摘要
本文对长松萝干燥枝状地衣体进行化学成分、药理活性研究。通过对长松萝干燥地衣枝状体进行95%的乙醇提取,用溶剂萃取、柱层析及制备高效液相色谱法分离、纯化获得26个化合物,并进行对核磁共振(NMR,2D-NMR)、质谱(HR-ESI-MS, EI-MS,ES-MS)、紫外、红外等光谱数据分析并结合文献报道,鉴定出24个化合物,包括(22E,24R)-5α,8α-过氧麦角甾-6,22-二烯-3β-醇(NMJL-1)、麦角甾醇(NMJL-3)等2个甾醇类；1个三萜类木栓酮(NMJL-26);黑茶渍素(NMJL-5)、巴尔巴酸(NMJL-6)、扁枝衣二酸(NMJL-7)、地弗地衣酸(NMJL-18)等4个缩酚类；Isomuronic acid(NMJL-4)、Murotic acid (NMJL-10)、 Lichesterinic acid (NMJL-11)、Neuropogolic acid (NMJL-12)、18R-Hydroxydihydroalloprotolichensterinic acid (NMJL-13)等5个脂肪酸类；苔黑酚(NMJL-8)、瑞藏酸(NMJL-9)、扁枝衣酸乙酯(NMJL-17)、4-O-methylorsellinic acid (NMJL-23)等4个苯酚类；1个黄酮苷类：芹菜素-7-0-β-D-葡萄糖醛酸苷(NMJL-22);正十八烷醇(NMJL-16)和阿拉伯糖醇(NMJL-21)2个醇类；(+)-松萝酸(NMJL-2)、2-(3,3-二(2-(7-乙酰基-4,6-二羟基-3,5-二甲基苯骈呋喃基))-丙烯酰基)-丙二酸二乙酯(NMJL-14)、(4aR,9bS)-2,6-二乙酰基-3,4a,7,9-四羟基-8,9b-二甲基-1-氧代-1,4,4a,9b-四氢二苯骈呋喃酮(NMJL-15)、(Z)-2-乙酰基-5,5-二[2-(7-乙酰基-4,6-二羟基-3,5-二甲基苯骈呋喃基)]-4-羟基-2,4-戊二烯-1-醛(NMJL-24)、4-[3-(7-乙酰基-4,6-羟基-3,5-二甲基-2-氧代-2,3-二氢苯骈呋喃基)]-4-[2-(7-乙酰基-4,6-二羟基-3,5-二甲基苯骈呋喃基)]-3-氧代丁酸乙酯(NMJL-25)等苯骈呋喃类化合物。其中NMJL-14、NMJL-15、NMJL-24、 NMJL-25为新化合物,5个脂肪酸均为仅地衣类中发现的特殊结构,首次从松萝属地衣分离得到,此外NMJL-22也是松萝属首次分离得到的黄酮苷类化合物。对从石油醚相分离得到极性小的白色香味组分进行GC/MS分析,用数据库检索的方法计算其相似度,峰面积归一化法计算其相对含量,共鉴定出17个饱和长链脂肪烷。
选用两种与临床应用较一致的人肝癌HepG2细胞和人乳腺癌MCF-7细胞株进行细胞毒性试验,对(+).松萝酸、黑茶渍素、Isomuronic acid、巴尔巴酸、扁枝衣二酸、地费地衣酸、NMJL-14、NMJL-24、NMJL-25等9个成分进行了四甲基偶氮哇盐(MTT)法肿瘤细胞毒性测试,结果MTT处理48h后,与空白对照,9个化合物达到一定浓度时均有极显著(P<0.01)的抑制肿瘤细胞的增值活性,并呈现出随着浓度的升高抑制率提高的趋势。其中化合物NMJL-25对人乳腺癌MCF-7细胞株的半数抑制浓度(ICso)为49.3μmoL/L,已超过松萝酸的(IC50)90.1μmoL/L,除了黑茶渍素以外的其它化合物的活性均与松萝酸的相近。
采用ABTS和DPPH两种方法对(+)-松萝酸NMJL-2、Isomuronic acid、黑茶渍素、巴尔巴酸、扁枝衣二酸、苔黑酚、NMJL-15、扁枝衣酸乙酯、地弗地衣酸、芹菜素-7-O-β-D-葡萄糖醛酸苷、NMJL-25等11个化合物进行抗氧化活性测定。结果11种化合物均表现不同程度的抗氧化能力,两种方法测定结果中NMJL-25抗氧化能力特别明显。DPPH自由基半数清除浓度(IC50)为0.01mmoL/L,标准品2,6-二叔丁基-4-甲基苯酚(BHT)半数清除浓度(IC50)为0.16mmoL/L; ABTS法测试总抗氧化能力为12.39mM Trolox。
本研究通过对传统药材长松萝进行化学成分的提取、分离、鉴定研究,利用光谱法鉴定24个化合物,其中包括4个结构比较新颖的新化合物,并对其人肿瘤细胞毒性及抗氧化活性测定。结果表明,新化合物4-[3-(7-乙酰基-4,6-二羟基-3,5-二甲基-2-氧代-2,3-二氢苯骈呋喃基)]-4-[2-(7-乙酰基-4,6-二羟基-3,5-二甲基苯骈呋喃基)]-3-氧代丁酸乙酯具有较强的抗氧化、肿瘤细胞毒活性。本研究为传统药材长松萝在医药卫生、保健、食品领域中开发利用、药材的质量控制、发现新的活性物质及天然先导化合物提供了科学的依据。
Chemical components and pharmacological activities of Usnea longissima Ach. have been studied in this paper. U. longissima was extracted with95%ethnol,26compounds were isolated and purified by solution extraction, column chromatography and High Performance Liquid Chromatography. By UV, IR, NMR, MS assay and combining with literature,24compounds were identified, including two sterols named5a,8a-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (NMJL-1) and Ergosterol (NMJL-3), one triterpenoid named friedelin(NMJL-26), four depsides named atranorin (NMJL-5), barbatinic acid (NMJL-6), evernic acid (NMJL-7) and diffractaic acid (NMJL-18), five fatty acids named Isomuronic acid (NMJL-4), Murotic acid (NMJL-10), Lichesterinic acid (NMJL-11), Neuropogolic acid (NMJL-12) and18R-Hydroxydihydroalloprotolichensterinic acid (NMJL-13), four phenols named orcinol (NMJL-8), Rhizonic acid (NMJL-9), Ethyl everninate (NMJL-17) and4-O-methylorsellinic acid (NMJL-23), one flavonoid glycosides named Apigenin-7-O-β-D-Dglucuronide (NMJL-22), two alcohols named1-octadecanol (NMJL-16) and and arabitol (NMJL-21), many benzofuranone derivatives named (+)-usnic acid (NMJL-2),2-(3,3-bis(7-acetyl-4,6-dihydroxy-3,5-dimethylbenzofuran-2-yl) acryloyl) malonate (NMJL-14),1,1'-((4aR,9bS)-3,4a,7,9-tetrahydroxy-8,9b-dimethyl-1-oxo-1,4,4a,9b-tetrahydrodibenzo[b, d]furan-2,6-diyl)diethanone (NMJL-15),(Z)-2-acetyl-5,5-bis(7-acetyl-4,6-dihydroxy-3,5-dimeth-ylbenzofuran-2-yl)-4-hydroxypenta-2,4-dienal (NMJL-24) and ethyl4-(7-acetyl-4,6-dihydroxy-3,5-dimethyl-2-oxo-2,3-dihydrobenzofuran-3-yl)-4-(7-acetyl-4,6-dihydroxy-3,5-dimethylbenzofuran-2-yl)-3-oxobutanoate (NMJL-25). Four of them, NMJL-14, NMJL-15, NMJL-24and NMJL-25, were new compounds. The five fatty acids, special structures of lichens, were extracted from U. longissima. for the first time. NMJL-22, flavonoid glycosides, was also extracted for the first time. The white fragrant fraction with small polarity separated from petroleum ether was analysed by GC/MS. The relative contents was calculated by similarity and peak area based on database and17saturated long-chain fatty alkanes were identified.
Cytotoxicity tests was determined by human hepatic cancer cells HepG2and human breast cancer cells MCF-7which keeping consistency with clinical application. MTT test were assayed by NMJL-2, NMJL-4, NMJL-5, NMJL-6, NMJL-7, NMJL-18, NMJL-14, NMJL-24and NMJL-25, the results showed that all components with certain concentration had significant tumor cells inhibition effects after48hours compared with blank control (P<0.01). the dosage-dependent effect between concentration and inhibition effects was existed. The median inhibitory concentration (IC50) of NMJL-25to MCF-7was49.3μ moL/L surpassed usnic acid (90.1μ moL/L). and other compounds were showed similar activities with usnic acid except atranorin.
The anti-oxidant activities of11compounds named NMJL-2, NMJL-4, NMJL-5、NMJL-6、 NMJL-7, NMJL-8,NMJL-15,NMJL-17,NMJL-18,NMJL-22and NMJL-25were measured by DPPH and ABTS assay. The results showed that all compounds had anti-oxidant effects in different degree. The anti-oxidant effect of NMJL-25was the best, the half radicals scavenging concentration (IC50) of DPPH was0.01mmoL/L and the standard sample (BHT) was0.16mmoL/L. The total anti-oxidant activity of NMJL-25was12.39mM Trolox measured by ABTS method.
As a conclusion, the chemical components and bioactivities of U. longissima were studied in this thesis. Compounds were identified by spectrum analysis, including four new compounds which were used as materials to determine anti-tumor and antioxygen activitives. The results showed that the anti-oxidant and anti-tumor activities of NMJL-25were strong. The study provide scientific basis for traditional medicine U. longissima. of development and utilization in medicine, health and food field, quality assurance and new active substance discovery.
引文
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