【作者】 徐佐旗；

【导师】 仰榴青；

【作者基本信息】 江苏大学 ， 生药学， 2006， 硕士

【摘要】 五味子为著名中药，其应用已有两千多年历史。五味子含有木脂素、多糖和挥发油等成分，但目前对五味子的利用主要是木脂素，对多糖和挥发油研究少；五味子提取木脂素后残渣一般作为废物丢弃，造成药用资源的浪费。因此本文就综合利用五味子活性成分展开研究，主要研究工作分以下几部分：第一部分五味子活性成分木脂素的分析对五味子木脂素的定性定量分析方法进行了研究和比较。五味子木脂素的TLC定性分析条件：采用GF₂₅₄薄层板，展开剂为正己烷：乙酸乙酯（8：3），五味子醇甲、酯甲、甲素和乙素的R_f值分别为0.21、0.32、0.66和0.71。五味子总木脂素紫外分光光度法分析条件：检测波长243nm，浓度在0.0026-0.0156mg／mL内具有良好的线性关系（r=0.99996）；平均回收率100.86％，RSD为2.19％（n=5）。五味子木脂素可见分光光度法分析条件：检测波长570nm，浓度在0.0015-0.0210mg／mL内具有良好的线性关系（r=0.99991）；平均回收率98.64％，RSD为4.95％（n=5）。结果表明，紫外分光光度法测得值高于可见分光光度法，本文选择药典记载的可见分光光度法作为五味子木脂素提取工艺研究的定量检测方法。建立了同时测定五味子提取物中四种主要木脂素（醇甲、酯甲、甲素和乙素）的薄层扫描分析方法，四种主要木脂素薄层扫描法分析条件：采用GF₂₅₄板，展开剂为正己烷-醋酸乙酯（8：3），双波长薄层扫描（λ_S=220nm，λ_R=340nm），五味子醇甲点样量于0.44～1.54μg、酯甲点样量于0.60～2.10μg、甲素点样量于0.52～1.82μg、乙素点样量于0.48～1.68μg范围内与峰面积呈良好线性关系。平均回收率分别为104.5％、95.0％、96.1％和102.1％，5次测定的RSD分别为4.0％、2.8％、2.6％和1.9％，薄层扫描法可作为五味子提取物中四种主要木脂素的定量分析方法。第二部分超临界CO₂萃取五味子中木脂素的工艺研究研究超临界CO₂萃取木脂素的工艺，以总木脂素提取率为指标，采用单因素和正交实验优化五味子木脂素超临界CO₂萃取工艺，最佳萃取条件为萃取压力25Mpa、萃取温度35℃、CO₂流量2L／min、萃取时间3h，此条件下南、北五味子总木脂素提取率分别为95.5％和93.2％，提取物中木脂素含量分别为9.2％和13.0％。与传统醇提法相比，超临界CO₂萃取法的木脂素提取率约为醇提法的1.5倍，提取物中木脂素含量约为醇提法的3～4倍，北五味子提取物中五味子乙素含量接近醇提法的7倍，因此，超临界CO₂萃取五味子木脂素明显优于醇提法，该方法工艺简单，选择性好，操作温度低，无污染，具有工业应用前景。第三部分萃余物中五味子多糖提取及活性初步研究对五味子超临界CO₂萃余物中多糖的提取工艺和多糖的抗氧化活性进行了研究。以多糖提取率为指标，采用单因素实验优化了萃余物中五味子多糖提取工艺，最佳提取条件为料液比1：20、温度100℃、时间6h、提取1次，此条件下南、北五味子超临界CO₂萃余物中多糖得率分别为13.5％和19.6％，多糖含量分别为33.6％和28.5％。与五味子醇提残渣及原料中多糖得率和含量相比较，结果表明超临界CO₂萃取未造成多糖损失，而60％醇提造成了部分多糖损失，说明超临界CO₂萃取法更有利于五味子的综合利用。初步抗氧化活性实验结果表明，南五味子粗多糖有较强的清除羟自由基作用，其IC₅₀为208.80μg／mL(抗坏血酸IC₅₀为132.57μg／mL)，北五味子粗多糖清除羟自由基效果大大弱于南五味子粗多糖，其IC₅₀为2672.30μg／mL，两者对超氧自由基的清除效果均不明显，南、北五味子多糖值得进一步研究。第四部分五味子挥发油提取及成分初步分析对五味子挥发油的超临界CO₂萃取工艺和组成成分进行了研究。以挥发油得率为指标，采用正交实验研究了超临界CO₂萃取五味子挥发油的工艺，最佳萃取条件为温度40℃、压力15Mpa、时间3h，此条件下南、北五味子挥发油平均得率分别为8.2％和8.5％，水蒸气蒸馏法南、北五味子挥发油得率分别为1.4％和2.0％（v／w％），超临界CO₂萃取法挥发油得率高于水蒸气蒸馏法。采用GC-MS法初步分析了挥发油的组成成分，分析条件为：HP-5MS柱；载气氦；进样口温度220℃；柱温80℃程序升温至220℃；质谱EI；电离能量70eV。超临界CO₂萃取的南、北五味子挥发油均被鉴定出16个成分，南、北五味子水蒸气蒸馏的挥发油分别被鉴定出24和26个成分。本论文研究建立了五味子的综合利用新方法：首先采用超临界CO₂萃取法提取五味子木脂素，萃取条件为压力25Mpa、萃取温度35℃、CO₂流量2L／min、萃取时间3h，南、北五味子木脂素提取率均达90％以上：然后从萃余物中提取五味子多糖，其工艺条件为料液比1：20、温度100℃、时间6h、提取1次，南、北五味子粗多糖的得率和含量与五味子原料的相近，南五味子粗多糖具有较强的清除自由基作用。更多还原

【Abstract】 Schisandra is a famous Traditional Chinese Medicine and has wide application for more than 2,000 years. It has the active components such as the lignans, volatile oils and polysaccharides, but only lignans were utilized while volatile oils and polysaccharides were seldom researched. After lignans were extracted from Schisandra, the rest was abandoned as wastes, and the medical resources were wasted severely. So the comprehensive utilization of the active components in the Schisandra was studied in this paper. The main contents are as follows:Part one: Analysis methods on lignans in SchisandraQuantitative and qualitative determination methods were studied and compared for the lignans in Schisandra. The conditions of TLC method for qualitative determination of lignans were as follows: The n-hexane-ethyl acetate （8:3） and self-made GF₂₅₄ plates were used and Rf values of Schisandrin , Wuweizi ester A, Schisandrin A and Schisandrin B were 0.21,0.32,0.66 and 0.71 respectively.The conditions of UV-spectrophotometry for quantitative determination of lignans were as follows: The detection wavelength was 243nm. The calibration curve was linear in range of 0.0026-0.0156mg/mL（r=0.99996）. The average recovery was 100.86% （RSD=2.19%）（n=5）.The conditions of visible iight-spectrophotometry for quantitative determination of lignans wrere as follows: The detection wavelength was 570nm. The calibration curve was linear in range of 0.0015-0.0210 mg/mL （r=0.99991）. The average recovery was 98.64% （RSD=4.95%）（n=5）. The result showed that values determined by UV-spectrophotometry method are higher than that determined by visible light-spectrophotometry method. The latter that was recorded in Pharmacopeias was chosen as the quantitative determination method for extraction technology.To establish a method for determining the content of the four lignans （Schisandrin, Wuweizi ester A. Schisandrin A, Schisandrin B） in extract of Schisandra simultaneously.The condition of TLCS for quantitative determination of four major lignans was: The contents of four major lignans could be determined by dual wavelength TLC-Scanning （λ_S=220nm,λ_R=340nm） after the extracts were developed by n-hexane- ethyl acetate （8: 3） on self-made GF₂₅₄ plates. The calibration curves were linear in range of 0.44～1.54μg for Schisandrin, 0.60～2.10μg for Wuweizi ester A, 0.52～1.82μg for Schisandrin A and 0.48～1.68μg for Schisandrin B respectively. The average recoveries were 104.5% for Schisandrin （RSD=4.0%）, 95.0% for Wuweizi ester A（RSD=2.8%）, 96.1% for Schisandrin A （RSD=2.6%） and 102.1% for Schisandrin B（RSD=1.9%）（n=5）. TLCS method was applied to the quantitative determination method for four major lignans in extracts.Part two: Study on the technology of SFE-CO₂ of lignans in SchisandraThe technology of SFE-CO₂ was optimized by single factor tests and orthogonal experimental with recoveries of lignans as indexes. The average recoveries of lignans in S. sphenanthera and Schisandra chinensis were 95.5%, 93.2% and the lignans’ contents in extracts ofSchisandra chinensis and S. sphenanthera were 9.2%, 13.0% at the optimum condition（25MPa as extraction pressure, 35℃as extraction temperature, 2L/rain as flow rate of CO₂ and 3h as extraction time）. The average recovery of SFE-CO₂ was 1.5 times, the content of extracts of SFE-CO₂ was 3～4 times and the content ofγ-schisandrin was nearly 7 times compared to that of extract by alcohol so that it was considered to be simple, good selectivity, low operation temperature and no pollution technology, and had industrial application value.Part three: Study on the extraction and activitiy of polysaccharides in residuesBoth the extraction technology of polysaccharides in residues of SFE-CO₂ and antioxidant activity of the polysaccharides were studied. The technology of polysaccharides extraction from residues of SFE-CO₂ was optimized by single factor tests with recoveries of polysaccharides as indexes. The recoveries of polysaecharides from S. sphenanthera’ and Schisandra chinensis’ residues of SFE-CO₂ were 96.2% and 96.07%. the contents of polysaccharides of S. sphenanthera and Schisandra chinensis’ were 33.6% and 28.5% at the optimum condition （1: 20 as the solid-liquid ratio, 100℃as the temperature, 6h as the time, one time extraction）. The results showed that polysaccharides were partly lost by the technology of 60% alcohol extraction while none by technology of SFE-CO₂ so that the latter is better for comprehensive utilization compared with the former.These polysaccharides’ pharmacology was studied elementarily. The results showed that S. sphenanthera polysaccharides have good antioxygenation to the hydroxyl free radicasl that IC₅₀ was 208.80μg/mL (IC₅₀ of Vitamin C was 132.57μg/mL), while that of Schisandra chinensis polysaccharides (IC₅₀ was 2672.30μg/mL) is much weaker than that of S. sphenanthera polysaccharides. Both of their effects on anti-oxygen free radicals were very weak. So polysaccharides of S. sphenanthera and Schisandra chinensis are worth to be further researched.Part four: The extraction of volatile oil in Schisandra and preliminary analysis of its constitutesSFE technology and chemical constitute of volatile oil were studied. The technology of SFE-CO₂ was optimized by orthogonal experimental with the yield of volatile oil as indexes. The average yield rate of volatile oils in S. sphenanthera and Schisandra chinensis were 8.2% and 8.5% at the optimum condition （15MPa as pressure, 40℃as temperature, 3h as time） while that by steam distillation were 1.4% and 2.0% （v/w%）, which were lower than the former. GC-MS method was established for qualitative determination of volatile oil: HP-5 capillary column, carrier gas: He, front inlet temperature 220℃, oven temperature: 80℃, temperature programmed to 220℃, electron impact ionization, ionization energy 70EV. 16 chemical compounds were identified in both S. sphenanthera’s and Schisandra chinensis’ volatile oils extracted by SFE-CO₂, 26 and 24 chemical compounds were identified in Schisandra chinensis’and S. sphenanthera’s volatile oils distilled by steam respectively.The assay indicates that Schisandra is worthy of comprehensive utilization. And a new technology was built for comprehensive utilization of lignans and polysaccharides in Schisandra. The new technology is as follows: lignans was extracted by SCF-CO₂ firstly at 25MPa as extraction pressure, 35℃as extraction temperature, 2L/min as flow rate of CO₂ and 3h as extraction time, 90% of the lignans were extracted, then polysaccharides were extracted at 1: 20 as the solid-liquid ratio, 100℃as the temperature, 6h as the time, one time extraction. The yield and content of polysaccharides were approximately equal to that of raw material. S. sphenanthera polysaccharides have good antioxygenation to the hydroxyl free radicals.更多还原