石榴叶多酚的提取富集工艺及其药理作用研究
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摘要
石榴(Punica granatum L.)为石榴科(Punicaceae)石榴属(Punica L.)落叶灌木或小乔木。石榴性温涩,既润燥又收敛,可治疗多种疾病。石榴叶不仅含有丰富的营养成分,同时还含有多种药效成分,具有非常重要的药用价值,然而通常情况下除石榴果被食用外,石榴叶多被白白废弃,造成了严重的资源浪费。因此系统的研究石榴叶的有效成分与药理作用,充分挖掘其潜在的利用价值,对石榴叶资源的合理开发和综合利用具有重要意义。本课题对石榴叶多酚的提取富集工艺与药理作用进行了基础研究,主要研究成果如下：
分别比较了不同季节、不同温度处理下石榴叶中各有效成分(总酚、鞣质、总黄酮、鞣花酸)含量的变化情况。实验结果表明,所有采样季节-温度组合中,11月份收集的落叶在25℃干燥下各有效成分含量最高,抗氧化活性最强。该研究结果为石榴叶茶的制备工艺提供了一定的理论依据。对抗氧化活性与各有效成分的相关性研究结果表明,石榴叶的抗氧化活性与多酚含量密切相关,尤其是与多酚中的鞣质含量呈显著相关,而与黄酮含量、鞣花酸含量无显著相关性。
通过单因素法、析因设计和响应面法对石榴叶多酚的提取工艺进行了优化,研究结果表明,热回流法为石榴叶多酚的最佳提取方法；四种提取溶剂中,50%乙醇为最适提取溶剂；响应面法优化得到石榴叶多酚的最佳提取条件为：以61%乙醇为提取溶剂,80℃下提取60min,所得粗提物得率以及石榴叶总酚含量依次为46.683%和19.991g GAE/100g。
通过大孔树脂吸附分离法对石榴叶多酚的富集工艺进行了优化,研究结果表明：以HPD-100树脂装柱,以偏酸性的样品溶液上样,以50%乙醇为洗脱溶剂时,石榴叶多酚的富集效果最佳。在此条件下,粗提液中82.13%的多酚可被富集,且所得富集产物(Fr.Ⅲ)抗氧化活性最强。
通过二甲苯致小鼠耳肿胀炎症模型和角叉菜胶致小鼠足肿胀炎症模型实验发现,石榴叶多酚提取物具有较好的抗炎症作用,当Fr.Ⅲ给药剂量达0.4g/kg时,其对小鼠耳肿胀与足肿胀的抑制效果与20mg/kg的阳性药吲哚美辛相当；石榴叶多酚提取物急性毒性实验结果表明,石榴叶多酚提取物属实际无毒物质。
Pomegranate is a kind of deciduous shrub or small tree, which belongs to the genus Punica in the family Punicaceae. Pomegranate fruit is warm in nature, has functions of moistening dryness, astriction and combating some diseases. Pomegranate leaf is rich in many active ingredients, such as phenolics, tannins, flavonoids, alkaloids and so on. Researches have shown that pomegranate leaf has many kinds of pharmacological activites. Usually, as the edible part, pomegranate fruit attracted much attention, while the leaf was wasted. The overall objective of the current study was to find out the potential capacity of pomegranate leaf, to develop and utilize pomegranate leaf resources rationally, and to improve the comprehensive value of pomegranate. In this study, extraction, enrichment, and pharmacological of phenolics from pomegranate leaf were investigated, and main results were as follows:
The contents of phenolics, tannins, flavonoids and ellagic acid of pomegranate leaf collected during different seasons and dried under different temperatures were compared. The results showed that pomegranate leaf sampled in November and dried under25℃exhibited the highest content of active ingredients, also presented the highest antioxidant activities. The results provides a certain theoretical basis for the preparation of pomegranate leaf tea. Correlation analysis results showed that antioxidant activity was closely related to polyphenol level, especially significantly correlated with tannins.
The extraction process of phenolics from pomegranate leaf was optimized by single factor experiment, factorial design and response surface method. Hot reflux method was found to be the best extraction method, and50%ethanol was optimum in the four extraction solvents. Further research has found that the optimum extraction solvent, temperature and time were61%ethanol,80℃and60min, respectively. Accordingly, the extract yeild and the phenolics contents in pomegranate leaf were as high as46.683%and19.991g GAE/100g, respectively.
The enrichment process of phenolics from pomegranate leaf was optimized by macroporous resin adsorption method. HPD-100resin was found to have the best adsorption and desorption capacities for phenolics from pomegranate leaf. As the pH of sample solution increased, the adsorption and desorption capacities of HPD-100resin for phenolics from pomegranate leaf decreased. Ethanol solutions of50%was found to be the best solvents to elute phenolics and the eluate (Fr.Ⅲ) exhibited the highest antioxidant activity.
The anti-inflammatory activity of ethanol extract was examined using inflammatory models including both the ear edema induced by xylene and the paw edema induced by carrageenan in mice. Results showed that pomegranate leaf extract presented strong anti-inflammatory activity. Fr.Ⅲ at a dose of0.4g/kg exhibited obvious anti-inflammatory activity, similar to indomethacin at a dose of20mg/kg. In acute toxicity test, no obvious symptom of poisoning and death was observed within seven days after Fr.Ⅲ was orally administrated to mice, indicating that pomegranate leaf extract has no acute toxicity effect.
分别比较了不同季节、不同温度处理下石榴叶中各有效成分(总酚、鞣质、总黄酮、鞣花酸)含量的变化情况。实验结果表明,所有采样季节-温度组合中,11月份收集的落叶在25℃干燥下各有效成分含量最高,抗氧化活性最强。该研究结果为石榴叶茶的制备工艺提供了一定的理论依据。对抗氧化活性与各有效成分的相关性研究结果表明,石榴叶的抗氧化活性与多酚含量密切相关,尤其是与多酚中的鞣质含量呈显著相关,而与黄酮含量、鞣花酸含量无显著相关性。
通过单因素法、析因设计和响应面法对石榴叶多酚的提取工艺进行了优化,研究结果表明,热回流法为石榴叶多酚的最佳提取方法；四种提取溶剂中,50%乙醇为最适提取溶剂；响应面法优化得到石榴叶多酚的最佳提取条件为：以61%乙醇为提取溶剂,80℃下提取60min,所得粗提物得率以及石榴叶总酚含量依次为46.683%和19.991g GAE/100g。
通过大孔树脂吸附分离法对石榴叶多酚的富集工艺进行了优化,研究结果表明：以HPD-100树脂装柱,以偏酸性的样品溶液上样,以50%乙醇为洗脱溶剂时,石榴叶多酚的富集效果最佳。在此条件下,粗提液中82.13%的多酚可被富集,且所得富集产物(Fr.Ⅲ)抗氧化活性最强。
通过二甲苯致小鼠耳肿胀炎症模型和角叉菜胶致小鼠足肿胀炎症模型实验发现,石榴叶多酚提取物具有较好的抗炎症作用,当Fr.Ⅲ给药剂量达0.4g/kg时,其对小鼠耳肿胀与足肿胀的抑制效果与20mg/kg的阳性药吲哚美辛相当；石榴叶多酚提取物急性毒性实验结果表明,石榴叶多酚提取物属实际无毒物质。
Pomegranate is a kind of deciduous shrub or small tree, which belongs to the genus Punica in the family Punicaceae. Pomegranate fruit is warm in nature, has functions of moistening dryness, astriction and combating some diseases. Pomegranate leaf is rich in many active ingredients, such as phenolics, tannins, flavonoids, alkaloids and so on. Researches have shown that pomegranate leaf has many kinds of pharmacological activites. Usually, as the edible part, pomegranate fruit attracted much attention, while the leaf was wasted. The overall objective of the current study was to find out the potential capacity of pomegranate leaf, to develop and utilize pomegranate leaf resources rationally, and to improve the comprehensive value of pomegranate. In this study, extraction, enrichment, and pharmacological of phenolics from pomegranate leaf were investigated, and main results were as follows:
The contents of phenolics, tannins, flavonoids and ellagic acid of pomegranate leaf collected during different seasons and dried under different temperatures were compared. The results showed that pomegranate leaf sampled in November and dried under25℃exhibited the highest content of active ingredients, also presented the highest antioxidant activities. The results provides a certain theoretical basis for the preparation of pomegranate leaf tea. Correlation analysis results showed that antioxidant activity was closely related to polyphenol level, especially significantly correlated with tannins.
The extraction process of phenolics from pomegranate leaf was optimized by single factor experiment, factorial design and response surface method. Hot reflux method was found to be the best extraction method, and50%ethanol was optimum in the four extraction solvents. Further research has found that the optimum extraction solvent, temperature and time were61%ethanol,80℃and60min, respectively. Accordingly, the extract yeild and the phenolics contents in pomegranate leaf were as high as46.683%and19.991g GAE/100g, respectively.
The enrichment process of phenolics from pomegranate leaf was optimized by macroporous resin adsorption method. HPD-100resin was found to have the best adsorption and desorption capacities for phenolics from pomegranate leaf. As the pH of sample solution increased, the adsorption and desorption capacities of HPD-100resin for phenolics from pomegranate leaf decreased. Ethanol solutions of50%was found to be the best solvents to elute phenolics and the eluate (Fr.Ⅲ) exhibited the highest antioxidant activity.
The anti-inflammatory activity of ethanol extract was examined using inflammatory models including both the ear edema induced by xylene and the paw edema induced by carrageenan in mice. Results showed that pomegranate leaf extract presented strong anti-inflammatory activity. Fr.Ⅲ at a dose of0.4g/kg exhibited obvious anti-inflammatory activity, similar to indomethacin at a dose of20mg/kg. In acute toxicity test, no obvious symptom of poisoning and death was observed within seven days after Fr.Ⅲ was orally administrated to mice, indicating that pomegranate leaf extract has no acute toxicity effect.
引文
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