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白术挥发油的提取、氧化分解及抗肿瘤研究
Study on the Extraction、Oxidative Decomposition and Anti-Tumor Activities in Vitro of Volatile Oil from Atractylodes Macrocephala Koidz.
【作者】 王虹;
【作者基本信息】 山西大学 , 生药学, 2011, 硕士
【摘要】 白术挥发油是白术的主要活性成分,因其抗肿瘤作用确切、不良反应小,具有开发成中药抗肿瘤新药的良好前景。挥发油性质不稳定、易氧化分解,给新药研发带来困难。针对上述,我们解决挥发油稳定性的研究思路是,让其尽快氧化分解,获得性质稳定的、具有抗肿瘤作用的白术挥发油。具体研究内容如下:1.筛选出白术挥发油最佳提取溶剂和不同提取方法的提取率,分别是乙酸乙酯(2.56%)和超临界CO2萃取(安徽产2.76%);筛选出最佳氧化分解条件为室外光照(19-27℃),HPLC跟踪检测苍术酮的分解率为98.08%。2.通过HPLC测定氧化分解前后白术挥发油中苍术酮、γ-榄香烯、β-桉叶醇、白术内酯Ⅰ和白术内酯Ⅲ五种抗肿瘤成分的含量。结果表明,氧化后苍术酮和γ-榄香烯的含量降为零,其它三种成分含量显著增加,其中β-桉叶醇的含量是超临界CO2萃取安徽产最高,是氧化分解前的1527.56倍,白术内酯Ⅰ、白术内酯Ⅲ的含量是水蒸气蒸馏提取河北产最高,是氧化分解前的4.30倍。3.采用GC-MS技术,对氧化分解前后及不同提取方法的白术挥发油化学成分及含量变化进行了分析比较。研究表明,氧化分解后主要抗肿瘤成分苍术酮和γ-榄香稀的含量平均分别降低了95%和98%,而白术内酯Ⅰ、白术内酯Ⅲ、广木香内酯、β-桉叶醇和Isovelleral五种成分的总增加量是氧化分解前的3.68-11.04倍。4.采用电感耦合等离子体发射光谱(ICP-AES)对白术生药、挥发油和氧化分解挥发油中Mg, Al、Fe、Cr、Cu、Mn、Ni、Sr、Ti和Zn10种微量元素的含量进行了测定。结果在不同提取方法中,各微量元素含量变化较大。5.采用细胞生物学方法研究氧化分解前后白术挥发油对人源卵巢癌细胞SKOV-3的抗肿瘤作用。结果表明,抑制细胞增殖与诱导凋亡呈时间和剂量依赖关系,MTT法测定表明以挥发油浓度200mg/L抑制率最高,不同浓度以作用72h抑制率最明显;采用流式细胞仪测定氧化分解前后白术挥发油对SKOV-3细胞凋亡和细胞周期的影响,结果挥发油浓度为150mg/L凋亡率最高,细胞周期分别阻滞在G2/M期和S期。
【Abstract】 Volatile oil is the main active ingredient of the Atractylodes macrocephala Koidz. Because of its exact antitumor effects and small side effects, it has a good prospect in antitumor drugs. However, the unstableness and easy oxidation make the drug discovery difficult. In response to these, the approach we are taking to solve the stability problem of volatile oil is to let it go through quick oxidation and decomposition, turning into volatile oil with anti-tumor effects. The specific contents of the research are as follows.1. It was found that the best extraction solvent of Volatile oil is ethyl acetate (2.56%) and the best extraction methods is supercritical CO2 extraction (2.76%). The optimum conditions of oxidation and decomposition is outdoor light (19-27℃), The decomposition rate of atractylone is 98.08%.2. Five anti-tumor components of Volatile Oil before and after oxidative decomposition from Atractylodes macrocephala Koidz were determinated by HPLC. The results showed that atractylone andγ-elemene content was zero after oxidation, the other three components were significantly increased,β-eudesmol content in Volatile Oil by supercritical CO2 extraction was the highest from Anhui,1527.56 times higher than that before oxidative decomposition. AtractylenolideⅠ、Ⅲcontent was the highest by steam distillation from Hebei and 4.30 times higher than that before oxidation.3. GC-MS were employed to analsis ingredients and contents of Volatile Oil before and after oxidative from Atractylodes macrocephala Koidz with different method. The results show that the main anti-tumor component of Volatile Oil after oxidative decomposition, the content of atractylone andγ-elemene was decreased by 95% and 98%, while the increment of atractylenolideⅠ, atractylenolideⅢ, costunolide,β-eudesmene and Isovelleral is 3.68-11.04 times more than that before oxidation Volatile Oil.4. Content of 10 kinds of trace elements(Mg,Al,Fe,Cr,Cu,Mn,Ni,Sr,Ti,Zn)in Atractylodes macrocephala Koidz, volatile oils and volatile oil of Oxidative decomposition was determined by ICP-AES. The results indicate that there existed greatly difference in different extraction conditions.5. This experiment researched the effect on the ovarian cancer cell SKOV-3 of Volatile Oil before and after oxidative decomposition from Atractylodes macrocephala Koidz. The volatile oil has the function of proliferation inhibition of ovarian cancer cell, and this effect is in dose and time dependent manner. Affer dealing with the concentrations of 200mg/L, it had highest inhibition rate,and it were significant after 72 hours at different concentrations. Effects on skov-3 apoptosis and cell cycle induced by volatile oil of atractylodes macrocephala koidz before and after oxidative decomposition was studied by Flow Cytometry. Affer dealing with the concentrations of 200mg/L it had highest apoptosis rate, a cell cycle-specific in G2/M and S phase, respectively.