolide(8);two diarylheptanoids:dicurcuminol (13),and 1,5-epoxy-3α-hydroxyl-1-(3,4-dihydroxy-5-methoxyphenyl)-7-(4-hydroxy-3-meth -oxy-phenyl)heptane(35);two phenolic acid derivatives:ferulic acid(36),and p-hydroxy cinnamic acid(37);one flavonoid:5,7,4’-trihydroxy-flavanone(38);one sterol and its glycoside:daucosterol(39),andβ-sitosterol(40).Among them,Thirteen compounds(1-13) were new compounds,13 was a novel compound with new skeleton;11,a novel nitrogen-containing germacrane-type sesquiterpene,has been isolated from natural sources for the first time.Four compounds(29,32,35,38) were isolated for the first time from the plants of this genus.Six compounds(14.24,28,33,36,37) were isolated for the first time from the plants.The investigation enriched the constituent-types of C.wenyujin and could provide material basic for further activity screening.The inorganic free radical NO has been implicated in physiological and pathological processes,such as vasodilation,nonspecific host defense,ischemia reperfusion injury,and chronic or acute inflammation.NO is produced by the oxidation of L-arginine by NO synthase(NOS).In the NOS family,inducible NOS in particular is involved in a pathological aspect with overproduction of NO,and can be expressed in response to pro-inflammatory agents such as interleukin-1β,tumor necrosis factor-α,and LPS in various cells including macrophages,endothelial cells,and smooth muscle cells.In this thesis,we investigated the inhibitory effects of the 38 compounds on NO production in LPS-activated macrophages by Griess method.Among the tested compounds,6 sesquiterpenes(22,26,28,31,34 and 35) displayed potent inhibitory effects,compound 25 exhibited moderate activities,which were close to that of hydrocortisone.Compounds 1,5,29 and 32 showed very weak activities.And other compounds showed no activity.Curcumol is one of the representative index components in the quality standard of the essential oil which is currently used as an anti-cancer and anti-virus remedy,and embodied in the Pharmacopoeia of the P.R.China(2005).Recent studies suggested that the pharmacological activities of curcumol were consistent with those of the essential oil. Therefore,curcumol is one of the major bioactive components of the essential oil.Although a variety of biological activities have been reported for curcumol,there is so far only one research report on its metabolism.In order to characterize its biotransformation in mammalian animals,curcumol was orally administered in rats,and the urinary metabolites were investigated.Dr.Hui Zhang in our group previously isolated and identified 10 metabolites.In our continuous study,17 metabolites were isolated from the urine,and the structures of the 14 ones were elucidated as:10α,14,15-trihydroxy-(1αH,7βH)-guai-4-en-3,8-dione(M-1), 10α-hydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxy-4-en-3-one(M-2),10β-hydroxy-(1αH,7βH) -guai-4-en-3,8-dioxo- 12-oic acid(M-3),(1αH,7βH)-guai-4,10(14)-dien-3,8-dioxo -12-oic acid(M-4),5β,10β-dihydroxy-(1αH,7βH,11αH)-guai-8(12),8(14)-diepoxide(M-5), 5α,10β-dihydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxide(M-6),8β-hydroxy-(1αH,7βH,11βH)-guai-8(12)-epoxy-4,10(14)-dien-3-one(M-7),10β,14-dihydroxy curcumol(M-8), 5β,10β,14-trihydroxy-(1αH,7βH)-guai-8-one(M-9),10β-hydroxy-(1αH,7βH,11αH)-guai-8 (12),8(14)-diepoxy-4-en-3-one(M-10),10β-hydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxy -4-en-3-one(M-11),10α-hydroxy-(1αH,7βH,11αH)-guai-8(12),8(14)-diepoxy-4-en-3 -one(M-12),10β,14-dihydroxy-(1αH,7βH)-guai-4-en-3,8-dione(M-13),10α,14-dihydroxy -(1αH,7βH)-guai-4-en-3,8-dione(M-14),respectively.All the metabolites are phase 1 metabolites,and nine metabolites are new compounds(M-1-M-9),among which M-2,M-10, M-11,and M-12;M-13 and M-14;M-5 and M-6 are three pairs of epimeric metabolites. Based on the identified metabolites,we could find that hydroxylation,epoxidation,alcohol oxidation,dehydration,ketalization and epoxy-ring opening reaction are the main phase 1 metabolic passways of curcumol in rats.The study on the metabolism of curcumol in rats would clarify its metabolic pathway and metabolic rules,and provide the important information for its development and further investigation.更多还原