胡柚皮化学成分及其活性研究
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摘要
常山胡柚Citrus Changshan-huyou Y. B. Chang为芸香科植物柚Citrus grandis (L.) Osbeck与甜橙Citrus sinensis (L.) Osbeck的杂交品种,主产于浙江省的常山、衢州、龙游、江山、开化等地,以常山为重点产区。本研究目的在于探明胡柚皮的化学成分的组成,对其药理及生物活性进行筛选,为第三代保健食品的开发提供可靠的理论基础,并为今后胡柚的分类及其皮的质量标准评价以及药理作用提供化学成分方面的依据,以便更好地综合开发和利用柑桔资源及发展我国传统中药。本研究的主要结果如下:
     采用层析法(硅胶柱吸附层析、薄层层析、大孔树脂吸附层析、C-18反相柱层析和Sephadex LH-20凝胶柱层析)从该种植物的果皮提取物中分离得到了33个化合物,通过1D-NMR,2D-NMR,Mass及IR光谱分析,鉴定了其中31个化合物的结构,2个为新化合物,其余29个化合物为首次从该种植物中分得,以二氢黄酮及其甙类——橙皮甙和柚皮甙为主要成分,其次是多甲氧基黄酮和柠檬苦素类化合物。从胡柚皮中分得的β-紫罗酮为具有13个碳原子的化合物,作为天然产物它们在自然界中不仅稀有而且含量也不丰富。
     另外,通过采用气相色谱/质谱/计算机联用(GC/MS/DS)技术,对胡柚皮中挥发性成分进行了分析,共检测鉴定出49种化合物,其中萜烯类化合物占主导地位,优势成分为苧烯(相对含量35.292%,分子量为136)。
     对其挥发油及不同极性溶剂的提取物的药理活性研究表明胡柚皮挥发油具有较好的抗真菌活性,对石膏样毛癣菌和红色毛癣菌表现出了很高的敏感性,它们的MIC值分别为O.025μl/ml和0.00625μl/ml;胡柚皮中某些化合物对T、B淋巴细胞的增殖反应有抑制作用,且对T淋巴细胞增殖反应的抑制作用比对B淋巴细胞增殖反应的抑制作用具有广谱性,有效部位为氯仿部位;抗凝血作用效果不明显,不同浓度的测试物对血小板聚集无抑制效果,在临床上表现为无抗凝血作用。通过对胡柚皮粗提物、纯化合物及标准品的对照试验表明,黄酮类化合物作为自由基清除剂时存在构-效关系,不同样品对O_2~(?)的清除能力是清除·OH能力的10~50倍,胡柚皮中黄酮类化合物是一种良好的自由基清除剂。
     通过利用二次旋转组合设计的方法探讨了常山胡柚皮中黄酮类化合物的最
    
     佳提取工艺参数。结果表明:提取时间、料:液比、提取温度和乙醇浓度对提取
     效果都有显著影响,影响最大的因素是提取时间,其次是料:液比,提取温度和
     乙醇浓度对提取效果影响最小。提取效果随温度的升高、乙醇浓度的增加及提取
     时间的延长而增加,随料:液比的增加而降低。最佳提取条件为乙醇浓度90%,
     料:液比 1:5,提取时间门 分钟,提取温度 20’C,最大提取量为 1.98mg/ffil。
     推荐的工业提取条件以乙醇浓度80%,料:液比为 1:8,提取温度50’C,提取
     时间 100分钟为宜。
Citrus Changshan-huyou Y. B. Chang is a hybrid of Citrus grandis (Linn.) Osbeck and C. sinensis (Linn.) Osbeck in genus Citrus. It is widely distributed in Changshan^ Quzhou, Longyou> Jiangshan and Kaihua regions of Zhejiang province; with the greatest population being found in Changshan. The study was conducted to understand the chemical constituents from the Huyou peels. The objective was to screen the biological and pharmacological activities, develop new healthy foods and offer a chemical basis for evaluation of quality standards and pharmacological activities. It would serve as a basis for value-added development and utilization of citrus resources, as well as promote Chinese traditional medicine. The main results were as follows:
    The chemical constituents of Huyou peels were studied systematically. 31 compounds were isolated by repeated column chromatography and elucidated on the basis of various spectral analysis (1D-NMR, 2D-NMR, MS, IR etc.). Among the isolated compounds were 2 new compounds that have never been reported in natural products and organic synthesis. The other 29 compounds although having been isolated from other plants, were however isolated for the first time in this plant The flavanones and their glycosides-hesperidin and naringin-were the primary components粀hile polymethoxylated flavones and limonoids were the subsidiary components. P -ionone anague isolated from the Huyou peels has 13 carbons. It is rare and not abundant as a natural product in nature. It has never been reported in Citrus, and was therefore being isolated for the first time in Citrus.
    The ideal extracting technology for flavonoids in peels was investigated by using quad ratio rotational combination designs. The results showed that extracting of flavonoids was significantly affected by time, ratio of material to solvent, temperature and alcohol contents. Extracting time was the primary condition, while the ratio of material to solvent was the second most important condition. Extracting was affected less by extracting temperature and alcohol contents than by other conditions. The
    
    
    yield increased as the temperature, alcohol content and extension of extracting time increase; but decreased as the ratio of material to solvent increases. The best extracting conditions were 90% alcohol content, a ratio of 1 '5 material to solvent, 115 minutes and 20 ℃. The maximal extraction rate of the flavonoids was above 1.98mg/ml. The industrial extracting conditions that we recommend are 80% alcohol content, a 1 '. 8 ratio of material to solvent, 100 minutes and 50 ℃. extracting time and temperature respectively.
    Another 49 chemical constituents of volatile oils (mostly terpenes) extracted from the Huyou peels were identified by using GC-MS-DS technique. The primary component was Limonene (relative content: 35.292%, molecular weight: 136).
    In vitro, that the volatile oils have a manifest antibacterial and antifungal activities had been established. Microsporum gypseum and Trichophyton rubrum are sensitive to the volatile oils. The MIC were 0.025Hl/ml and 0.00625W/ml. The results showed that some chemical constituents had good inhibitory effects for T cell and B cell proliferation reaction; making us conclude that the chloroform fraction was the immune bioactivities section. The effect of anti-cruor was not effective. Meanwhile, the study confirmed that the flavonoids extracted from the Huyou peels had the relationship of structure-effect as the scavenge reagents of free radicals. The ability of different samples scavenging O2- was 50 times that of scavenging ?OH . The flavonoids from the Huyou peels was effective scavenge reagents of free radicals.
    This study can not only stimulate the development of citrus processing, but also enhance the comprehensive utilization of citrus by-products. It can stimulate a new approach for the valued-added utilization and development of citrus residues.
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