芦笋质量控制的现代分析方法研究
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摘要
芦笋是百合科天门冬属植物,是世界十大名菜之一,味道鲜美、营养丰富,并具有很好的保健功效。研究表明,芦笋具有多种药理活性,如抗肿瘤、降血脂、增强免疫力、抗衰老、抗疲劳和保护肝脏等。目前,对芦笋化学成分的研究较少,仅停留在总黄酮和总皂苷的初步分离纯化上,因此,尚无法对芦笋进行全面有效的质量控制。本论文首先对芦笋的营养成分进行了较为全面的质量控制,然后对芦笋中的化学成分进行分离鉴定,并对芦笋黄酮部位进行富集纯化并作定量分析,再通过二维柱切换液相色谱法分离制备了芦笋中五种黄酮苷,并采用全二维液相色谱法对芦笋黄酮部位进行了分析,考察了各成分与脂质体膜之间的相互作用,最后对所得五种黄酮苷和芦笋黄酮部位进行了体外抗氧化活性研究,初步揭示并验证了芦笋发挥药理活性的物质基础和黄酮抗氧化活性的构效关系。
一、芦笋中营养成分的质量控制
1、氨基酸在pH5.5时与茚三酮反应形成蓝紫色化合物,颜色的深浅与溶液中氨基酸的含量成正比,且在570nm处有最大吸收,利用此原理,测定该波长处的吸光度即可计算氨基酸浓度。本实验以L-谷氨酸为标准品对方法学进行了全面考察,线性回归方程为A=0.0073C-0.1133(r=0.9998),线性范围30.30-151.50μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。十批不同来源的鲜芦笋样品中总游离氨基酸含量为17.43-22.43‰。
2、考马斯亮蓝G-250在游离状态下呈红色,在稀酸中与蛋白质结合变成蓝色,最大吸收波长也从465nm变成595nm,蛋白质与考马斯亮蓝G-250结合物在595nm波长下的吸光度与蛋白质含量成正比,因此,可以根据此原理测定蛋白质含量。本实验以牛血清白蛋白为标准品对方法学进行了全面的考察,线性回归方程为A=0.0077C+0.0524(r=0.9996),线性范围19.80-99.00μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。十批不同来源的鲜芦笋样品中总蛋白质含量为1.08-1.69‰。
3、芦笋中总多糖的含量测定采用的是硫酸蒽酮显色法,以葡萄糖为标准品,线性方程为A=0.0081C+0.0907(r=0.9990),线性范围20.00-100.00μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。十批不同来源的干燥芦笋样品中总多糖含量为1.16-1.64%。
4、选择AQC为衍生化试剂,以普通C18柱作为分离载体,建立了芦笋水解氨基酸的指纹图谱,确定了16个共有峰,鉴定了其中的11个氨基酸,并在指纹图谱条件的基础上测定了10批芦笋中11种氨基酸的含量,结果如下:组氨酸2.97-5.56‰,甘氨酸0.39-0.53‰,天冬氨酸1.64-3.63‰,谷氨酸0.89-1.55‰,苏氨酸0.32-0.46‰,丙氨酸0.32-0.57‰,胱氨酸0.47-1.01‰,酪氨酸0.23-0.28‰,缬氨酸0.36-0.65‰,亮氨酸0.37-0.60‰,苯丙氨酸0.35-0.44‰。
二、芦笋的化学成分研究
1、芦笋中两种嘌呤类化合物的分离制备
干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱,收集20%乙醇洗脱流分,再经葡聚糖凝胶柱脱色,纯化后的样品采用制备液相色谱法分离,得到两种嘌呤类化合物,经1HNMR和13CNMR鉴定为9-(2-甲硫基-3-磺酸基-4-羟基)-呋喃-6-氨基-嘌呤和腺苷。
2、芦笋中五个黄酮类化合物的分离制备
干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱,收集40%乙醇洗脱流分,再经葡聚糖凝胶柱脱色,纯化后的样品采用制备液相色谱法分离,得到五种黄酮苷,经HPLC-DAD-MS鉴定为槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷。
3、芦笋中一种有机酸的分离制备
干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱,收集60%乙醇洗脱流分,再经葡聚糖凝胶柱纯化,即得纯品,经1HNMR和13CNMR鉴定为肉桂酸。
三、芦笋黄酮部位的制备与定量分析
1、干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱(60×4cm),先静止吸附1h,再分别用水1500mL、20%乙醇2000mL和40%乙醇2000mL依次洗脱。收集40%乙醇部分并于50℃减压浓缩至干,加30%甲醇溶解后,过Sephadex LH-20型葡聚糖凝胶柱(100×3cm),分别用300mL水、200mL50%甲醇和1000mL甲醇依次洗脱,收集含黄酮的流分,合并后于50℃减压浓缩至干,即得芦笋黄酮部位。
2、采用紫外分光光度法,以芦丁为标准品,测定了芦笋黄酮部位中总黄酮含量,并对方法学作了详细考察,线性回归方程为A=0.0024C-0.0174 (r=0.9992),线性范围62.00-310.00μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。所测三批样品中总黄酮的含量均高于50%。
3、采用高效液相色谱法测定了芦笋黄酮部位中槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷的含量,色谱条件如下:色谱柱为Diamonsil○R (钻石)C18柱(200×4.6mm,5μm)(Dikma Technologies),流动相:乙腈-甲醇-0.1%醋酸水(8﹕16﹕76),流速1.0 mL/min,柱温30℃,检测波长260nm,进样量20μL,方法学考察结果符合要求。测定三批样品中槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷的含量分别为11.26-12.62%, 22.64-24.78%, 4.37-5.09%,
4.13-4.89%,3.22-3.63%。
四、芦笋中黄酮苷单体的二维色谱法制备及芦笋黄酮部位的全二维液相色谱分析
1、采用二维柱切换液相色谱分离制备了芦笋中五种黄酮苷单体(槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷),该系统维间切换没有用到定量环,而是用六通切换阀取而代之,这样可以确保组分被全部切换进入第二维,避免了样品损失,且切换体积不受定量环限制。与普通的一维制备液相色谱法相比,该法可以大大提高分离效率,并节约有机溶剂用量。
2、采用HSCCC-PHPLC二维方法从芦笋黄酮粗品中分离制备了五个黄酮苷单体(槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷)。该方法中,HSCCC作为一种预处理手段,分离得到两个含目标成分的流分,继而通过制备液相色谱进一步分离得到高纯度的目标化合物。该法避免了繁琐的样品柱色谱预处理过程,具有操作简便、制备效率高等优点。
3、采用全二维液相色谱法分析了芦笋黄酮部位,第一维采用的是脂质体柱(250×4.6mm,5μm,自制),流动相为10mmol/L pH 7.4的醋酸铵缓冲液,流速0.1mL/min;第二维采用Chromolith Performance RP-18e (100×4.6mm)整体柱,流动相采用A:乙腈,B:10%乙腈梯度洗脱,0-2min 3%A,2-4min 3%A-17% A,4-4.5min,17% A -3%A,4.5-5.0min,3%A,流速3.0 mL/min。通过考察芦笋中不同成分在脂质体柱上的色谱行为,可以判断各成分与脂质体膜之间的相互作用强弱,从而推断药物在生物体内的ADME。
五、芦笋总黄酮及其黄酮苷单体体外抗氧化活性研究
以VitC为阳性对照药物,系统研究了芦笋总黄酮及其黄酮苷单体的体外抗氧化活性。结果表明,芦笋中的黄酮苷单体有较强的抗氧化活性:能明显清除·OH和O2-,对DPPH自由基也有较强的清楚作用,具有较强的抑制大鼠红细胞氧化溶血的作用。芦笋总黄酮也有相应的体外抗氧化活性,强度与总黄酮含量相关。本实验验证并揭示了芦笋发挥多种药理活性的物质基础和黄酮类化合物抗氧化活性的构效关系。
综上所述,本论文以芦笋的质量控制为目的,建立了总游离氨基酸、总蛋白质和总多糖三大营养成分的定量分析方法,建立了芦笋水解氨基酸的指纹图谱并对其中11种氨基酸作了定量分析。为了进一步了解芦笋发挥药理活性的物质基础,本论文对芦笋的化学成分作了初步研究,首次从芦笋中分离出两种嘌呤类化合物、五种黄酮苷和一种有机酸。建立了芦笋黄酮部位的制备方法和定量分析方法,为今后进一步开发芦笋黄酮打下了基础。本论文还建立了芦笋中五种黄酮苷的二维柱切换液相色谱制备方法和HSCCC-PHPLC二维制备方法,与传统制备液相色谱法相比,这两种方法具有制备效率高、溶剂消耗少等优点。本论文采用全二维液相色谱法分离分析了芦笋黄酮部位,考察了芦笋中不同成分与脂质体膜之间的相互作用。最后,本论文对芦笋黄酮部位和芦笋中五种黄酮苷的体外抗氧化活性作了系统考察,验证并揭示了芦笋发挥药理活性的物质基础和黄酮类化合物抗氧化活性的构效关系。
Asparagus officinalis L. is one of the ten most famous vegetables of the world. It is delicious and eutrophy, and is good for health. Recent studies have found that Asparagus officinalis L. can be used to cure cancer, lower blood-lipid, enhance immunity, anti-aging, anti-fatigue and protect liver. So far as we know, there are only a few papers about the chemical composition of Asparagus officinalis L. These papers are about the separation and purification of total flavonoids and total saponins. So, it is difficult to control the quality of Asparagus officinalis L. fully and efficiently. This study detected the nutritional ingredients in Asparagus officinalis L. and then identified the purified compounds in Asparagus officinalis L. Next, total flavonoids of Asparagus officinalis L. was prepared and analyzed. The preparation method of the five flavonoid glycosides from Asparagus officinalis L. was optimized using two dimensional prep-HPLC and the total flavonoids of Asparagus officinalis L. was analyzed with comprehensive two dimensional HPLC to study the interactions between the components in Asparagus officinalis L. and the liposome membrane. Finally, the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L. were studied. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated.
1. Quality control of the nutritional ingredients in Asparagus officinalis L.
1.1 Amino acids react with ninhydrin to generate a kind of hepatic complex at pH 5.5 and the shade of the color varies directly with the contents of the amino acids. The complex has the strongest absorption at the wavelength of 570 nm. So, the contents of the amino acids can be determined with this principle. This paper fully studied the technology of this method using L-Glu as reference substance. The equation of the linear regression was A=0.0073C-0.1133 (r=0.9998) and the linear range was 30.30-151.50μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of amino acids in ten batches of Asparagus officinalis L. were 17.43-22.43‰.
1.2. Coomassie brilliant blue G-250 is red at free state. It changes into blue when combines with proteins in dilute acid and the wavelength with the strongest absorption changes into 595 nm instead of 465 nm. The absorbance of the conjugate at 595 nm varies directly with the contents of proteins. This paper fully studied the technology of this method using BSA as reference substance. The equation of the linear regression was A=0.0077C+0.0524 (r=0.9996) and the linear range was 19.80-99.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of proteins in ten batches of Asparagus officinalis L. were 1.08-1.69‰.
1.3. The total polysaccharides in dry Asparagus officinalis L. was determined by coloration with sulphuric acid and anthrone. This paper fully studied the technology of this method using glucose as reference substance. The equation of the linear regression was A=0.0081C+0.0907 (r=0.9990) and the linear range was 20.00-100.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of total polysaccharides in ten batches of dry Asparagus officinalis L. were 1.16-1.64 %.
1.4 The fingerprint of hydrolytic amino acids in Asparagus officinalis L. was established on a common C18 column using AQC as the derivatization reagent. Sixteen peaks were defined as common peaks and 11 of them were identified. Eleven amino acids in ten batches of Asparagus officinalis L. were determined. The results were as follows: histidine 2.97-5.56‰, glycine 0.39-0.53‰, aspartic acid 1.64-3.63‰, glutamic acid 0.89-1.55‰, threonine 0.32-0.46‰, alanine 0.32-0.57‰, cystine 0.47-1.01‰, tyrosine 0.23-0.28‰, valine 0.36-0.65‰, leucine 0.37-0.60‰, phenylalanine 0.35-0.44‰.
2. Studies on the chemical constituents from Asparagus officinalis L.
2.1 Separation and purification of two purines from Asparagus officinalis L. The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 20% ethanol was collected and decolored with polydextran gel. The purified sample was separated with prep-HPLC and two compounds were prepared. They were identified as 9-(2-methylthio-3-sulfo-4-hydroxy)-furan-6-aminopurine and adenosine by 1HNMR and 13CNMR.
2.2 Separation and purification of five flavonoid glycosides from Asparagus officinalis L.
The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 40% ethanol was collected and decolored with polydextran gel. The purified sample was separated with prep-HPLC and five compounds were prepared. They were identified as quercetin-3-O-glucosyl- rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin by HPLC-DAD-MS.
2.3 Separation and purification of an organic acid from Asparagus officinalis L.
The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 60% ethanol was collected and purified with polydextran gel. The compound was identified as cinnamic acid by 1HNMR and 13CNMR.
3. Preparation of purified flavonoids extract of Asparagus officinalis L. and quantitative analysis
3.1. The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins (60×4cm). The extract was adsorbed by the resins for 1 h, and then eluted with 1500 mL of water, 2000 mL of 20% ethanol and 2000 mL of 40% ethanol. The fraction of 40% ethanol was collected and evaporated to dryness by rotary evaporation at 50℃under reduced pressure. The extract was dissolved in 30% methanol and then processed with polydextran gel (100×3cm). It was eluted with 300 mL of water, 200 mL of 50% methanol and 1000 mL of methanol. The fractions containing flavonoids were combined and evaporated to dryness by rotary evaporation at 50℃under reduced pressure. That was the purified flavonoids extract of Asparagus officinalis L..
3.2. The contents of total flavonoids in the purified flavonoids extract of Asparagus officinalis L. were determined with ultraviolet spectrophotometry. This paper fully studied the technology of this method using rutin as reference substance. The equation of the linear regression was A=0.0024C-0.0174 (r=0.9992) and the linear range was 62.00-310.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of total flavonoids in three batches of samples were >50 %.
3.3. The contents of quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin in the purified flavonoids extract of Asparagus officinalis L. were determined with HPLC. Diamonsil○R C18 column (200×4.6mm,5μm, Dikma Technologies) was used for separation. The mobile phase was acetonitrile-methanol-0.1% HAc (8﹕16﹕76). The flue rate was 1.0 mL/min. The column temperature was set at 30℃and the detection was set at 260 nm. The injection volume was 20μL. This paper fully studied the technology of this method and all the results were within the criteria. The contents of quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl-rutinoside, nicotiflorin and narcissin in three batches of samples were 11.26-12.62%, 22.64-24.78%, 4.37-5.09%, 4.13-4.89% and 3.22-3.63%, respectively.
4. Preparation of five flavonoid glycosides from Asparagus officinalis L. by two-dimensional methods and the separation of the flavonoids using comprehensive two dimensional HPLC
4.1. Five flavonoid glycosides (quercetin-3-O-glucosyl- rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin) were purified from Asparagus officinalis L. by two-dimensional prep-HPLC with column switch technology. A six-port two-position switching valve instead of a sample loop was used in this system. This approach assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension were not restricted. Compared with traditional prep-HPLC, this method enhanced the production rate greatly and saved much organic solvent.
4.2. Five flavonoid glycosides (quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl-rutinoside, nicotiflorin and narcissin) were purified from Asparagus officinalis L. by HSCCC-PHPLC. In this method, HSCCC was used as a pretreatment process. Two fractions containing the target compounds were obtained by HSCCC. Then, they were further separated using PHPLC and the target compounds with high purity were finally obtained. This method was simple and high-performance without tedious pretreatment process using column chromatography.
4.3. The purified flavonoids extract of Asparagus officinalis L. was analyzed by comprehensive two dimensional HPLC. In the first dimension, a liposome column was used with a flow rate of 0.1 mL/min. And in the second dimension, a Chromolith Performance RP-18e (100×4.6mm) column was used. The mobile phase consisted of A: acetonitrile, B: 10% acetonitrile. The gradient progress was as follows: 0-2min 3%A,2-4min 3%A-17% A,4-4.5min,17% A -3%A,4.5-5.0min,3%A, and the flow rate was 3.0 mL/min. The interactions between the components in Asparagus officinalis L. and liposome membrane could be judged according to the chromatographic behavior of these components on a liposome column. And the ADME of the drugs could also be concluded.
5. Studies on the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L.
The in vitro scavenging activities of total flavonoids of Asparagus officinalis L. and five flavonoid glycosides on superoxide radical, hydroxyl radical and DPPH, and their protective effects on H2O2-induced hemolysis of rat erythrocytes were investigated. The results suggested that total flavonoids and five flavonoid glycosides from Asparagus officinalis L. showed significant antioxidant activities in vitro. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated. In a word, this study was carried out to control the quality of Asparagus officinalis L. The quantitation methods were developed to determine total free amino acids, total proteins and total polysaccharides in Asparagus officinalis L.. The fingerprint of hydrolytic amino acids in Asparagus officinalis L. was established and 11 amino acids were quantitated. To further understand the material basis for the efficiency of Asparagus officinalis L., the chemical constituents in Asparagus officinalis L. were studied. Two purines, five flavonoid glycosides and one organic acid were separated from Asparagus officinalis L. for the first time. The methods for preparation of total flavonoids of Asparagus officinalis L. and its quantitative analysis were developed. It was very meaningful for the further study of flavonoids in Asparagus officinalis L.. This paper also established the preparation method for five flavonoid glycosides from Asparagus officinalis L. with two-dimensional column switch technology or HSCCC-PHPLC method. The two methods were superior to the traditional ones for its higher efficiency, less reagent consumption et al.. The purified flavonoids extract of Asparagus officinalis L. was analyzed by comprehensive two dimensional HPLC. The interactions between the components in Asparagus officinalis L. and liposome membrane were studied. Finally, the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L. were studied. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated.
一、芦笋中营养成分的质量控制
1、氨基酸在pH5.5时与茚三酮反应形成蓝紫色化合物,颜色的深浅与溶液中氨基酸的含量成正比,且在570nm处有最大吸收,利用此原理,测定该波长处的吸光度即可计算氨基酸浓度。本实验以L-谷氨酸为标准品对方法学进行了全面考察,线性回归方程为A=0.0073C-0.1133(r=0.9998),线性范围30.30-151.50μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。十批不同来源的鲜芦笋样品中总游离氨基酸含量为17.43-22.43‰。
2、考马斯亮蓝G-250在游离状态下呈红色,在稀酸中与蛋白质结合变成蓝色,最大吸收波长也从465nm变成595nm,蛋白质与考马斯亮蓝G-250结合物在595nm波长下的吸光度与蛋白质含量成正比,因此,可以根据此原理测定蛋白质含量。本实验以牛血清白蛋白为标准品对方法学进行了全面的考察,线性回归方程为A=0.0077C+0.0524(r=0.9996),线性范围19.80-99.00μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。十批不同来源的鲜芦笋样品中总蛋白质含量为1.08-1.69‰。
3、芦笋中总多糖的含量测定采用的是硫酸蒽酮显色法,以葡萄糖为标准品,线性方程为A=0.0081C+0.0907(r=0.9990),线性范围20.00-100.00μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。十批不同来源的干燥芦笋样品中总多糖含量为1.16-1.64%。
4、选择AQC为衍生化试剂,以普通C18柱作为分离载体,建立了芦笋水解氨基酸的指纹图谱,确定了16个共有峰,鉴定了其中的11个氨基酸,并在指纹图谱条件的基础上测定了10批芦笋中11种氨基酸的含量,结果如下:组氨酸2.97-5.56‰,甘氨酸0.39-0.53‰,天冬氨酸1.64-3.63‰,谷氨酸0.89-1.55‰,苏氨酸0.32-0.46‰,丙氨酸0.32-0.57‰,胱氨酸0.47-1.01‰,酪氨酸0.23-0.28‰,缬氨酸0.36-0.65‰,亮氨酸0.37-0.60‰,苯丙氨酸0.35-0.44‰。
二、芦笋的化学成分研究
1、芦笋中两种嘌呤类化合物的分离制备
干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱,收集20%乙醇洗脱流分,再经葡聚糖凝胶柱脱色,纯化后的样品采用制备液相色谱法分离,得到两种嘌呤类化合物,经1HNMR和13CNMR鉴定为9-(2-甲硫基-3-磺酸基-4-羟基)-呋喃-6-氨基-嘌呤和腺苷。
2、芦笋中五个黄酮类化合物的分离制备
干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱,收集40%乙醇洗脱流分,再经葡聚糖凝胶柱脱色,纯化后的样品采用制备液相色谱法分离,得到五种黄酮苷,经HPLC-DAD-MS鉴定为槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷。
3、芦笋中一种有机酸的分离制备
干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱,收集60%乙醇洗脱流分,再经葡聚糖凝胶柱纯化,即得纯品,经1HNMR和13CNMR鉴定为肉桂酸。
三、芦笋黄酮部位的制备与定量分析
1、干燥芦笋药材的60%乙醇回流提取液浓缩后过大孔吸附树脂柱(60×4cm),先静止吸附1h,再分别用水1500mL、20%乙醇2000mL和40%乙醇2000mL依次洗脱。收集40%乙醇部分并于50℃减压浓缩至干,加30%甲醇溶解后,过Sephadex LH-20型葡聚糖凝胶柱(100×3cm),分别用300mL水、200mL50%甲醇和1000mL甲醇依次洗脱,收集含黄酮的流分,合并后于50℃减压浓缩至干,即得芦笋黄酮部位。
2、采用紫外分光光度法,以芦丁为标准品,测定了芦笋黄酮部位中总黄酮含量,并对方法学作了详细考察,线性回归方程为A=0.0024C-0.0174 (r=0.9992),线性范围62.00-310.00μg/mL,样品经显色反应后在1.5h小时内稳定,精密度、重现性和回收率结果均符合要求。所测三批样品中总黄酮的含量均高于50%。
3、采用高效液相色谱法测定了芦笋黄酮部位中槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷的含量,色谱条件如下:色谱柱为Diamonsil○R (钻石)C18柱(200×4.6mm,5μm)(Dikma Technologies),流动相:乙腈-甲醇-0.1%醋酸水(8﹕16﹕76),流速1.0 mL/min,柱温30℃,检测波长260nm,进样量20μL,方法学考察结果符合要求。测定三批样品中槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷的含量分别为11.26-12.62%, 22.64-24.78%, 4.37-5.09%,
4.13-4.89%,3.22-3.63%。
四、芦笋中黄酮苷单体的二维色谱法制备及芦笋黄酮部位的全二维液相色谱分析
1、采用二维柱切换液相色谱分离制备了芦笋中五种黄酮苷单体(槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷),该系统维间切换没有用到定量环,而是用六通切换阀取而代之,这样可以确保组分被全部切换进入第二维,避免了样品损失,且切换体积不受定量环限制。与普通的一维制备液相色谱法相比,该法可以大大提高分离效率,并节约有机溶剂用量。
2、采用HSCCC-PHPLC二维方法从芦笋黄酮粗品中分离制备了五个黄酮苷单体(槲皮素-3-O-葡萄糖-芸香糖苷、芦丁、异鼠李素-3-O-葡萄糖-芸香糖苷、烟花苷和水仙苷)。该方法中,HSCCC作为一种预处理手段,分离得到两个含目标成分的流分,继而通过制备液相色谱进一步分离得到高纯度的目标化合物。该法避免了繁琐的样品柱色谱预处理过程,具有操作简便、制备效率高等优点。
3、采用全二维液相色谱法分析了芦笋黄酮部位,第一维采用的是脂质体柱(250×4.6mm,5μm,自制),流动相为10mmol/L pH 7.4的醋酸铵缓冲液,流速0.1mL/min;第二维采用Chromolith Performance RP-18e (100×4.6mm)整体柱,流动相采用A:乙腈,B:10%乙腈梯度洗脱,0-2min 3%A,2-4min 3%A-17% A,4-4.5min,17% A -3%A,4.5-5.0min,3%A,流速3.0 mL/min。通过考察芦笋中不同成分在脂质体柱上的色谱行为,可以判断各成分与脂质体膜之间的相互作用强弱,从而推断药物在生物体内的ADME。
五、芦笋总黄酮及其黄酮苷单体体外抗氧化活性研究
以VitC为阳性对照药物,系统研究了芦笋总黄酮及其黄酮苷单体的体外抗氧化活性。结果表明,芦笋中的黄酮苷单体有较强的抗氧化活性:能明显清除·OH和O2-,对DPPH自由基也有较强的清楚作用,具有较强的抑制大鼠红细胞氧化溶血的作用。芦笋总黄酮也有相应的体外抗氧化活性,强度与总黄酮含量相关。本实验验证并揭示了芦笋发挥多种药理活性的物质基础和黄酮类化合物抗氧化活性的构效关系。
综上所述,本论文以芦笋的质量控制为目的,建立了总游离氨基酸、总蛋白质和总多糖三大营养成分的定量分析方法,建立了芦笋水解氨基酸的指纹图谱并对其中11种氨基酸作了定量分析。为了进一步了解芦笋发挥药理活性的物质基础,本论文对芦笋的化学成分作了初步研究,首次从芦笋中分离出两种嘌呤类化合物、五种黄酮苷和一种有机酸。建立了芦笋黄酮部位的制备方法和定量分析方法,为今后进一步开发芦笋黄酮打下了基础。本论文还建立了芦笋中五种黄酮苷的二维柱切换液相色谱制备方法和HSCCC-PHPLC二维制备方法,与传统制备液相色谱法相比,这两种方法具有制备效率高、溶剂消耗少等优点。本论文采用全二维液相色谱法分离分析了芦笋黄酮部位,考察了芦笋中不同成分与脂质体膜之间的相互作用。最后,本论文对芦笋黄酮部位和芦笋中五种黄酮苷的体外抗氧化活性作了系统考察,验证并揭示了芦笋发挥药理活性的物质基础和黄酮类化合物抗氧化活性的构效关系。
Asparagus officinalis L. is one of the ten most famous vegetables of the world. It is delicious and eutrophy, and is good for health. Recent studies have found that Asparagus officinalis L. can be used to cure cancer, lower blood-lipid, enhance immunity, anti-aging, anti-fatigue and protect liver. So far as we know, there are only a few papers about the chemical composition of Asparagus officinalis L. These papers are about the separation and purification of total flavonoids and total saponins. So, it is difficult to control the quality of Asparagus officinalis L. fully and efficiently. This study detected the nutritional ingredients in Asparagus officinalis L. and then identified the purified compounds in Asparagus officinalis L. Next, total flavonoids of Asparagus officinalis L. was prepared and analyzed. The preparation method of the five flavonoid glycosides from Asparagus officinalis L. was optimized using two dimensional prep-HPLC and the total flavonoids of Asparagus officinalis L. was analyzed with comprehensive two dimensional HPLC to study the interactions between the components in Asparagus officinalis L. and the liposome membrane. Finally, the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L. were studied. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated.
1. Quality control of the nutritional ingredients in Asparagus officinalis L.
1.1 Amino acids react with ninhydrin to generate a kind of hepatic complex at pH 5.5 and the shade of the color varies directly with the contents of the amino acids. The complex has the strongest absorption at the wavelength of 570 nm. So, the contents of the amino acids can be determined with this principle. This paper fully studied the technology of this method using L-Glu as reference substance. The equation of the linear regression was A=0.0073C-0.1133 (r=0.9998) and the linear range was 30.30-151.50μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of amino acids in ten batches of Asparagus officinalis L. were 17.43-22.43‰.
1.2. Coomassie brilliant blue G-250 is red at free state. It changes into blue when combines with proteins in dilute acid and the wavelength with the strongest absorption changes into 595 nm instead of 465 nm. The absorbance of the conjugate at 595 nm varies directly with the contents of proteins. This paper fully studied the technology of this method using BSA as reference substance. The equation of the linear regression was A=0.0077C+0.0524 (r=0.9996) and the linear range was 19.80-99.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of proteins in ten batches of Asparagus officinalis L. were 1.08-1.69‰.
1.3. The total polysaccharides in dry Asparagus officinalis L. was determined by coloration with sulphuric acid and anthrone. This paper fully studied the technology of this method using glucose as reference substance. The equation of the linear regression was A=0.0081C+0.0907 (r=0.9990) and the linear range was 20.00-100.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of total polysaccharides in ten batches of dry Asparagus officinalis L. were 1.16-1.64 %.
1.4 The fingerprint of hydrolytic amino acids in Asparagus officinalis L. was established on a common C18 column using AQC as the derivatization reagent. Sixteen peaks were defined as common peaks and 11 of them were identified. Eleven amino acids in ten batches of Asparagus officinalis L. were determined. The results were as follows: histidine 2.97-5.56‰, glycine 0.39-0.53‰, aspartic acid 1.64-3.63‰, glutamic acid 0.89-1.55‰, threonine 0.32-0.46‰, alanine 0.32-0.57‰, cystine 0.47-1.01‰, tyrosine 0.23-0.28‰, valine 0.36-0.65‰, leucine 0.37-0.60‰, phenylalanine 0.35-0.44‰.
2. Studies on the chemical constituents from Asparagus officinalis L.
2.1 Separation and purification of two purines from Asparagus officinalis L. The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 20% ethanol was collected and decolored with polydextran gel. The purified sample was separated with prep-HPLC and two compounds were prepared. They were identified as 9-(2-methylthio-3-sulfo-4-hydroxy)-furan-6-aminopurine and adenosine by 1HNMR and 13CNMR.
2.2 Separation and purification of five flavonoid glycosides from Asparagus officinalis L.
The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 40% ethanol was collected and decolored with polydextran gel. The purified sample was separated with prep-HPLC and five compounds were prepared. They were identified as quercetin-3-O-glucosyl- rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin by HPLC-DAD-MS.
2.3 Separation and purification of an organic acid from Asparagus officinalis L.
The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 60% ethanol was collected and purified with polydextran gel. The compound was identified as cinnamic acid by 1HNMR and 13CNMR.
3. Preparation of purified flavonoids extract of Asparagus officinalis L. and quantitative analysis
3.1. The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins (60×4cm). The extract was adsorbed by the resins for 1 h, and then eluted with 1500 mL of water, 2000 mL of 20% ethanol and 2000 mL of 40% ethanol. The fraction of 40% ethanol was collected and evaporated to dryness by rotary evaporation at 50℃under reduced pressure. The extract was dissolved in 30% methanol and then processed with polydextran gel (100×3cm). It was eluted with 300 mL of water, 200 mL of 50% methanol and 1000 mL of methanol. The fractions containing flavonoids were combined and evaporated to dryness by rotary evaporation at 50℃under reduced pressure. That was the purified flavonoids extract of Asparagus officinalis L..
3.2. The contents of total flavonoids in the purified flavonoids extract of Asparagus officinalis L. were determined with ultraviolet spectrophotometry. This paper fully studied the technology of this method using rutin as reference substance. The equation of the linear regression was A=0.0024C-0.0174 (r=0.9992) and the linear range was 62.00-310.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of total flavonoids in three batches of samples were >50 %.
3.3. The contents of quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin in the purified flavonoids extract of Asparagus officinalis L. were determined with HPLC. Diamonsil○R C18 column (200×4.6mm,5μm, Dikma Technologies) was used for separation. The mobile phase was acetonitrile-methanol-0.1% HAc (8﹕16﹕76). The flue rate was 1.0 mL/min. The column temperature was set at 30℃and the detection was set at 260 nm. The injection volume was 20μL. This paper fully studied the technology of this method and all the results were within the criteria. The contents of quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl-rutinoside, nicotiflorin and narcissin in three batches of samples were 11.26-12.62%, 22.64-24.78%, 4.37-5.09%, 4.13-4.89% and 3.22-3.63%, respectively.
4. Preparation of five flavonoid glycosides from Asparagus officinalis L. by two-dimensional methods and the separation of the flavonoids using comprehensive two dimensional HPLC
4.1. Five flavonoid glycosides (quercetin-3-O-glucosyl- rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin) were purified from Asparagus officinalis L. by two-dimensional prep-HPLC with column switch technology. A six-port two-position switching valve instead of a sample loop was used in this system. This approach assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension were not restricted. Compared with traditional prep-HPLC, this method enhanced the production rate greatly and saved much organic solvent.
4.2. Five flavonoid glycosides (quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl-rutinoside, nicotiflorin and narcissin) were purified from Asparagus officinalis L. by HSCCC-PHPLC. In this method, HSCCC was used as a pretreatment process. Two fractions containing the target compounds were obtained by HSCCC. Then, they were further separated using PHPLC and the target compounds with high purity were finally obtained. This method was simple and high-performance without tedious pretreatment process using column chromatography.
4.3. The purified flavonoids extract of Asparagus officinalis L. was analyzed by comprehensive two dimensional HPLC. In the first dimension, a liposome column was used with a flow rate of 0.1 mL/min. And in the second dimension, a Chromolith Performance RP-18e (100×4.6mm) column was used. The mobile phase consisted of A: acetonitrile, B: 10% acetonitrile. The gradient progress was as follows: 0-2min 3%A,2-4min 3%A-17% A,4-4.5min,17% A -3%A,4.5-5.0min,3%A, and the flow rate was 3.0 mL/min. The interactions between the components in Asparagus officinalis L. and liposome membrane could be judged according to the chromatographic behavior of these components on a liposome column. And the ADME of the drugs could also be concluded.
5. Studies on the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L.
The in vitro scavenging activities of total flavonoids of Asparagus officinalis L. and five flavonoid glycosides on superoxide radical, hydroxyl radical and DPPH, and their protective effects on H2O2-induced hemolysis of rat erythrocytes were investigated. The results suggested that total flavonoids and five flavonoid glycosides from Asparagus officinalis L. showed significant antioxidant activities in vitro. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated. In a word, this study was carried out to control the quality of Asparagus officinalis L. The quantitation methods were developed to determine total free amino acids, total proteins and total polysaccharides in Asparagus officinalis L.. The fingerprint of hydrolytic amino acids in Asparagus officinalis L. was established and 11 amino acids were quantitated. To further understand the material basis for the efficiency of Asparagus officinalis L., the chemical constituents in Asparagus officinalis L. were studied. Two purines, five flavonoid glycosides and one organic acid were separated from Asparagus officinalis L. for the first time. The methods for preparation of total flavonoids of Asparagus officinalis L. and its quantitative analysis were developed. It was very meaningful for the further study of flavonoids in Asparagus officinalis L.. This paper also established the preparation method for five flavonoid glycosides from Asparagus officinalis L. with two-dimensional column switch technology or HSCCC-PHPLC method. The two methods were superior to the traditional ones for its higher efficiency, less reagent consumption et al.. The purified flavonoids extract of Asparagus officinalis L. was analyzed by comprehensive two dimensional HPLC. The interactions between the components in Asparagus officinalis L. and liposome membrane were studied. Finally, the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L. were studied. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated.
引文
[1]方幼兰,刘艳如,林少琴,等.芦笋多糖的研究.福建师范大学学报(自然科学版),1995,11(2):69-73
[2]赵静,安利国,张福森,等. RP—HPLC法测定芦笋中黄酮类化合物芦丁的含量.中国野生植物资源,2007,26(3):61-63
[3]张素华,王正云.大孔树脂纯化芦笋黄酮工艺的研究.食品科学,2006,27(2)182-186
[4]孙春艳,赵伯涛,郁志芳,等.芦笋的化学成分及药理作用研究进展.中国野生植物资源,2004,23(5)1-5
[5]顾关云,蒋昱.芦笋的化学成分和生物活性.国外医药·植物药分册,2007,22(2):47-50
[6]安玉会,吴铁汉,郑香梅,等.芦笋口服液中氨基酸含量分析.河南医科大学学报,1998,33(4):12-13
[7]夏俊,陈治文,石莹.绿芦笋提取液抑制肿瘤细胞核酸生物合成的研究.癌变·畸变·突变,2003,15(4):212-214
[8]毕肖林,郭胜伟,狄留庆.中华芦荟多糖提取和粗多糖中总糖的含量测定.南京中医药大学学报,2005,21(4):269
[9]赵英永,戴云,崔秀明,等.考马斯亮蓝G-250染色法测定草乌中可溶性蛋白质含量.云南民族大学学报(自然科学版),2006,15(3):235-237
[10]曲春香,沈颂东,王雪峰,等.用考马斯亮蓝测定植物粗提液中可溶性蛋白质含量方法的研究.苏州大学学报(自然科学版),2006,22(2):82-85
[11]谢培山.中药现代化的取向与质量控制模式.中药新药与临床药理,2002,13(4):201-203
[12]朱立中.中药指纹图谱对药品法规修订的影响.世界科学技术——中药现代化,2001,3(1):35-36
[13]谢培山.中药色谱指纹图谱质量控制模式的研究和应用.世界科学技术——中药现代化,2001,3(6):28-32
[14] Kang J, Sun JH, Zhou L, et al. Characterization of compounds from the roots of Saposhnikovia divaricata by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Rapid Commun MassSpectrom. 2008, 22(12):1899-1911
[15] Han J, Ye M, Qiao X, et al. Characterization of phenolic compounds in the Chinese herbal drug Artemisia annua by liquid chromatography coupled to electrospray ionization mass spectrometry. J Pharm Biomed Anal. 2008, 47(3): 516-525
[16] Zhao HY, Sun JH, Fan MX, et al. Analysis of phenolic compounds in Epimedium plants using liquid chromatography coupled with electrospray ionization mass spectrometry. J Chromatogr A. 2008, 1190 (1-2): 157-181
[17] Wang K, Liu Z, Huang JA, et al. Preparative isolation and purification of theaflavins and catechins by high-speed countercurrent chromatography. J Chromatogr. B , 2008, 867(2): 282-286
[18] Liu R, Wu S, Sun A. Separation and purification of four chromones from radix saposhnikoviae by high-speed counter-current chromatography. Phytochem Anal., 2008, 19(3): 206-211
[19] Shi S, Zhang Y, Zhao Y, et al. Preparative isolation and purification of three flavonoid glycosides from Taraxacum mongolicum by high-speed counter-current chromatography. J Sep Sci., 2008, 31(4): 683-688
[20] Wang X, Shi X, Li F, et al. Application of analytical and preparative high-speed counter-current chromatography for the separation of Z-ligustilide from a crude extract of Angelica sinensis. Phytochem Anal., 2008, 19(3):193-197
[21] Luo Y, Xu Y, Chen L, et al. Preparative purification of anti-tumor derivatives of honokiol by high-speed counter-current chromatography.J Chromatogr A., 2008, 1178 (1-2): 160-165
[22] Zhou T, Zhao W, Fan G, et al. Isolation and purification of iridoid glycosides from Gardenia jasminoides Ellis by isocratic reversed-phase two-dimensional preparative high-performance liquid chromatography with column switch technology. J Chromatogr B, 2007, 858 (1-2): 296-301
[23] Zhao W, Zhou T, Fan G, et al. Isolation and purification of lignans from Magnolia biondii Pamp by isocratic reversed-phase two-dimensional liquid chromatography following microwave-assisted extraction. J Sep Sci., 2007, 30 (15): 2370-2381
[24] Xu M, Wang G, Xie H, et al. Pharmacokinetic comparisons of schizandrin after oral administration of schizandrin monomer, Fructus Schisandrae aqueous extract and Sheng-Mai-San to rats. J Ethnopharmacol. 2008, 115 (3): 483-488
[25] Dong L, Deng C, Wang B, et al. Fast determination of Z-ligustilide in plasma by gas chromatography/mass spectrometry following headspace single-drop microextraction. J Sep Sci. 2007, 30 (9):1318-1325
[26] Li X, Wang G, Sun J, et al. Pharmacokinetic and absolute bioavailability study of total panax notoginsenoside, a typical multiple constituent traditional chinese medicine (TCM) in rats. Biol Pharm Bull. 2007, 30 (5): 847-851
[27] Yu Z, Gao X, Zhao Y, et al. HPLC determination of safflor yellow A and three active isoflavones from TCM Naodesheng in rat plasma and tissues and its application to pharmacokinetic studies. Biomed Chromatogr. 2007, 21(6): 577-584
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[2]黄晓德,赵伯涛,钱骅.芦笋茎叶游离氨基酸的提取及含量测定.中国野生植物资源,2006,25(4):61-63
[3]彭丽萍,蒋伯成,周浩然,等.芦笋叶中氨基酸含量的测定.齐齐哈尔大学学报,2000,16(4):52-53
[4]叶春勇,林媚.芦笋营养成分分析研究.中国果菜,2005,2:37-38
[5]邵金良,黎其万,董宝生,等.茚三酮比色法测定茶叶中游离氨基酸总量.中国食品添加剂,2008,2:162-165
[6]范婉萍,吴婕.用分光光度法测定含氨基酸类制品中的氨基酸含量.中国测试技术,2007,33(3):117-119
[7]林文津,徐榕青,张亚敏.正交实验优化中药太子参氨基酸超声提取工艺.海峡药学,2006,18(1):33-35
[8]陈景勇,彭奇均.紫外-可见分光法定量测定谷氨酸的比较.光谱实验室,2008,25(3):394-397
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[14]王文平,郭祀远,李琳,等.考马斯亮蓝法测定野木瓜多糖中蛋白质的含量.食品研究与开发,2008,29(1):115-117
[15]陈晓梅,刘雅文,程熠,等.考马斯亮蓝法蛋白定量标准曲线稳定性观察.中国公共卫生,2006,22(3):380-381
[16]陆建良,梁月荣,张凌云.考马斯亮蓝法在茶汤可溶性蛋白含量分析中的应用和改良.茶叶,2002,28(2):89-93
[17]曹稳根,焦庆才,刘茜,等.考马斯亮蓝显色剂变色反应机理的研究.化学学报,2002,60(9):1656-1661
[18]李娟,张耀庭,曾伟,等.应用考马斯亮蓝法测定总蛋白含量.中国生物制品学杂志,2000,13(2):118-120
[19]曲春香,沈颂东,王雪峰,等.用考马斯亮蓝测定植物粗提液中可溶性蛋白质含量方法的研究.苏州大学学报(自然科学版),2006,22(2):82-85
[20]毕肖林,郭胜伟,狄留庆.中华芦荟多糖提取和粗多糖中总糖的含量测定.南京中医药大学学报,2005,21(4):269
[21]李金忠,马海乐,吴沿友.超声波提取山药多糖的研究.粮油食品科技,2005,13(5):14-15
[22]周静华,汪祖芳,李芬.大理百合中多糖的提取与总糖含量的测定.医药产业资讯,2006,5(6):735
[23]王华付.黄芪中黄芪多糖含量的测定.安徽农业科学,2007,35(34):10948,11021
[24]陈平,陈新,王珲,等.硫酸-蒽酮法测定鄂产竹节参多糖含量.中国医院药学杂志,2007,27(12):1654-1656
[25]季宇彬,陈学军,汲晨锋,等.芦笋多糖提取、单糖组分分析及定量测定.中草药,2006,37(8):1159-1161
[26]黄晓德,赵伯涛,钱骅,等.芦笋茎叶多糖的提取纯化研究.江西农业学报,2006,18(1):15-18
[27]黄莺莺.芦笋中17种氨基酸反相高压液相色谱分析法的快速测定,仪器仪表与分析检测,2001,3:32-34
[1]赵静,安利国,张福森,等. RP—HPLC法测定芦笋中黄酮类化合物芦丁的含量.中国野生植物资源,2007,26(3):61-63
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[3]刘树兴,魏丽娜,李红.超声法提取芦笋老茎中黄酮类物质的研究.食品科学,2006,27(11):360-363
[4]张素华,王正云.大孔树脂纯化芦笋黄酮工艺的研究.食品科学,2006,27(2):182-186
[5]朱立华,孙萍,曹国红,等.反相高效液相色谱法测定芦笋各段芦丁的含量.济南大学学报(自然科学版),2007,21(1):53-55
[6]宋元清,王艳平,毛远菁.分光光度法测定芦笋中总黄酮的含量.化学分析计量,2005,14(4):52-53
[7]李素珍,张贵生.芦笋不同色泽和部位嫩茎的化学成分分析.浙江农业学报,1994,6(1):54-57
[8]顾关云,蒋昱.芦笋的化学成分和生物活性.国外医药·植物药分册,2007,22(2):47-50
[9]周利亘,王春辉,王君虹,等.芦笋的活性成分及其生物学功能.安徽农学通报,2006,12(2):23-25,116
[10]李长秀,张铁垣,李华民.芦笋根部化学成份的分析研究I.非皂化物及脂肪酸成份分析.质谱学报,1995,16(4):31-36
[11]孙建华,左春旭,杨尚军,等.芦笋茎皮化学成分的研究.中草药,1999,30(12):888-890
[12]孙春艳,赵伯涛,郁志芳,等.芦笋茎叶中黄酮类化合物的提取工艺研究.食品工业科技,2006,27(4):141-145
[13]张素华,王正云,葛庆丰.芦笋中活性物质提取技术参数及组分鉴定的研究.食品科学,2005,26(2):138-141
[14]安玉会,吴铁汉,郑香梅,等.芦笋中二种皂角甙类化合物的提取和分离.河南医科大学学报,1998,33(4):14-15
[15]王祝举,唐力英,付梅红,等.夏枯草中的黄酮类化合物研究.时珍国医国药,2008,19(8):1966-1967
[16]陈鸿雁,程伟贤,冯宇,等.单叶蔓荆子黄酮类化学成分研究.天然产物研究与开发,2008,20(4):582-584
[17]辛文波,侴桂新,王峥涛.毛钩藤叶的化学成分.中国天然药物,2008,6(4):262-264.
[18]刘玉峰,杨秀伟,武滨.款冬花化学成分的研究.中国中药杂志,2007,32(22):2378-2381
[19]陈雏,张浩,顾恒,等.中国沙棘果实中的黄酮苷类成分.华西药学杂志,2007,22(4):367-370
[20]唐柳怡,罗明华,蒋宁,等.血满草化学成分的研究.中药材,2007,30(5):549-551
[21]吴霞,刘净,王志斌,等.薰衣草中黄酮类化学成分的研究.中国中药杂志,2007,32(9):821-823
[22]陈君,许小方,柴兴云,等.灰毡毛忍冬花蕾的化学成分.中国天然药物,2006,4(5):347-351
[23]陈改敏,张建业,张向沛,等.马鞭草黄酮类化学成分的研究.中药材,2006,29(7):677-679
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[25]许琼明,石峰,徐丽珍,等.瘤果紫玉盘叶中的黄酮类成分.沈阳药科大学学报,2006,23(5):277-279
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[2]于鹏飞,王建平,蒋海强,等.甘草中黄酮类抗氧化成分的提取工艺研究.食品与药品,2008,10(5):25-27
[3]姚石祥,李百强,崔晓彪,等.淫羊藿总黄酮大孔树脂精制工艺研究.中外健康文摘:医药月刊,2008,5(8):927-928
[4]杜晖,陈晓青,黄冰.大孔吸附树脂分离纯化夏枯草总黄酮的工艺研究.中成药,2008,30(9):1294-1298
[5]王薇,康亚国.葛根总黄酮提取纯化工艺研究.陕西中医学院学报,2007,30(6):53-54
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[7]花蕾,张文清,夏玮.桑叶总黄酮的大孔树脂纯化工艺.中成药,2007,29(12):1758-1761
[8]张书燕.蒲公英总黄酮的提取工艺研究.山东中医杂志,2007,26(12):858-859
[9]黄芸,崔力剑,窦玉红,等.紫外分光光度法测定大豆中异黄酮的含量.大豆科学,2007,2:273-275
[10]闻永举,陈春艳,申秀丽.知母总黄酮含量的测定研究.宜春学院学报,2007,29(6):79-80
[11]邓卫萍,解成喜,艾尔肯·艾米尔.新疆阿魏中总黄酮的提取分离及含量测定.天然产物研究与开发,2007,19(B11):450-452,464
[12]李伟荣,刘昌辉,洪馨,等.广东梅县柚果中总黄酮和柚皮苷的含量测定.中国高校科技与产业化,2006,z1:220-221
[13]周惠燕,李士敏.竹叶中麦黄酮的分离鉴定及含量测定.中药材,2006,29(12):1301-1302
[14]郑岩,王邠,王京丽,等.高效液相测定不同产地鸡血藤药材中黄酮类化合物的含量.中国中药杂志,2008,33(15):1920-1922
[15]陈彦,蔡垠,贾晓斌,等.高效液相色谱法测定仙灵脾配方颗粒中5种主要黄酮类成分的含量.中国实验方剂学杂志,2007,13(11):1-3
[16]王东凯,崔福德,杨丽,等. RP—HPLC法测定醋柳黄酮固体脂质纳米粒的含量及包封率.药物分析杂志,2007,27(1):33-35
[1] Wong V., Shalliker R. A., Isolation of the active constituents in natural materials by‘heart-cutting’isocratic reversed-phase two-dimensional liquid chromatography. J. Chromatogr. A, 2004, 1036(1): 15-24
[2] Gray M. J., Dennis G. R., Slonecker P. J., et al. Separation of oligostyrene isomers in a complex mixture using two-dimensional heart-cutting reversed-phased liquid chromatography. J. Chromatogr. A, 2004, 1028(2): 247-257
[3] Gray M. J., Dennis G. R., Slonecker P. J., et al. Comprehensive two-dimensional separations of complex mixtures using reversed-phase reversed-phase liquid chromatography. J. Chromatogr. A, 2004, 1041 (1-2): 101-110
[4] Matsui T., Tamaya K., Kawasaki T., et al. Determination of angiotensin metabolites in human plasma by fluorimetric high-performance liquid chromatography using a heart-cut column-switching technique. J. Chromatogr. B, 1999, 729 (1-2): 89-96
[5] Cortes H. J. (Ed.), L. D. Rothman. Multidimensional Chromatography, Marcel Dekker, New York 1990, pp. 219-248
[6] Kang S. W., Kim M. C., Kim C. Y., et al. The rapid identification of isoflavonoids from Belamcanda chinensis by LC-NMR and LC-MS. Chem. Pharm. Bull., 2008, 56 (10): 1452-1454
[7] Liu H, Berger SJ, Chakraborty AB, et al., Multidimensional chromatography coupled to electrospray ionization time-of-flight mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins. J. Chromatogr. B, 2002, 782 (1-2): 267-289
[8] Machtejevas E, John H, Wagner K, et al., Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate. J. Chromatogr. B, 2004, 803 (1): 121-130
[9] Winther B, Reubsaet JL. Application of supplementary flow in comprehensive 2D liquid chromatography combining SEC and RPC. J. Sep.Sci., 2005, 28(5): 477-482.
[10] Haefliger OP. Universal two-dimensional HPLC technique for the chemical analysis of complex surfactant mixtures. Anal. Chem. 2003, 75(3): 371-378
[11] Kammerer B, Kahlich R, Ufer M, et al., Achiral–chiral LC/LC–MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism. Analytical Biochemistry 2005, 339(2): 297-309
[12] Kimura H, Tanigawa T, Morisaka H, et al., Simple 2D-HPLC using a monolithic silica column for peptide separation. J. Sep.Sci., 2004, 27(10-11): 897-904
[13] Sheldon EM. Development of a LC–LC–MS complete heart-cut approach for the characterization of pharmaceutical compounds using standard instrumentation. J. Pharm. Biomed. Anal. 2003, 31(6): 1153-1166
[14] Gevaert K, Van Damme P, Martens L, et al., Diagonal reverse-phase chromatography applications in peptide-centric proteomics: Ahead of catalogue-omics? J. Anal. Biochem. 2005, 345(1): 18-29
[15] Fraga CG, Bruckner CA, Synovec RE. Increasing the Number of Analyzable Peaks in Comprehensive Two-Dimensional Separations through Chemometrics.Anal. Chem. 2001, 73(3): 675-683
[16] Gross GM, Prazen BJ, Synovec RE. Parallel column liquid chromatography with a single multi-wavelength absorbance detector for enhanced selectivity using chemometric analysis. Analytica Chimica Acta 2003, 490(1-2): 197-210
[17] Hata K., Morisaka H., Hara K., et al., Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns. Anal. Biochem., 2006, 350(2): 292-297
[18] Millea K. M., Kass I. J., Cohen S. A., et al., Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension. J. Chromatogr. A, 2005, 1079 (1-2): 287-298
[19] Kok S. J., Hankemeier Th., Schoenmakers P. J. Comprehensive two-dimensional liquid chromatography with on-line Fourier-transform-infrared-spectroscopy detection for the characterization of copolymers. J. Chromatogr. A, 2005, 1098 (1-2): 104-110
[20] Jandera P., Fischer J., Lahovska H., et al., Ionic liquids versus triethylamine as mobile phase additives in the analysis ofβ-blockers. J. Chromatogr. A, 2006, 1119 (1-2): 3-10
[21] Chen X., Kong L., Su X., et al., Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry. J. Chromatogr. A, 2004, 1040 (2): 169-178
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[3]田京伟,杨建雄.白藜芦醇苷的体外抗氧化活性.中草药,2001,32(10)918-920
[4]赵芳,边丽,胡栋梁.茄子皮红色素抗氧化活性研究.食品与机械,2008,24(2)62-64
[5]徐大量,林辉,李盛青,等.玉竹水提液体内外抗氧化的实验研究.中药材,2008,31(5):729-731
[6]陈琪,王伯初,唐春红,等.黄酮类化合物抗氧化性与其构效的关系.重庆大学学报,2003,26(11):48-51,55
[7]龙春,高志强,陈凤鸣,等.黄酮类化合物的结构—抗氧化活性关系研究进展.重庆文理学院学报(自然科学版),2006,5(2):13-17
[8]秦德安,苏丹,王晓玲.橙皮苷对羟自由基的清除作用.中国药学杂志,1996,31(7):396-398.
[9]高志强,徐强,宋仲容,等.黄酮类化合物抗氧化活性构效关系的密度泛函理论研究.化学世界,2008,49(7):439-443
[10]姜玉兰,朴惠善,李镐.桑叶抗氧化活性成分的研究.中药材,2008,31(4):519-522
[11]焦士蓉,马力,黄承钰,等.枳实提取物的体外抗氧化作用研究.中药材,2008,31(1):113-116
[12]张琳,陆维敏.黄酮类化合物抗氧化性能与其结构的关系.浙江大学学报(理学版),2006,33(2):187-191
[13]周欣,范国荣,吴玉田.短瓣金莲花总黄酮及指标性成分的体外抗氧化活性研究.中药材,2007,30(8):1000-1002
[14]汪河滨,周忠波,罗锋,等.甘草黄酮提取方法及抗氧化活性研究.时珍国医国药,2008,19(9):2106-2107
[15]李伟,张应团.火棘多酚类物质的体外抗氧化作用.食品工业科技,2008,29(9):121-123
[1]王智聪,张庆合,赵中一,等.分析化学,2005,33(5):722-728
[2]田晶,路鑫,杨军,等.色谱,2005,23(1):32-36
[3] Y. Mao and PW. Carr. Anal. Chem., 2000, 72: 110.
[4] P. J. Slonecker, X. Li, T. H. Ridgway, et al., Anal. Chem., 1996, 68: 682
[5] P. Jandera, K. Novotna, L. Kolarova, et al., Chromatographia, 2004; 60: S27
[6] F. Cacciola, P. Jandera, E. Blahova, et al., J. Sep.Sci., 2006, 29: 2500-2513
[7] J. C. Giddings, J. Chromatogr. A, 1995; 703: 3
[8] M. Gilar, P. Olivova, A. E. Daly, et al., Anal. Chem., 2005, 77: 6426
[9] Sai Ma, Lingxin Chen, Guoan Luo, et al., J. Chromatogr. A, 2006, 1127: 207
[10] J. M. Davis. Anal. Chem., 1993, 65: 2014
[11] K. Rowe and J. M. Anal. Chem., 1995, 67: 2981
[12] K. Rowe, D. Bowlin, M. Zou, et al., Anal. Chem. 1995, 67: 2994
[13] Schure MR. J. Pharma. Biomed. Anal., 1997, 9: 169
[14] S. P. Dixon, I. D. Pitfield and D. Perrett. Biomed. Chromatogr., 2006, 20: 508-529
[15] G. J. Opiteck and J. W. Jorgenson. Anal. Chem., 1997, 69: 2283
[16] A. Van der Horst and P. J. Schoenmakers. J. Chromatogr. A, 2003, 1000: 693.
[17] Mondello L, Tranchida PQ, Stanek V, et al., J. Chromatogr. A, 2005; 1086: 91.
[18] Dugo P, Favoino O, Luppino R, et al., Anal. Chem. 2004; 76: 2525.
[19] Stoll DR and Carr PW. Journal of the American Chemical Society 2005; 127: 5034.
[20] Ikegami T, Hara T, Kimura H, et al., J. Chromatogr. A, 2006; 1106: 112.
[21] Liu H, Berger SJ, Chakraborty AB, et al., J. Chromatogr. B, 2002; 782: 267.
[22] Machtejevas E, John H, Wagner K, et al., J. Chromatogr. B, 2004; 803: 121.
[23] Wagner K, Miliotis T, Marko-Varga G, et al., Anal. Chem. 2002; 74: 809.
[24] Winther B and Reubsaet JL. J. Sep.Sci., 2005; 28: 477.
[25] Haefliger OP. Anal. Chem. 2003; 75: 371.
[26] Venkatramani CJ and Zelechonok Y. Anal. Chem. 2003; 75: 3152.
[27] Kammerer B, Kahlich R, Ufer M, et al., Analytical Biochemistry 2005; 339: 297.
[28] Kimura H, Tanigawa T, Morisaka H, et al., J. Sep.Sci., 2004; 27: 897.
[29] Sheldon EM. J. Pharm. Biomed. Anal. 2003; 31: 1153.
[30] Gevaert K, Van Damme P, Martens L, et al., J. Anal. Biochem. 2005; 345: 18.
[31] Fraga CG, Bruckner CA and Synovec RE. Anal. Chem. 2001; 73: 675.
[32] Gross GM, Prazen BJ and Synovec RE. Analytica Chimica Acta 2003; 490: 197.
[33] K. Hata, H. Morisaka, K. Hara, et al., Anal. Biochem., 2006, 350: 292-297
[34] K. M.Millea, I. J. Kass, S. A. Cohen, et al., J. Chromatogr. A, 2005, 1079: 287-298
[35] S. J. Kok, Th. Hankemeier, P. J. Schoenmakers, J. Chromatogr. A, 2005,1098: 104-110
[36] P. Jandera, J. Fischer, H. Lahovska, et al., J. Chromatogr. A, 2006, 1119: 3-10
[37] X. Chen, L. Kong, X. Su, et al., J. Chromatogr. A, 2004, 1040: 169-178
[38] X. Chen, L. Kong, X. Su, et al., J. Chromatogr. A, 2005, 1089: 87-100
[39] X. Chen, L. Hu, X. Su, et al., J. Pharm. Biomed. Anal., 2006, 40: 559-570
[40] P. Dugo, O. Favoino, R. Luppino, et al., Anal. Chem., 2004, 76: 2525-2530
[2]赵静,安利国,张福森,等. RP—HPLC法测定芦笋中黄酮类化合物芦丁的含量.中国野生植物资源,2007,26(3):61-63
[3]张素华,王正云.大孔树脂纯化芦笋黄酮工艺的研究.食品科学,2006,27(2)182-186
[4]孙春艳,赵伯涛,郁志芳,等.芦笋的化学成分及药理作用研究进展.中国野生植物资源,2004,23(5)1-5
[5]顾关云,蒋昱.芦笋的化学成分和生物活性.国外医药·植物药分册,2007,22(2):47-50
[6]安玉会,吴铁汉,郑香梅,等.芦笋口服液中氨基酸含量分析.河南医科大学学报,1998,33(4):12-13
[7]夏俊,陈治文,石莹.绿芦笋提取液抑制肿瘤细胞核酸生物合成的研究.癌变·畸变·突变,2003,15(4):212-214
[8]毕肖林,郭胜伟,狄留庆.中华芦荟多糖提取和粗多糖中总糖的含量测定.南京中医药大学学报,2005,21(4):269
[9]赵英永,戴云,崔秀明,等.考马斯亮蓝G-250染色法测定草乌中可溶性蛋白质含量.云南民族大学学报(自然科学版),2006,15(3):235-237
[10]曲春香,沈颂东,王雪峰,等.用考马斯亮蓝测定植物粗提液中可溶性蛋白质含量方法的研究.苏州大学学报(自然科学版),2006,22(2):82-85
[11]谢培山.中药现代化的取向与质量控制模式.中药新药与临床药理,2002,13(4):201-203
[12]朱立中.中药指纹图谱对药品法规修订的影响.世界科学技术——中药现代化,2001,3(1):35-36
[13]谢培山.中药色谱指纹图谱质量控制模式的研究和应用.世界科学技术——中药现代化,2001,3(6):28-32
[14] Kang J, Sun JH, Zhou L, et al. Characterization of compounds from the roots of Saposhnikovia divaricata by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Rapid Commun MassSpectrom. 2008, 22(12):1899-1911
[15] Han J, Ye M, Qiao X, et al. Characterization of phenolic compounds in the Chinese herbal drug Artemisia annua by liquid chromatography coupled to electrospray ionization mass spectrometry. J Pharm Biomed Anal. 2008, 47(3): 516-525
[16] Zhao HY, Sun JH, Fan MX, et al. Analysis of phenolic compounds in Epimedium plants using liquid chromatography coupled with electrospray ionization mass spectrometry. J Chromatogr A. 2008, 1190 (1-2): 157-181
[17] Wang K, Liu Z, Huang JA, et al. Preparative isolation and purification of theaflavins and catechins by high-speed countercurrent chromatography. J Chromatogr. B , 2008, 867(2): 282-286
[18] Liu R, Wu S, Sun A. Separation and purification of four chromones from radix saposhnikoviae by high-speed counter-current chromatography. Phytochem Anal., 2008, 19(3): 206-211
[19] Shi S, Zhang Y, Zhao Y, et al. Preparative isolation and purification of three flavonoid glycosides from Taraxacum mongolicum by high-speed counter-current chromatography. J Sep Sci., 2008, 31(4): 683-688
[20] Wang X, Shi X, Li F, et al. Application of analytical and preparative high-speed counter-current chromatography for the separation of Z-ligustilide from a crude extract of Angelica sinensis. Phytochem Anal., 2008, 19(3):193-197
[21] Luo Y, Xu Y, Chen L, et al. Preparative purification of anti-tumor derivatives of honokiol by high-speed counter-current chromatography.J Chromatogr A., 2008, 1178 (1-2): 160-165
[22] Zhou T, Zhao W, Fan G, et al. Isolation and purification of iridoid glycosides from Gardenia jasminoides Ellis by isocratic reversed-phase two-dimensional preparative high-performance liquid chromatography with column switch technology. J Chromatogr B, 2007, 858 (1-2): 296-301
[23] Zhao W, Zhou T, Fan G, et al. Isolation and purification of lignans from Magnolia biondii Pamp by isocratic reversed-phase two-dimensional liquid chromatography following microwave-assisted extraction. J Sep Sci., 2007, 30 (15): 2370-2381
[24] Xu M, Wang G, Xie H, et al. Pharmacokinetic comparisons of schizandrin after oral administration of schizandrin monomer, Fructus Schisandrae aqueous extract and Sheng-Mai-San to rats. J Ethnopharmacol. 2008, 115 (3): 483-488
[25] Dong L, Deng C, Wang B, et al. Fast determination of Z-ligustilide in plasma by gas chromatography/mass spectrometry following headspace single-drop microextraction. J Sep Sci. 2007, 30 (9):1318-1325
[26] Li X, Wang G, Sun J, et al. Pharmacokinetic and absolute bioavailability study of total panax notoginsenoside, a typical multiple constituent traditional chinese medicine (TCM) in rats. Biol Pharm Bull. 2007, 30 (5): 847-851
[27] Yu Z, Gao X, Zhao Y, et al. HPLC determination of safflor yellow A and three active isoflavones from TCM Naodesheng in rat plasma and tissues and its application to pharmacokinetic studies. Biomed Chromatogr. 2007, 21(6): 577-584
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[2]黄晓德,赵伯涛,钱骅.芦笋茎叶游离氨基酸的提取及含量测定.中国野生植物资源,2006,25(4):61-63
[3]彭丽萍,蒋伯成,周浩然,等.芦笋叶中氨基酸含量的测定.齐齐哈尔大学学报,2000,16(4):52-53
[4]叶春勇,林媚.芦笋营养成分分析研究.中国果菜,2005,2:37-38
[5]邵金良,黎其万,董宝生,等.茚三酮比色法测定茶叶中游离氨基酸总量.中国食品添加剂,2008,2:162-165
[6]范婉萍,吴婕.用分光光度法测定含氨基酸类制品中的氨基酸含量.中国测试技术,2007,33(3):117-119
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[8]陈景勇,彭奇均.紫外-可见分光法定量测定谷氨酸的比较.光谱实验室,2008,25(3):394-397
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[14]王文平,郭祀远,李琳,等.考马斯亮蓝法测定野木瓜多糖中蛋白质的含量.食品研究与开发,2008,29(1):115-117
[15]陈晓梅,刘雅文,程熠,等.考马斯亮蓝法蛋白定量标准曲线稳定性观察.中国公共卫生,2006,22(3):380-381
[16]陆建良,梁月荣,张凌云.考马斯亮蓝法在茶汤可溶性蛋白含量分析中的应用和改良.茶叶,2002,28(2):89-93
[17]曹稳根,焦庆才,刘茜,等.考马斯亮蓝显色剂变色反应机理的研究.化学学报,2002,60(9):1656-1661
[18]李娟,张耀庭,曾伟,等.应用考马斯亮蓝法测定总蛋白含量.中国生物制品学杂志,2000,13(2):118-120
[19]曲春香,沈颂东,王雪峰,等.用考马斯亮蓝测定植物粗提液中可溶性蛋白质含量方法的研究.苏州大学学报(自然科学版),2006,22(2):82-85
[20]毕肖林,郭胜伟,狄留庆.中华芦荟多糖提取和粗多糖中总糖的含量测定.南京中医药大学学报,2005,21(4):269
[21]李金忠,马海乐,吴沿友.超声波提取山药多糖的研究.粮油食品科技,2005,13(5):14-15
[22]周静华,汪祖芳,李芬.大理百合中多糖的提取与总糖含量的测定.医药产业资讯,2006,5(6):735
[23]王华付.黄芪中黄芪多糖含量的测定.安徽农业科学,2007,35(34):10948,11021
[24]陈平,陈新,王珲,等.硫酸-蒽酮法测定鄂产竹节参多糖含量.中国医院药学杂志,2007,27(12):1654-1656
[25]季宇彬,陈学军,汲晨锋,等.芦笋多糖提取、单糖组分分析及定量测定.中草药,2006,37(8):1159-1161
[26]黄晓德,赵伯涛,钱骅,等.芦笋茎叶多糖的提取纯化研究.江西农业学报,2006,18(1):15-18
[27]黄莺莺.芦笋中17种氨基酸反相高压液相色谱分析法的快速测定,仪器仪表与分析检测,2001,3:32-34
[1]赵静,安利国,张福森,等. RP—HPLC法测定芦笋中黄酮类化合物芦丁的含量.中国野生植物资源,2007,26(3):61-63
[2]黄雷芳,陈波.本地芦笋中芦丁的提取及其反相色谱鉴定.食品科技,2006,31(7):244-248
[3]刘树兴,魏丽娜,李红.超声法提取芦笋老茎中黄酮类物质的研究.食品科学,2006,27(11):360-363
[4]张素华,王正云.大孔树脂纯化芦笋黄酮工艺的研究.食品科学,2006,27(2):182-186
[5]朱立华,孙萍,曹国红,等.反相高效液相色谱法测定芦笋各段芦丁的含量.济南大学学报(自然科学版),2007,21(1):53-55
[6]宋元清,王艳平,毛远菁.分光光度法测定芦笋中总黄酮的含量.化学分析计量,2005,14(4):52-53
[7]李素珍,张贵生.芦笋不同色泽和部位嫩茎的化学成分分析.浙江农业学报,1994,6(1):54-57
[8]顾关云,蒋昱.芦笋的化学成分和生物活性.国外医药·植物药分册,2007,22(2):47-50
[9]周利亘,王春辉,王君虹,等.芦笋的活性成分及其生物学功能.安徽农学通报,2006,12(2):23-25,116
[10]李长秀,张铁垣,李华民.芦笋根部化学成份的分析研究I.非皂化物及脂肪酸成份分析.质谱学报,1995,16(4):31-36
[11]孙建华,左春旭,杨尚军,等.芦笋茎皮化学成分的研究.中草药,1999,30(12):888-890
[12]孙春艳,赵伯涛,郁志芳,等.芦笋茎叶中黄酮类化合物的提取工艺研究.食品工业科技,2006,27(4):141-145
[13]张素华,王正云,葛庆丰.芦笋中活性物质提取技术参数及组分鉴定的研究.食品科学,2005,26(2):138-141
[14]安玉会,吴铁汉,郑香梅,等.芦笋中二种皂角甙类化合物的提取和分离.河南医科大学学报,1998,33(4):14-15
[15]王祝举,唐力英,付梅红,等.夏枯草中的黄酮类化合物研究.时珍国医国药,2008,19(8):1966-1967
[16]陈鸿雁,程伟贤,冯宇,等.单叶蔓荆子黄酮类化学成分研究.天然产物研究与开发,2008,20(4):582-584
[17]辛文波,侴桂新,王峥涛.毛钩藤叶的化学成分.中国天然药物,2008,6(4):262-264.
[18]刘玉峰,杨秀伟,武滨.款冬花化学成分的研究.中国中药杂志,2007,32(22):2378-2381
[19]陈雏,张浩,顾恒,等.中国沙棘果实中的黄酮苷类成分.华西药学杂志,2007,22(4):367-370
[20]唐柳怡,罗明华,蒋宁,等.血满草化学成分的研究.中药材,2007,30(5):549-551
[21]吴霞,刘净,王志斌,等.薰衣草中黄酮类化学成分的研究.中国中药杂志,2007,32(9):821-823
[22]陈君,许小方,柴兴云,等.灰毡毛忍冬花蕾的化学成分.中国天然药物,2006,4(5):347-351
[23]陈改敏,张建业,张向沛,等.马鞭草黄酮类化学成分的研究.中药材,2006,29(7):677-679
[24]张体灯,潘瑞娥,毕志明,等.金花忍冬地上部分的黄酮类成分研究.中国药学杂志,2006,41(10):741-743
[25]许琼明,石峰,徐丽珍,等.瘤果紫玉盘叶中的黄酮类成分.沈阳药科大学学报,2006,23(5):277-279
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[2]于鹏飞,王建平,蒋海强,等.甘草中黄酮类抗氧化成分的提取工艺研究.食品与药品,2008,10(5):25-27
[3]姚石祥,李百强,崔晓彪,等.淫羊藿总黄酮大孔树脂精制工艺研究.中外健康文摘:医药月刊,2008,5(8):927-928
[4]杜晖,陈晓青,黄冰.大孔吸附树脂分离纯化夏枯草总黄酮的工艺研究.中成药,2008,30(9):1294-1298
[5]王薇,康亚国.葛根总黄酮提取纯化工艺研究.陕西中医学院学报,2007,30(6):53-54
[6]廖森泰,何雪梅,吴娱明,等.桑枝总黄酮的提取工艺研究.天然产物研究与开发,2007,19(B11):503-507
[7]花蕾,张文清,夏玮.桑叶总黄酮的大孔树脂纯化工艺.中成药,2007,29(12):1758-1761
[8]张书燕.蒲公英总黄酮的提取工艺研究.山东中医杂志,2007,26(12):858-859
[9]黄芸,崔力剑,窦玉红,等.紫外分光光度法测定大豆中异黄酮的含量.大豆科学,2007,2:273-275
[10]闻永举,陈春艳,申秀丽.知母总黄酮含量的测定研究.宜春学院学报,2007,29(6):79-80
[11]邓卫萍,解成喜,艾尔肯·艾米尔.新疆阿魏中总黄酮的提取分离及含量测定.天然产物研究与开发,2007,19(B11):450-452,464
[12]李伟荣,刘昌辉,洪馨,等.广东梅县柚果中总黄酮和柚皮苷的含量测定.中国高校科技与产业化,2006,z1:220-221
[13]周惠燕,李士敏.竹叶中麦黄酮的分离鉴定及含量测定.中药材,2006,29(12):1301-1302
[14]郑岩,王邠,王京丽,等.高效液相测定不同产地鸡血藤药材中黄酮类化合物的含量.中国中药杂志,2008,33(15):1920-1922
[15]陈彦,蔡垠,贾晓斌,等.高效液相色谱法测定仙灵脾配方颗粒中5种主要黄酮类成分的含量.中国实验方剂学杂志,2007,13(11):1-3
[16]王东凯,崔福德,杨丽,等. RP—HPLC法测定醋柳黄酮固体脂质纳米粒的含量及包封率.药物分析杂志,2007,27(1):33-35
[1] Wong V., Shalliker R. A., Isolation of the active constituents in natural materials by‘heart-cutting’isocratic reversed-phase two-dimensional liquid chromatography. J. Chromatogr. A, 2004, 1036(1): 15-24
[2] Gray M. J., Dennis G. R., Slonecker P. J., et al. Separation of oligostyrene isomers in a complex mixture using two-dimensional heart-cutting reversed-phased liquid chromatography. J. Chromatogr. A, 2004, 1028(2): 247-257
[3] Gray M. J., Dennis G. R., Slonecker P. J., et al. Comprehensive two-dimensional separations of complex mixtures using reversed-phase reversed-phase liquid chromatography. J. Chromatogr. A, 2004, 1041 (1-2): 101-110
[4] Matsui T., Tamaya K., Kawasaki T., et al. Determination of angiotensin metabolites in human plasma by fluorimetric high-performance liquid chromatography using a heart-cut column-switching technique. J. Chromatogr. B, 1999, 729 (1-2): 89-96
[5] Cortes H. J. (Ed.), L. D. Rothman. Multidimensional Chromatography, Marcel Dekker, New York 1990, pp. 219-248
[6] Kang S. W., Kim M. C., Kim C. Y., et al. The rapid identification of isoflavonoids from Belamcanda chinensis by LC-NMR and LC-MS. Chem. Pharm. Bull., 2008, 56 (10): 1452-1454
[7] Liu H, Berger SJ, Chakraborty AB, et al., Multidimensional chromatography coupled to electrospray ionization time-of-flight mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins. J. Chromatogr. B, 2002, 782 (1-2): 267-289
[8] Machtejevas E, John H, Wagner K, et al., Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate. J. Chromatogr. B, 2004, 803 (1): 121-130
[9] Winther B, Reubsaet JL. Application of supplementary flow in comprehensive 2D liquid chromatography combining SEC and RPC. J. Sep.Sci., 2005, 28(5): 477-482.
[10] Haefliger OP. Universal two-dimensional HPLC technique for the chemical analysis of complex surfactant mixtures. Anal. Chem. 2003, 75(3): 371-378
[11] Kammerer B, Kahlich R, Ufer M, et al., Achiral–chiral LC/LC–MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism. Analytical Biochemistry 2005, 339(2): 297-309
[12] Kimura H, Tanigawa T, Morisaka H, et al., Simple 2D-HPLC using a monolithic silica column for peptide separation. J. Sep.Sci., 2004, 27(10-11): 897-904
[13] Sheldon EM. Development of a LC–LC–MS complete heart-cut approach for the characterization of pharmaceutical compounds using standard instrumentation. J. Pharm. Biomed. Anal. 2003, 31(6): 1153-1166
[14] Gevaert K, Van Damme P, Martens L, et al., Diagonal reverse-phase chromatography applications in peptide-centric proteomics: Ahead of catalogue-omics? J. Anal. Biochem. 2005, 345(1): 18-29
[15] Fraga CG, Bruckner CA, Synovec RE. Increasing the Number of Analyzable Peaks in Comprehensive Two-Dimensional Separations through Chemometrics.Anal. Chem. 2001, 73(3): 675-683
[16] Gross GM, Prazen BJ, Synovec RE. Parallel column liquid chromatography with a single multi-wavelength absorbance detector for enhanced selectivity using chemometric analysis. Analytica Chimica Acta 2003, 490(1-2): 197-210
[17] Hata K., Morisaka H., Hara K., et al., Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns. Anal. Biochem., 2006, 350(2): 292-297
[18] Millea K. M., Kass I. J., Cohen S. A., et al., Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension. J. Chromatogr. A, 2005, 1079 (1-2): 287-298
[19] Kok S. J., Hankemeier Th., Schoenmakers P. J. Comprehensive two-dimensional liquid chromatography with on-line Fourier-transform-infrared-spectroscopy detection for the characterization of copolymers. J. Chromatogr. A, 2005, 1098 (1-2): 104-110
[20] Jandera P., Fischer J., Lahovska H., et al., Ionic liquids versus triethylamine as mobile phase additives in the analysis ofβ-blockers. J. Chromatogr. A, 2006, 1119 (1-2): 3-10
[21] Chen X., Kong L., Su X., et al., Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry. J. Chromatogr. A, 2004, 1040 (2): 169-178
[1]黄池宝,罗宗铭,宾丽英.黄酮类化合物抗氧化性与其结构关系的研究.广东工业大学学报,2000,17(2):71-75
[2]庞素秋,王国权,秦路平,等.楮实子红色素体外抗氧化作用研究.中药材,2006,29(3):262-265
[3]田京伟,杨建雄.白藜芦醇苷的体外抗氧化活性.中草药,2001,32(10)918-920
[4]赵芳,边丽,胡栋梁.茄子皮红色素抗氧化活性研究.食品与机械,2008,24(2)62-64
[5]徐大量,林辉,李盛青,等.玉竹水提液体内外抗氧化的实验研究.中药材,2008,31(5):729-731
[6]陈琪,王伯初,唐春红,等.黄酮类化合物抗氧化性与其构效的关系.重庆大学学报,2003,26(11):48-51,55
[7]龙春,高志强,陈凤鸣,等.黄酮类化合物的结构—抗氧化活性关系研究进展.重庆文理学院学报(自然科学版),2006,5(2):13-17
[8]秦德安,苏丹,王晓玲.橙皮苷对羟自由基的清除作用.中国药学杂志,1996,31(7):396-398.
[9]高志强,徐强,宋仲容,等.黄酮类化合物抗氧化活性构效关系的密度泛函理论研究.化学世界,2008,49(7):439-443
[10]姜玉兰,朴惠善,李镐.桑叶抗氧化活性成分的研究.中药材,2008,31(4):519-522
[11]焦士蓉,马力,黄承钰,等.枳实提取物的体外抗氧化作用研究.中药材,2008,31(1):113-116
[12]张琳,陆维敏.黄酮类化合物抗氧化性能与其结构的关系.浙江大学学报(理学版),2006,33(2):187-191
[13]周欣,范国荣,吴玉田.短瓣金莲花总黄酮及指标性成分的体外抗氧化活性研究.中药材,2007,30(8):1000-1002
[14]汪河滨,周忠波,罗锋,等.甘草黄酮提取方法及抗氧化活性研究.时珍国医国药,2008,19(9):2106-2107
[15]李伟,张应团.火棘多酚类物质的体外抗氧化作用.食品工业科技,2008,29(9):121-123
[1]王智聪,张庆合,赵中一,等.分析化学,2005,33(5):722-728
[2]田晶,路鑫,杨军,等.色谱,2005,23(1):32-36
[3] Y. Mao and PW. Carr. Anal. Chem., 2000, 72: 110.
[4] P. J. Slonecker, X. Li, T. H. Ridgway, et al., Anal. Chem., 1996, 68: 682
[5] P. Jandera, K. Novotna, L. Kolarova, et al., Chromatographia, 2004; 60: S27
[6] F. Cacciola, P. Jandera, E. Blahova, et al., J. Sep.Sci., 2006, 29: 2500-2513
[7] J. C. Giddings, J. Chromatogr. A, 1995; 703: 3
[8] M. Gilar, P. Olivova, A. E. Daly, et al., Anal. Chem., 2005, 77: 6426
[9] Sai Ma, Lingxin Chen, Guoan Luo, et al., J. Chromatogr. A, 2006, 1127: 207
[10] J. M. Davis. Anal. Chem., 1993, 65: 2014
[11] K. Rowe and J. M. Anal. Chem., 1995, 67: 2981
[12] K. Rowe, D. Bowlin, M. Zou, et al., Anal. Chem. 1995, 67: 2994
[13] Schure MR. J. Pharma. Biomed. Anal., 1997, 9: 169
[14] S. P. Dixon, I. D. Pitfield and D. Perrett. Biomed. Chromatogr., 2006, 20: 508-529
[15] G. J. Opiteck and J. W. Jorgenson. Anal. Chem., 1997, 69: 2283
[16] A. Van der Horst and P. J. Schoenmakers. J. Chromatogr. A, 2003, 1000: 693.
[17] Mondello L, Tranchida PQ, Stanek V, et al., J. Chromatogr. A, 2005; 1086: 91.
[18] Dugo P, Favoino O, Luppino R, et al., Anal. Chem. 2004; 76: 2525.
[19] Stoll DR and Carr PW. Journal of the American Chemical Society 2005; 127: 5034.
[20] Ikegami T, Hara T, Kimura H, et al., J. Chromatogr. A, 2006; 1106: 112.
[21] Liu H, Berger SJ, Chakraborty AB, et al., J. Chromatogr. B, 2002; 782: 267.
[22] Machtejevas E, John H, Wagner K, et al., J. Chromatogr. B, 2004; 803: 121.
[23] Wagner K, Miliotis T, Marko-Varga G, et al., Anal. Chem. 2002; 74: 809.
[24] Winther B and Reubsaet JL. J. Sep.Sci., 2005; 28: 477.
[25] Haefliger OP. Anal. Chem. 2003; 75: 371.
[26] Venkatramani CJ and Zelechonok Y. Anal. Chem. 2003; 75: 3152.
[27] Kammerer B, Kahlich R, Ufer M, et al., Analytical Biochemistry 2005; 339: 297.
[28] Kimura H, Tanigawa T, Morisaka H, et al., J. Sep.Sci., 2004; 27: 897.
[29] Sheldon EM. J. Pharm. Biomed. Anal. 2003; 31: 1153.
[30] Gevaert K, Van Damme P, Martens L, et al., J. Anal. Biochem. 2005; 345: 18.
[31] Fraga CG, Bruckner CA and Synovec RE. Anal. Chem. 2001; 73: 675.
[32] Gross GM, Prazen BJ and Synovec RE. Analytica Chimica Acta 2003; 490: 197.
[33] K. Hata, H. Morisaka, K. Hara, et al., Anal. Biochem., 2006, 350: 292-297
[34] K. M.Millea, I. J. Kass, S. A. Cohen, et al., J. Chromatogr. A, 2005, 1079: 287-298
[35] S. J. Kok, Th. Hankemeier, P. J. Schoenmakers, J. Chromatogr. A, 2005,1098: 104-110
[36] P. Jandera, J. Fischer, H. Lahovska, et al., J. Chromatogr. A, 2006, 1119: 3-10
[37] X. Chen, L. Kong, X. Su, et al., J. Chromatogr. A, 2004, 1040: 169-178
[38] X. Chen, L. Kong, X. Su, et al., J. Chromatogr. A, 2005, 1089: 87-100
[39] X. Chen, L. Hu, X. Su, et al., J. Pharm. Biomed. Anal., 2006, 40: 559-570
[40] P. Dugo, O. Favoino, R. Luppino, et al., Anal. Chem., 2004, 76: 2525-2530
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