山茶属植物果皮多酚的分离制备及抗氧化研究
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摘要
在我国,山茶属植物资源非常丰富,分布很广,但是对其多酚的研究尚属起步阶段。生物酚具有多种生物活性,对提高植物油质量、稳定性和保健效果起着重要的作用。本文研究了几种山茶属植物果皮多酚含量的测定、分离、成分分析、制备、抗氧化活性,主要实验结论如下：
1.采用Folin-Ciocalteu法测定油茶果皮多酚的含量,对其进行方法评价发现,稳定性较好,60min后吸光度基本稳定,重现性实验RSD为0.539%,精密度实验RSD为0.386%,样品回收率在97.05%-100.1%之间。所以该方法可作为油茶果皮多酚含量的测定方法,结果准确可靠。
2.采用固相萃取-高效液相法对几种山茶属植物果皮多酚提取液进行了初步分离鉴定分析,经优化后的条件为：采用Supel cleanTM LC-18SPE Tubes(3mL,500mg)小柱,依次使用6mL甲醇、6mL乙腈、10mL水活化小柱后,上样完全吸附后直接收集流出液进行液相色谱分析。4种山茶属植物果皮多酚提取液进行液相色谱定性分析,结果表明：通过与标液对比发现,山茶和浙江红花油茶可能含有单宁、没食子酸、儿茶素、芦丁,多齿红山茶可能含有单宁、儿茶素,广宁红花油茶可能含有儿茶素。
3.油茶果皮多酚提取液经固相萃取预处理后,利用半制备液相色谱法分离制备,经优化后的半制备条件为：流动相A(水：乙酸=100：1)和B(乙腈)以2.5mL/min的流速进行梯度洗脱；进样量：0.4mL。制备得到8种组分,收集时间在18.30-18.80min的E组分多酚含量最高,为13.54mg/g。
4.通过DPPH法和TEAC法两种方法对半制备液相色谱法收集到的8种物质进行抗氧化活性测定,实验结果表明,两种方法测定的抗氧化能力结果一致,且有5种组分活性较高,其结构需进一步研究。对总多酚含量与抗氧化活性的相关性进行了分析,结果表明总多酚含量与抗氧化活性呈正相关。
In China, Camellia has abundant resources and abroad distribution, but the report about Camellia pericarp polyphenol was rare. Biophenol has various bioactive, it has funtions in improving plant oil quality, steady, health protection. First the content of Camellia pericarp polyphenol was determined, then isolated and made component analysis, after that collected fractions and carried out their anti-oxidation, the main results were as follows:
1. Folin-ciocalteu method was used for determination of polyphenol content in Camellia pericarp polyphenol, The result of method evaluation showed that, stability was well, absorbance was close to steady after60min, RSD of reproducibility experiment was0.539%, RSD of precision experiment was0.386%, the recovery was between97.05%-100.1%. So this method can be used for determination of polyphenol content in Camellia pericarp polyphenol, the result was accurate and reliable. The method was used to determine four kinds of Camellia pericarp polyphenol which were grew up in the same place and picked at the same time, the results showed that the polyphenol content was different, it declared that there was diversity between the varieties.
2. SPE technic and HPLC method were used to isolate and determinate crude extraction of Camellia pericarp polyphenol, the optimization condition was as follows:Supel clean TM LC-18SPE Tubes(3mL,500mg) were choosed to isolate polyphenol, the solid-phase extraction column was activated successively by6mL methanol、6mL acetonitrile、10mL water, the sample was absorbed completely, then collected the elution, analysed by HPLC. According to the HPLC analysis of four kinds of Camellia pericarp polyphenol was different which purified by SPE technic, the results showed that: compared with the sample, Camellia japonica and Camellia chekiangoleosa Hu. may have tannic acid, gallic acid, catechin and rutin, Camellia polyodonta How. Eχ Hu. may have tannic acid and catechin, Camellia semiserrata Chi. may have catechin.
3. The crude elution of Camellia pericarp polyphenol was isolated and prepared through semi-preparation HPLC after purified by SPE technic, the optimization condition of semi-preparation HPLC was as follows:gradient elution was carried on by mobile phase A (water:acetic acid=100:1) and B (acetonitrile) at2.5mL/min flow rate, the injection volume was0.4mL. Eight fractions were collected, the content of fraction E which collection time was between18.30-18.80min has the highest polyphenol content, was13.54mg/g.
4. Anti-oxidation of eight fractions collected with semi-preparation HPLC were determined through DPPH and ABTS methods, the results showed that, the results of two methods were consistent, there were five fractions had high oxidation resistance, the structure of them needed further studies. The relevance of polyphenol content and anti-oxidation was studied, the result showed that they presented straight relevance.
1.采用Folin-Ciocalteu法测定油茶果皮多酚的含量,对其进行方法评价发现,稳定性较好,60min后吸光度基本稳定,重现性实验RSD为0.539%,精密度实验RSD为0.386%,样品回收率在97.05%-100.1%之间。所以该方法可作为油茶果皮多酚含量的测定方法,结果准确可靠。
2.采用固相萃取-高效液相法对几种山茶属植物果皮多酚提取液进行了初步分离鉴定分析,经优化后的条件为：采用Supel cleanTM LC-18SPE Tubes(3mL,500mg)小柱,依次使用6mL甲醇、6mL乙腈、10mL水活化小柱后,上样完全吸附后直接收集流出液进行液相色谱分析。4种山茶属植物果皮多酚提取液进行液相色谱定性分析,结果表明：通过与标液对比发现,山茶和浙江红花油茶可能含有单宁、没食子酸、儿茶素、芦丁,多齿红山茶可能含有单宁、儿茶素,广宁红花油茶可能含有儿茶素。
3.油茶果皮多酚提取液经固相萃取预处理后,利用半制备液相色谱法分离制备,经优化后的半制备条件为：流动相A(水：乙酸=100：1)和B(乙腈)以2.5mL/min的流速进行梯度洗脱；进样量：0.4mL。制备得到8种组分,收集时间在18.30-18.80min的E组分多酚含量最高,为13.54mg/g。
4.通过DPPH法和TEAC法两种方法对半制备液相色谱法收集到的8种物质进行抗氧化活性测定,实验结果表明,两种方法测定的抗氧化能力结果一致,且有5种组分活性较高,其结构需进一步研究。对总多酚含量与抗氧化活性的相关性进行了分析,结果表明总多酚含量与抗氧化活性呈正相关。
In China, Camellia has abundant resources and abroad distribution, but the report about Camellia pericarp polyphenol was rare. Biophenol has various bioactive, it has funtions in improving plant oil quality, steady, health protection. First the content of Camellia pericarp polyphenol was determined, then isolated and made component analysis, after that collected fractions and carried out their anti-oxidation, the main results were as follows:
1. Folin-ciocalteu method was used for determination of polyphenol content in Camellia pericarp polyphenol, The result of method evaluation showed that, stability was well, absorbance was close to steady after60min, RSD of reproducibility experiment was0.539%, RSD of precision experiment was0.386%, the recovery was between97.05%-100.1%. So this method can be used for determination of polyphenol content in Camellia pericarp polyphenol, the result was accurate and reliable. The method was used to determine four kinds of Camellia pericarp polyphenol which were grew up in the same place and picked at the same time, the results showed that the polyphenol content was different, it declared that there was diversity between the varieties.
2. SPE technic and HPLC method were used to isolate and determinate crude extraction of Camellia pericarp polyphenol, the optimization condition was as follows:Supel clean TM LC-18SPE Tubes(3mL,500mg) were choosed to isolate polyphenol, the solid-phase extraction column was activated successively by6mL methanol、6mL acetonitrile、10mL water, the sample was absorbed completely, then collected the elution, analysed by HPLC. According to the HPLC analysis of four kinds of Camellia pericarp polyphenol was different which purified by SPE technic, the results showed that: compared with the sample, Camellia japonica and Camellia chekiangoleosa Hu. may have tannic acid, gallic acid, catechin and rutin, Camellia polyodonta How. Eχ Hu. may have tannic acid and catechin, Camellia semiserrata Chi. may have catechin.
3. The crude elution of Camellia pericarp polyphenol was isolated and prepared through semi-preparation HPLC after purified by SPE technic, the optimization condition of semi-preparation HPLC was as follows:gradient elution was carried on by mobile phase A (water:acetic acid=100:1) and B (acetonitrile) at2.5mL/min flow rate, the injection volume was0.4mL. Eight fractions were collected, the content of fraction E which collection time was between18.30-18.80min has the highest polyphenol content, was13.54mg/g.
4. Anti-oxidation of eight fractions collected with semi-preparation HPLC were determined through DPPH and ABTS methods, the results showed that, the results of two methods were consistent, there were five fractions had high oxidation resistance, the structure of them needed further studies. The relevance of polyphenol content and anti-oxidation was studied, the result showed that they presented straight relevance.
引文
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