【作者】 曾建红；

【导师】 徐刚标；

【作者基本信息】 中南林业科技大学 ， 植物学， 2012， 博士

【摘要】 本学位论文利用气相色谱.质谱联用仪(GC-MS)分析技术对广西道地产区的10批次广西莪术样品进行比较分析,采用国家药品监督管理局推荐的“中药色谱指纹图谱计算机相似性评价系统”进行模式分析及相似度计算,建立了广西莪术油GC-MS特征指纹图谱,得到广西莪术油GC-MS指纹图谱特征峰面积值；以不同浓度的广西莪术油分别处理鼻咽癌CEN-2细胞,噻唑蓝(MTT)法检测莪术油对CNE-2细胞的毒性和不同浓度的莪术油对CNE-2细胞生长增殖抑制作用；Hochest33342荧光染色法观察凋亡细胞的发生及形态学变化,流式细胞术检测其凋亡率,蛋白免疫印迹法检测核转录因子κB (NF-κB)蛋白的表达；将获得的10批次不同产地的广西莪术油特征峰面积值和莪术油对鼻咽癌CEN-2细胞的增殖抑制率进行灰关联度分析,对原始数据进行规格化,根据规格化值计算参考数列与比较数列的绝对差值、各特征峰与对肿瘤细胞的增殖抑制率的关联系数、各特征峰与对肿瘤细胞的增殖抑制率的关联度。结果如下：(1)通过GC-MS检测了广西莪术油含有52种化学成分,鉴定了39种成分,主要为萜类及倍半萜衍生物；广西莪术油指纹图谱检测出20个共有特征峰,特征峰峰面积之和占总峰面积的93.0%以上,10批次样品具有较高的相似度(>0.90)；10批次广西莪术油的指纹图谱中各特征成分均得到了较好分离,具有特征性和唯一性。(2)莪术油对鼻咽癌CNE-2细胞有较强的体外细胞毒性作用,明显的抑制鼻咽癌CNE-2细胞的生长,存在着剂量.时间-效应的关系；莪术油能诱导鼻咽癌CNE-2细胞NF-κB的凋亡,其分子机制之一可能是通过下调NF-κB的表达,从而达到抑制鼻咽癌细胞的增殖,促进凋亡,发挥抑制肿瘤生长的作用。(3)莪术油中主要的抗肿瘤活性成分为莪术醇、莪术二酮、β-榄香烯、吉马酮、莪术酮,其中莪术醇与肿瘤细胞抑制作用具有高度关联(关联度>0.90),莪术二酮、β-榄香烯、吉马酮、莪术酮与肿瘤细胞抑制作用的关联度均在0.70以上。其它各特征峰代表的化学成分与其增殖抑制作用的关联度均在0.60-0.80之间；依据关联度的大小,各成分对抗肿瘤作用贡献的大小顺序为：莪术醇>莪术二酮>β-榄香烯>吉马酮>莪术酮>α-丁子香烯>δ-瑟林烯>β-石竹烯>β-榄香烯酮>a-蒎烯>α-石竹烯>桉油素>樟脑>2-壬醇>异龙脑莪术烯>δ-榄香烯>莰烯>D-柠檬烯>龙脑。广西莪术抗肿瘤作用是其“有效组分群”共同作用的结果。本研究建立了广西莪术油GC-MS指纹图谱,确定了广西莪术油“有效组分群”指纹特征对药效贡献的大小,这为中药的“谱效关联”的指纹图谱的建立、中药的质量控制和药效评价标准的制定提供了新的思路和方法。更多还原

【Abstract】 In this paper, gas chromatography-mass spectrometry (GC-MS) analysis was performed for10samples of genuine producing areas in guangxi from curcuma kwangsiensis. The TIL profiles were evaluated by "Computer Aided Similarity Evaluation System"(developed by Research Center for Modernization of Chinese Medicine, Central South University), the fingerprint of essential oil from Curcuma kwangsiensis was established, and the characteristic peak area of the GC-MS fingerprint was acquired. The nasopharyngeal carcinoma CNE-2cells were treated with curcumol oil at different concentration12.5,25.0,50.0and100.0μg/ml), negative control (Ethanol medium) and positive control (5-Fu). The toxicity of Curcuma oil to CNE-2cells and the effect of inhibiting proliferation were detected by MTT method, the apoptosis was observed and analyzed by hoechst33342flourescence staning and flow cytometry, the expression of NF-κB was detected with western blotting; The characteristic peak area from10sample of genuine producing areas and inhibiting proliferation rate to CNE-2cells were analyzed by grey relational methods, the original data was standardized. At the premise of the standardized data, we calculated the absolute difference of reference series and comparison series, the correlation coefficient and correlation of the characteristic peaks and inhibiting proliferation rate to tumor cells. The results as follows:(1) Study on fingerprint of essential oil from Curcuma kwangsiensis:We separated52kinds of components in Curcuma kwangsiensis essential oil and identified39different compounds by GC-MS, mainly terpenoids and sesquiterpene derivatives. The20feature peaks in fingerprints of Curcuma kwangsiensi were identified over93%of the total area of the peaks, the resemblance was higher among10samples(>90%). The characteristic peak area from10sample of genuine producing areas had good separation, characteristics and uniqueness.(2) Study on pharmacodynamic of anti-tumor effect by Curcuma kwangsiensis essential oil:The Curcuma oil had strong toxicity to CNE-2cells in vitro, and was capable of significantly inhibiting proliferation of CNE-2, and the effect of inhibiting proliferation was dose-effect relationship. The Curcuma oil induced the apoptosis of NF-κB in the CNE-2cells, the molecular mechanisms may be related to the down regulated of the NF-κB protein level, and thus inhibit the proliferation of nasopharyngeal carcinoma CNE-2cells and promote apoptosis, and inhibit the growth of tumor cells.(3) Study on fingerprint’s spectrum-effect from Curcuma kwangsiensis essential oil:The main anti-tumor active ingredient in curcumol oil was curcumol, curdione, β-elemene, germacrone and curzerenone. The curcumol was highly correlated with the effect of inhibitiing proliferation to nasopharyngeal carcinoma cell (relational garde>0.90). The relational grade was more than0.70between the curdione, β-elemene, germacrone, curzerenone and the effect of inhibiting proliferation to nasopharyngeal carcinoma cell. The relational grade was between0.60-0.80between other characteristic peaks represent the chemical composition and the effect of inhibitiing proliferation to nasopharyngeal carcinoma cell. According to the relational grade, the sequence of contribution to inhibiting proliferation to nasopharyngeal carcinoma cell was Curcumol> Curdione>β-Elemene> Germacrone> Curzerenone>a-Caryophyllene>δ-Selinene>β-Caryophyllene>β-Elemenone> a-Pinene>a-Caryophyllene> Eucalyptol> Camphor>2-Nonanol> Isoborneol>Curzerene>δ-Elemene> Camphene> D-Limonene> Borneol. The anti-tumor effect of Curcuma kwangsiensis was the result of combined effect of "effective component group".This study established the fingerprint of essential oil from Curcuma kwangsiensis, identified the effective component group represented by fingerprint contributed to pharmacodynamic, and provide a reference for establishing fingerprint of TCM’s spectrum-effect relationship, TCM quality control and develop evaluation criteria of pharmacodynamics.更多还原