苦参中生物碱以及彩色小麦中B族维生素的提取检测
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摘要
亚临界水萃取是一种在较高的温度和较大的压力下用纯水萃取固体或半固体的新颖样品前处理方法,通过改变萃取温度改变水的极性，从而萃取不同极性的有机化合物。萃取速度快、样品回收率高、不使用或很少使用有机溶剂,是一种绿色的样品提取方法。毛细管电泳作为一种新兴分离技术，具有高效、快速、微量、成本低等特点。本文正是基于毛细管电泳和亚临界水萃取的优点，建立了苦参中生物碱的提取测定方法；根据小麦中B族维生素的特点，使用酶解法和高效液相色谱荧光检测提取测定彩色小麦中的B族维生素。工作重点主要有以下两个方面：
1.本实验采用亚临界水萃取-毛细管电泳场强放大堆积法对苦参中的金雀花碱、槐果碱、苦参碱、槐定碱和氧化苦参碱五种生物碱进行提取和检测。系统的考察了萃取温度、萃取压力、萃取时间和萃取循环次数对乙醇加速溶剂萃取和亚临界水萃取的收率的影响。对亚临界水萃取、加速溶剂萃取、水超声提取和氯仿浸泡提取四种方法进行了比较。电泳分离缓冲液为磷酸二氢钠(pH3.0;110mM)-异丙醇(85:15; v/v)。考察了样品基质中的磷酸浓度对分析物峰高重现性的影响。金雀花碱、槐定碱和氧化苦参碱在125-4000ng/mL内线性良好（R2>0.999），槐果碱和苦参碱在62.5-2000ng/mL内线性良好（R2>0.998），检测限在1.3-4ng/mL的范围内。检测并比较了几种不同来源的苦参药材以及苦参配方颗粒中各目标生物碱的含量。本研究为药用植物中生物碱的质量控制提供了一种新的、高效的、环境友好的分析方法。
2.彩色小麦是珍贵的种质资源，研究其营养价值对于育种具有重要意义。本研究以12个不同品种的彩色小麦为材料，通过酶解法进行B族维生素的提取、高效液相色谱-荧光检测法精确测定不同品种小麦中维生素B1、B2以及B6的含量，并对结果进行了显著性差异分析。测定了小麦的千粒重及小麦籽粒色素含量，研究了B族维生素的含量与千粒重以及小麦籽粒色素含量的关系。结果表明，不同品种之间B族维生素的差异显著，VB1的变幅为2.85-4.08μg/g，VB2的变幅为0.710-0.857μg/g，VB6的变幅为3.91-5.26μg/g。不同品种小麦中维生素B1和B6含量呈显著正相关；维生素B1和B6的含量与千粒重和色素含量都呈负相关。
Subcritical water extraction (SWE) use pure water as the solvent and is an innovativegreen sample preparation technique. Subcritical water's polarity can be changed by controllingtemperature and pressure. When the temperature is increased, the polarity decreases, and thesolubility of organic compounds in the subcritical water increases dramatically. Due to itsoutstanding advantages including less use of organic solvent, short extraction time, highsample recovery rate, SWE has been applied to the sample preprapation in many fields.Capillary electrophoresis (CE) has many advantages, such as high efficiency, short analysistime, small sample size and inexpensive instrumentation. It has been widely used for the rapidand efficient determination of active compounds in complex samples. In this dissertation, ananalysis method for alkaloids in Sophora flavescens Ait. was investigated systematically andestablished by SWE-CE. And according to the characteristics of the B-vitamins in the wheat,the B-vitamin contents (vitamin B1(VB1), vitamin B2(VB2), and vitamin B6(VB6)) in thegrains of colored wheat were determined using high-performance liquid chromatography withfluorometric detection. The main results are presented as follows:
1. The extraction and determination of cytisine, sophocarpine, matrine, sophoridine andoxymatrine in Sophora flavescens Ait. were performed using subcritical water extraction andcapillary electrophoresis coupled with field-amplified stacking. The effects of extractiontemperature, pressure, time and cycle number on the extraction yields were investigatedsystematically for accelerated solvent extraction with ethanol (ASE) and accelerated solventextraction with water (subcritical water extraction, SWE). The extraction yields obtainedusing SWE, ASE, water ultrasonic extraction and chloroform soaking extraction methodswere compared. The subcritical water extraction exhibited the highest total alkaloid yields.The electrophoresis separation buffer was monosodium phosphate (pH3.0;110mM)-isopropanol (85:15; v/v). The effect of phosphoric acid added in the sample matrix on thereproducibility of the peak heights of the analytes was also examined. Cytisine, sophoridineand oxymatrine showed good linearity (R2>0.999) within125-4000ng/mL, and sophocarpine and matrine exhibited good linearity (R2>0.998) within62.5-2000ng/mL, with the detectionlimits in the range of4-1.3ng/mL. The five alkaloid contents in medicinal plants fromdifferent sources and Sophora instant granule were determined and compared. By integratingSWE and CE, we attempted to provide a new, efficient and environmentally friendly analysisapproach for the medicinal plant quality control.
2. Color wheat is a valuable germplast resource, as a nutritious composition study ofsuch has shown potential implications for plant breeding. In this study, the B-vitamin contents(vitamin B1(VB1), vitamin B2(VB2), and vitamin B6(VB6)) in the grains of12colored wheatvarieties were determined using high-performance liquid chromatography with fluorometricdetection. And the relationships between the kernel pigment content and the contents of the Bvitamins, the kernel weight and the contents of the B vitamins, as well as the relationshipsamong the contents of the B vitamins were studied. The results showed that in wheat kernels,the contents of VB1, VB2, and VB6were2.85-4.08μg/g,0.78-0.85μg/g, and3.94-5.26μg/g,respectively. For kernels of the same color, those with high pigment contents had lower Bvitamins contents; therefore, the pigment contents of the kernels were inversely correlatedwith the B vitamin contents. The kernel weight also showed an inverse correlation with theVB6content, while the VB1content showed a positive correlation with the VB6content,regardless of the kernel color. For kernels of the same color, the correlations between the VB2and VB1contents were positively correlated, and so was the correlations between the VB2andVB6contents.
1.本实验采用亚临界水萃取-毛细管电泳场强放大堆积法对苦参中的金雀花碱、槐果碱、苦参碱、槐定碱和氧化苦参碱五种生物碱进行提取和检测。系统的考察了萃取温度、萃取压力、萃取时间和萃取循环次数对乙醇加速溶剂萃取和亚临界水萃取的收率的影响。对亚临界水萃取、加速溶剂萃取、水超声提取和氯仿浸泡提取四种方法进行了比较。电泳分离缓冲液为磷酸二氢钠(pH3.0;110mM)-异丙醇(85:15; v/v)。考察了样品基质中的磷酸浓度对分析物峰高重现性的影响。金雀花碱、槐定碱和氧化苦参碱在125-4000ng/mL内线性良好（R2>0.999），槐果碱和苦参碱在62.5-2000ng/mL内线性良好（R2>0.998），检测限在1.3-4ng/mL的范围内。检测并比较了几种不同来源的苦参药材以及苦参配方颗粒中各目标生物碱的含量。本研究为药用植物中生物碱的质量控制提供了一种新的、高效的、环境友好的分析方法。
2.彩色小麦是珍贵的种质资源，研究其营养价值对于育种具有重要意义。本研究以12个不同品种的彩色小麦为材料，通过酶解法进行B族维生素的提取、高效液相色谱-荧光检测法精确测定不同品种小麦中维生素B1、B2以及B6的含量，并对结果进行了显著性差异分析。测定了小麦的千粒重及小麦籽粒色素含量，研究了B族维生素的含量与千粒重以及小麦籽粒色素含量的关系。结果表明，不同品种之间B族维生素的差异显著，VB1的变幅为2.85-4.08μg/g，VB2的变幅为0.710-0.857μg/g，VB6的变幅为3.91-5.26μg/g。不同品种小麦中维生素B1和B6含量呈显著正相关；维生素B1和B6的含量与千粒重和色素含量都呈负相关。
Subcritical water extraction (SWE) use pure water as the solvent and is an innovativegreen sample preparation technique. Subcritical water's polarity can be changed by controllingtemperature and pressure. When the temperature is increased, the polarity decreases, and thesolubility of organic compounds in the subcritical water increases dramatically. Due to itsoutstanding advantages including less use of organic solvent, short extraction time, highsample recovery rate, SWE has been applied to the sample preprapation in many fields.Capillary electrophoresis (CE) has many advantages, such as high efficiency, short analysistime, small sample size and inexpensive instrumentation. It has been widely used for the rapidand efficient determination of active compounds in complex samples. In this dissertation, ananalysis method for alkaloids in Sophora flavescens Ait. was investigated systematically andestablished by SWE-CE. And according to the characteristics of the B-vitamins in the wheat,the B-vitamin contents (vitamin B1(VB1), vitamin B2(VB2), and vitamin B6(VB6)) in thegrains of colored wheat were determined using high-performance liquid chromatography withfluorometric detection. The main results are presented as follows:
1. The extraction and determination of cytisine, sophocarpine, matrine, sophoridine andoxymatrine in Sophora flavescens Ait. were performed using subcritical water extraction andcapillary electrophoresis coupled with field-amplified stacking. The effects of extractiontemperature, pressure, time and cycle number on the extraction yields were investigatedsystematically for accelerated solvent extraction with ethanol (ASE) and accelerated solventextraction with water (subcritical water extraction, SWE). The extraction yields obtainedusing SWE, ASE, water ultrasonic extraction and chloroform soaking extraction methodswere compared. The subcritical water extraction exhibited the highest total alkaloid yields.The electrophoresis separation buffer was monosodium phosphate (pH3.0;110mM)-isopropanol (85:15; v/v). The effect of phosphoric acid added in the sample matrix on thereproducibility of the peak heights of the analytes was also examined. Cytisine, sophoridineand oxymatrine showed good linearity (R2>0.999) within125-4000ng/mL, and sophocarpine and matrine exhibited good linearity (R2>0.998) within62.5-2000ng/mL, with the detectionlimits in the range of4-1.3ng/mL. The five alkaloid contents in medicinal plants fromdifferent sources and Sophora instant granule were determined and compared. By integratingSWE and CE, we attempted to provide a new, efficient and environmentally friendly analysisapproach for the medicinal plant quality control.
2. Color wheat is a valuable germplast resource, as a nutritious composition study ofsuch has shown potential implications for plant breeding. In this study, the B-vitamin contents(vitamin B1(VB1), vitamin B2(VB2), and vitamin B6(VB6)) in the grains of12colored wheatvarieties were determined using high-performance liquid chromatography with fluorometricdetection. And the relationships between the kernel pigment content and the contents of the Bvitamins, the kernel weight and the contents of the B vitamins, as well as the relationshipsamong the contents of the B vitamins were studied. The results showed that in wheat kernels,the contents of VB1, VB2, and VB6were2.85-4.08μg/g,0.78-0.85μg/g, and3.94-5.26μg/g,respectively. For kernels of the same color, those with high pigment contents had lower Bvitamins contents; therefore, the pigment contents of the kernels were inversely correlatedwith the B vitamin contents. The kernel weight also showed an inverse correlation with theVB6content, while the VB1content showed a positive correlation with the VB6content,regardless of the kernel color. For kernels of the same color, the correlations between the VB2and VB1contents were positively correlated, and so was the correlations between the VB2andVB6contents.
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