【作者】 武晓丹；

【导师】 金哲雄；

【作者基本信息】 东北林业大学 ， 植物学， 2010， 博士

【摘要】 仙鹤草(Agrimonia Pilosa Ledeb)为蔷薇科多年生草本植物龙芽草的全草。传统主要用于治疗各种出血证,现代研究证明,仙鹤草还有抗肿瘤、降血糖、升压等作用。仙鹤草中鞣质类成分含量丰富,且鞣质的研究也日益受到国内外科研工作者的重视。本文首次采用傅立叶变换红外光谱法(FT-IR)对仙鹤草进行分析,对其鞣质成分进行体内、体外抗肿瘤作用研究,并初步探讨其作用机理。红外光谱研究结果表明,通过帽儿山仙鹤草主体成分红外图谱对比分析可知,仙鹤草在1659 cm-1、1569 cm-1、1168 cm-1和989 cm-1处有4个鞣质特征峰,并且在1643cm-1、1512 cm-1和1470cm-1波长附近也存在鞣质特征峰,说明仙鹤草中含有鞣质成分；通过红外光谱的三级鉴定可知,七种产地仙鹤草丙酮提取物乙酸乙酯和正丁醇层均含有鞣质成分,且鞣质量不同,并通过对其原药材和提取物的分析可将七种产地的仙鹤草区分开；通过对不同提取物的乙酸乙酯层和正丁醇层一维红外光谱和二阶导数红外光谱分析可知,不同提取物的乙酸乙酯层和正丁醇层中均含有鞣质成分,但鞣质量不同,只有以丙酮作为提取溶剂时才能充分提取出鞣质成分；通过乙酸乙酯层和正丁醇层精制前后的一维红外光谱和二阶导数红外光谱与单宁酸标准图谱对比分析可知,经过精制制备后,可以不同程度的提高样品的纯度；采用经干酪素法测定精制制备后的仙鹤草丙酮提取物乙酸乙酯层和正丁醇层的平均鞣质含量分别为19.91%和14.87%。所以,傅立叶变换红外光谱法作为鉴别不同产地的仙鹤草是一种有效的手段,且为仙鹤草利用红外光谱创建指纹图谱打下了基础。体内抗肿瘤实验结果表明,乙酸乙酯层和正丁醇层均可以抑制S180小鼠实体瘤的生长,与阴性组相比,各给药组具有显著性差异(P<0.05),均表现出良好的抑瘤作用,200 mg/kg的乙酸乙酯层的抑瘤率为50.44%,250mg/kg的正丁醇层的抑瘤率为57.52%；乙酸乙酯层和正丁醇层各剂量组均能不同程度的升高S180小鼠胸腺指数和脾指数,对荷瘤小鼠两大免疫器官具有良好的保护作用；仙鹤草丙酮提取物乙酸乙酯层和正丁醇层可提高抗氧化酶SOD、CAT和GSH活力,降低脂质过氧化产物MDA的含量,说明乙酸乙酯层和正丁醇层能够明显的提高荷瘤小鼠抗氧化系统的功能,抑制脂质过氧化,清除体内过剩的氧自由基,进而发挥抗肿瘤作用,且乙酸乙酯层的作用优于正丁醇层,仙鹤草的抗肿瘤作用可能通过抗氧化作用实现的,这也可能是其体内抗肿瘤作用机理之一。由体外抗肿瘤实验结果可知,乙酸乙酯层和正丁醇层均可显著抑制BEL-7402细胞和HepG2细胞的生长,并随着药物浓度的增大而增强,作用较明显。乙酸乙酯层对BEL-7402细胞的IC50为89.28 gg/mL,对HepG2细胞的IC50为127.85 gg/mL,正丁醇层对BEL-7402细胞的IC50为107.54μg/mL,对HepG2细胞的IC50为234.41 gg/mL,乙酸乙酯层的作用明显强于正丁醇层；通过流式细胞仪(FCM)进一步检测可知,乙酸乙酯层和正丁醇层均能诱导BEL-7402细胞和HepG2细胞发生细胞凋亡,且作用较明显；通过激光共聚焦显微镜检测可知,不同浓度的乙酸乙酯层和正丁醇层作用于BEL-7402细胞和HepG2细胞48 h后,肿瘤细胞明显减少,均不同程度的出现细胞核裂解,出现较明显的凋亡形态,且有明显的绿色荧光,表明肿瘤细胞内游离Ca2+水平升高；不同浓度的乙酸乙酯层和正丁醇层作用于BEL-7402细胞和HepG2细胞48 h后,细胞内活性氧(ROS)均随给药剂量的增加而增加,与空白组相比,均具有显著性差异(P<0.05)。所以,肿瘤细胞内游离Ca2+水平超载和活性氧水平的升高可能诱导BEL-7402细胞和HepG2细胞发生细胞凋亡,这也许是其体外抗肿瘤作用的机理之一。本文首次采用FT-IR对仙鹤草进行了分析,为其利用红外光谱创建指纹图谱奠定基础,并能确定仙鹤草丙酮提取物乙酸乙酯层和正丁醇层中均含有一定量的鞣质成分,且鞣质含量不尽相同。通过药理实验可确定仙鹤草中鞣质成分具有抗肿瘤作用,其作用机理可能是通过增强抗氧化系统功能和诱导肿瘤细胞凋亡来实现的。更多还原

【Abstract】 Agrimony (Agrimonia Pilosa Ledeb) is the Rosaceae perennial herb agrimony of the whole plant. The traditional major treatment is for the kinds of bleeding. The modern researches show that agrimony was used for the anti-tumor, reducing blood glucose, boosting pressure and so on. The tannin is abundant in the agrimony, and the study on tannin is thought more and more important in internal and abroad scientific researchers day by day. In this paper, Fourier Transform Infrared Spectroscopy (FT-IR) was the fist time used for the analysis of agrimony, and investigating its tannin components of anti-tumor effect in vitro and vivo and exploring its mechanisms.The results of FT-IR showed through the FT-IR analysis the Mao ershan agrimony had the four tannin characteristic peaks in 1659 cm-1,1569 cm-1,1168 cm-1 and 989 cm-1, and there were tannin characteristic peak near the 1643 cm-1,1512 cm-1 and 1470 cm-1. The results illustrated the agrimony had tannin components. Through the analysis of the multi-steps infrared maro-fingerprint method showed that the ethyl acetate and the n-butanol abstraction from the acetone extraction of seven habitats agrimony contained a certain amount of tannin component and the content was different. We could distinguish them by using the origin Chinese medicinal materials and the extracts of the agrimony from different habitats. Through the analysis of the one-dimensional infrared spectroscopy and the second derivative infrared spectroscopy of the ethyl acetate layer and the n-butanol layer of the different extractives, the ethyl acetate layer and the n-butanol layer of the different extractives both had the tannin component and the content was different. The tannin was extracted adequately only by using the acetone. The comparative analysis between the ethyl acetate layer, the n-butanol layer and tannin through one-dimensional infrared spectroscopy and second-derivative infrared spectra. After the purification, the purity of the ethyl acetate layer and the n-butanol layer were raised on the different degree. The average tannin contents were 19.91% and 14.87% of the ethyl acetate and the n-butanol layer of agrimony acetone extracts by casein method. So, Fourier Transform Infrared Spectroscopy was the useful method on the identification of different habitats agrimony. And this was the foundation of the finger print of agrimony by FT-IR.The vivo anti-tumor results showed that the ethyl acetate layer and the n-butanol layer could inhibit the growth of S180 entity-tumor mice, as compared with the control group, the administration groups were significant difference(P<0.05). The inhibitory rate was 50.44% by 200 mg/kg of the ethyl acetate layer, and the inhibitory rate was 57.52% by 250 mg/kg of the n-butanol layer. Both the ethyl acetate layer and the n-butanol layer increased the thymus index and spleen index of S180 entity-tumor mice in different level, and had a good protective effect for the two immune organs in entity-tumor mice. The ethyl acetate and the n-butanol layer of agrimony acetone extracts enhanced the antioxidant enzymes SOD, CAT and GSH activity, reduced lipid peroxidation MDA content.So the ethyl acetate extract layer and the n-butanol layer significantly increased the antioxidant functions of the system of the entity-tumor mice, inhibited lipid peroxidation, cleaned the of excess oxygen free radicals, and played an anti-tumor effect, which might be the one of the mechanisms in vitro anti-tumor effect.Through the vitro anti-tumor results, we could see that the ethyl acetate layer and the n-butanol layer both inhibited the growth of BEL-7402 cells and HepG2 cells significantly, and the effect was enhanced by the increasing of concentration apparently. The BEL-7402 cell’s IC50 was 89.28μg/mL and the HepG2 cell’s IC50 was 127.85μg/mL of the ethyl acetate layer; The BEL-7402 cell’s IC50 was 107.54μg/mL and the HepG2 cells’s IC50 was 234.41μg/mL of the n-butanol layer.The ethyl acetate layer was better obviously than the n-butanol layer. The ethyl acetate layer and the n-butanol layer could induce the BEL-7402 cells and the HepG2 cells apoptosis by flow cytometry (FCM) further, and had a good effect. Through the laser confocal microscopy, we saw that the tumor cells significantly reduced by different concentrations of the ethyl acetate layer and the n-butanol layer on the BEL-7402 cells and HepG2 cells after 48 h, and there were nuclear cracking, apparent apoptosis shape, and there was a clear green fluorescence. These showed that the overloading of tumor cells elevated levels of intracellular free Ca2+ might be increased. The different concentrations of the ethyl acetate layer and the n-butanol layer effected on the BEL-7402 cells and HepG2 cells after 48 h, the intracellular reactive oxygen species (ROS) were increased with the concentration increased by FCM. As compared with the control group, there was significant differences (P<0.05). Therefore, the increasing of tumor cells intracellular free Ca2+ and the ROS might induce the tumor cells apoptosis, this might be the one of the mechanisms in vivo anti-tumor effect.In summary, FT-IR was the fist time used for the analysis of agrimony and created the foundation of the agrimony’s finger print, and there were tannin component in the ethyl acetate and the n-butanol layer of agrimony acetone extracts, but the content was differnt. Through the pharmacology researches determined that the tannins of agrimony had anti-cancer effect, and its mechanisms might be enhancing the antioxidant functions and inducing the tumor apoptosis.更多还原