黄连中α-葡萄糖苷酶抑制剂的筛选分离及质谱分析

为筛选黄连中α-葡萄糖苷酶抑制剂,本研究采用高效液相色谱-电喷雾质谱联用技术(HPLC-DAD-MS)对黄连提取物中的化学成分进行分析鉴定,并采用高速逆流色谱分离其中的活性成分。选用反相C18色谱柱,以0.02%醋酸溶液(A)和甲醇(B)为流动相,进行梯度洗脱;利用电喷雾质谱(ESI-MS)正离子模式在线检测化学成分;以α-葡萄糖苷酶作为生物靶分子,以超滤质谱技术筛选酶抑制剂。再经高速逆流色谱分离纯化,以乙酸乙酯-正丁醇-乙醇-水(3.0∶1.7∶0.5∶6.0,v/v/v/v)为两相溶剂系统,所得分离收集液经高效液相色谱法检测。实验通过HPLC-DAD-MS共鉴定出5个化学成分,分别为药根碱、表小檗碱、黄连碱、巴马亭和小檗碱。通过HSCCC分离得到两种α-葡萄糖苷酶抑制剂巴马亭和小檗碱。利用液相色谱-超滤-质谱-高速逆流色谱联用技术可以快速分离鉴定黄连中的化合物。此方法对于筛选有效成分具有快速和灵敏等优势。     

牡丹籽饼粕中芍药苷类成分及其大孔吸附树脂纯化研究

采用乙醇提取,树脂纯化,HPLC制备以及LC-MS和1H NMR鉴定,从牡丹籽粕的醇提物中分离纯化了4种主要成分,分别为6'-O-β-D-葡萄糖芍药内酯苷、芍药内酯苷、β-gentiobiosylpaeoniflorin和芍药苷。对大孔吸附树脂法纯化芍药苷类成分的条件进行了试验,从4类11种树脂中筛选出HPD-200A型大孔吸附树脂,其较优的吸附分离条件为:上样液浓度(芍药苷)8.0 mg/mL,上样体积为4.5倍床体积(BV),流速为1/16 BV/min,洗脱剂乙醇溶液浓度为50%(v/v),洗脱体积为4 BV,流速为1/16 BV/min。此条件下所得提取物中含芍药苷32.3%、芍药内酯苷16.5%、6'-O-β-D-葡萄糖芍药内酯苷8.02%、β-gentiobiosylpaeoniflorin 6.63%。     

高速逆流色谱分离制备川西獐牙菜中两种苷类化合物

采用高速逆流色谱从川西獐牙菜中分离制备了两种高纯度苷类化合物.以正丁醇-氯仿-甲醇-水(3.4∶8∶5∶6,v/v)为溶剂系统,主机转速为800 rpm,流速:O～210 min,1.5mL/min;210 ～360m in,2.5 mL/min,检测波长254 nm的条件下进行分离制备,在360 min内从100 mg样品中一步分离制备得到1-O-樱草糖-3,7,8-三甲氧基(口山)酮(Ⅰ,11 mg)和异荭草苷(Ⅱ,24 mg).经HPLC检测,两个化合物的纯度均在99％以上,结构由UV、1H和13C NMR鉴定.     

高速逆流色谱技术分离纯化红葱中的萘醌类化合物

采用高速逆流色谱(HSCCC)技术从红葱中快速分离纯化得到红葱乙素和异红葱乙素,建立了快速分离制备红葱中萘酚类化合物的方法。首先采用95%乙醇加热回流提取得红葱提取物,再用乙酸乙酯萃取富集萘醌类成分,然后用高速逆流色谱分离纯化,以石油醚-乙酸乙酯-甲醇-水(6∶4∶5∶5,v/v)组成二元溶剂系统,上相为固定相,下相为流动相,仪器转速为850 rpm,流速为2.0 m L/min,检测波长为254 nm。从200 mg富集萘醌类成分的粗提物中,一次性分离制备得到60 mg异红葱乙素和49 mg红葱乙素,经高效液相色谱法(HPLC)分析,其纯度分别为97.3%和98.6%。通过核磁共振氢谱(~1H NMR)和核磁共振碳谱(~(13)C NMR)鉴定化合物为红葱乙素和异红葱乙素。研究结果表明,该方法快速、高效,适用于红葱中萘酚类化合物的分离纯化。     

RP-HPLC 法测定酸枣仁汤中甘草苷的含量

目的:建立测定酸枣仁汤中甘草苷含量的反相高效液相色谱分析方法。方法:色谱柱:WatersC18(250nm×4.6mm,5μm);流动相:乙腈-0.1%磷酸水溶液,梯度洗脱;流速:1.0ml/min,检测波长:276nm。结果:芒果苷在0.093μg-0.744μg范围内与峰面积线性关系良好(r=0.9999),平均加样回收率为98.04%,RSD为0.41%。结论:本方法简便、准确、可靠、重复性好,可作为酸枣仁汤中甘草苷的含量测定方法。     

反相高效液相色谱法同时测定车前子中的毛蕊花苷和异毛蕊花苷

采用反相高效液相色谱法在Waters Symmitry C18(5 μm,250 mmm×4.6 mm)色谱柱上以甲醇:体积分数0.1%甲酸溶液(40:60,v:v)为流动相同时测定车前子中毛蕊花苷和异毛蕊花苷的含量,流动相流速为0.6 mL/min,检测波长为330 nm.毛蕊花苷线性范围为0.2～2.0 μ,r=0.9982.异毛蕊花苷线性范围为0.2～2.0 μ,r=0.9957.样品的平均回收率分别为99.70%,101.18%.测得大粒车前子中毛蕊花苷含量为8.65 mg/g,异毛蕊花苷含量为1.36 mg/g.此方法准确、快速,适用于车前子中毛蕊花苷和异毛蕊花苷的定量分析.     

HPLC法测定甘草酸和甘草苷的含量

目的:建立以HPLC法同时测定甘草中甘草酸和甘草苷含量的方法.方法:采用C18柱(4.6mmx15cm,5μm)为分析柱;以乙腈-1%醋酸溶液为流动相;流速为0.6ml·min-1;柱温25℃;采用外表法定量测定.结果表明甘草酸在4.16-28.0μg/ml范围内,回归方程为Y=10562.8X+30963(r=0.9999);甘草苷在13.0-23.4μg/ml范围内,回归方程为Y=12857.4X+16437(r=0.9997),平均加样回收率均大于96.08%,日内、日间的RSD值分别小于1.41%和1.85%.结论本方法操作简单、准确、快速、重现性好,其它组分干扰少,可用于甘草酸和甘草苷的含量测定.     

酸水解结合高速逆流色谱分离制备高纯度牛蒡子苷元

建立酸水解结合高速逆流色谱法从牛蒡子中快速分离制备高纯度牛蒡子苷元的方法。采用醇提酸解法提取,再经氯仿萃取得牛蒡子粗提物;以石油醚-乙酸乙酯-甲醇-水(2∶5∶3∶4,v/v)作为两相溶剂系统,在流速10 m L/min、转速850 rpm、检测波长280 nm下实现对牛蒡子苷元的快速分离制备。80 min内从连续两次进样的1200 mg牛蒡子粗提物中分离得到牛蒡子苷元318 mg,其纯度达99.12%,得率达26.5%。该方法简便、快速、高效,可用于牛蒡子苷元的快速分离制备,为牛蒡子的开发利用提供了参考依据。     

离子液体优化高速逆流色谱法分离槐花中的黄酮类化合物

应用高速逆流色谱法首次从槐花中一步分离出2种黄酮类化合物,并利用离子液体提高分离效果。以正己烷-乙酸乙酯-乙醇-水-冰醋酸(1∶1∶1∶1∶0.05,v/v)为两相溶剂体系,从50 mg槐花粗提物中一步分离得到芦丁18.2 mg,槲皮素9.6 mg,其纯度均在97%以上。加入离子液体1-丁基-3-甲基咪唑六氟化硼酸([BMIM][PF6]),使出峰时间由原来的85 min提前到55 min,分离度由0.9提高到1.8,达到完全分离,分离效果得到明显提高,为离子液体在高速逆流色谱中的进一步应用提供依据。     

HPLC法测定刺囊毛霉微生物转化甘草次酸的主产物含量

采用刺囊毛霉AS3.3450对甘草次酸进行微生物转化,生成的主产物经分析鉴定是7β-羟基甘草次酸.采用高效液相色谱方法,以C18-ODS为色谱柱,甲醇-0.03%三氟乙酸水溶液(梯度洗脱)为流动相,检测波长为:254 nm,测得在160 r/min、27℃的转化条件下,底物加量为130 mg/L,转化时间为9 d时,主产物得率最高为712 mg/g甘草次酸,且在选定色谱条件下7β-羟基甘草次酸的线性范围良好,平均加样回收率为98.1%,平均标准偏差(RSD)为2.37%.     

高速逆流色谱法分离制备花生壳中的黄酮类化合物

应用高速逆流色谱法首次从花生壳中分离制备了3种黄酮类化合物。以正己烷-乙酸乙酯-甲醇-水-冰醋酸(5:3:3.5:5:0.25,v/v)为两相溶剂系统,在主机转速800 r/min、流速2 mL/min、检测波长275 nm条件下进行分离制备,纯度用HPLC法测定,各化合物结构经质谱和核磁共振氢谱、碳谱鉴定。结果表明,100 min内从70 mg花生壳粗提物中一步分离制备得到木犀草素11.0 mg,香叶木素2.2 mg,5,7-二羟基色原酮5.2 mg,其纯度均达96.0%以上。利用该方法可以对花生壳中的黄酮类化合物进行快速的分离和纯化。     

高速逆流色谱法分离制备丹酚酸B

采用高速逆流色谱法分离纯化丹参水溶性成分丹酚酸类物质,制备丹酚酸B化学对照品。分离采用的溶剂系统为正己烷-乙酸乙酯-水-甲醇(1.5:5:5:1.5),上相做固定相,下相做流动相,流速为1.7 mL/min,仪器转速850 rpm,进样量80 mg,纯度用HPLC方法测定。结果表明:一次分离可制备63.4 mg丹酚酸B,其纯度为98.6%。该方法操作简单,可作为高纯度丹酚酸B化学对照品的制备分离方法。     

Extraction and preparative purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography

A method for extraction and preparative separation of tanshinones from Salvia miltiorrhiza Bunge was successfully established in this paper. Tanshinones from Salvia miltiorrhiza Bunge were extracted using ethyl acetate as the extractant under reflux. The extracts were then purified by high speed counter-current chromatography (HSCCC) with light petroleum-ethyl acetate-methanol-water (6:4:6.5:3.5, v/v) as the two phase solvent system. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 8.2mg of dihydrotanshinone I, 5.8 mg of 1,2,15,16-tetrahydrotanshiquinone, 26.3mg of cryptotanshinone, 16.2mg of tanshinone I, 25.6 mg of neo-przewaquinone A, 68.8 mg of tanshinone IIA and 9.3mg of miltirone were obtained from 400mg of extracts from Salvia miltiorrhiza Bunge in one-step HSCCC separation, with the purity of 97. 6%, 95.1%, 99.0%, 99.1%, 93.2%, 99.3% and 98.7%, respectively, as determined by HPLC area normalization method. Their chemical structures were identified by 1H NMR.     

应用高速逆流色谱分离桑枝酚类成分

建立了高速逆流色谱(HsCCC)分离制备高纯度的桑枝酚类成分的新方法.分离条件如下:溶剂系统为正己烷-乙酸乙酯-甲醇冰(1∶1∶1∶2,v/v),上相为固定相,下相为流动相;流速2.0 mL/min;转速900rpm;进样量75 mg.收集得到三个高纯度化合物,经HPLC、MS、1H和13C NMR等分别鉴定为反式氧化白藜芦醇(25.2mg),反式白藜芦醇(7.4 mg)和桑辛素M(29.1 mg).高速逆流色谱可以高效分离桑枝成分,方法简便,技术可行,优于传统的柱色谱法.     

Preparative isolation and purification of two new isomeric diterpenoid alkaloids from Aconitum coreanum by high-speed counter-current chromatography

Preparative high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) was used to isolate and separate bioactive constituents from the roots of Aconitum coreanum. Two new diterpenoid alkaloid isomers were successfully separated for the first time by HSCCC with an optimized two-phase solvent system composed of ethyl acetate-n-butanol-methanol-2% acetic acid (3.5:1.5:2:4.5, v/v/v/v), 25.4mg of GFT (1) and 18.3mg of GFU (2) were isolated form 1g crude extract in one step HSCCC experiment. The purities of the two new compounds were all over 95% as analyzed by HPLC and their structures were identified by ESI-MS, (1)H NMR, (13)C NMR, and 2D NMR analysis.     

Two-step purification of scutellarin from Erigeron breviscapus (vant.) Hand. Mazz. by high-speed counter-current chromatography

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate-n-butanol-acetonitrile-0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.     

The isolation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from Acer truncatum Bunge by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from the ethyl acetate extract of the leaves of Acer truncatum Bunge using a two-phase system composed of n-hexane-ethyl acetate-methanol-water at a volume ratio of (0.25:5:1:5, v/v/v/v) for the first time. Each injection of 80 mg crude extract yielded 7.25 mg of pure 1,2,3,4,6-penta-O-galloyl-beta-D-glucose. High-performance liquid chromatography (HPLC) analyses of the CCC fraction revealed that the purity of 1,2,3,4,6-penta-O-galloyl-beta-D- glucose was over 95%.     

On-line coupling of dynamic microwave-assisted extraction with high-speed counter-current chromatography for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim

An on-line method based upon dynamic microwave-assisted extraction (DMAE) coupled with high-speed counter-current chromatography (HSCCC) was developed for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim. The DMAE parameters were optimized by means of the Box-Behnken design. The maximum extraction yield was achieved using 30:1 ml/g of liquid-solid ratio, 10 ml/min of solvent flow rate and 200 W of microwave power. The crude extracts were then separated by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:5:5, v/v/v/v). 13.0mg of nevadensin was isolated from 15.0 g original sample by HSCCC with five times sample injection in 12h, and the isolation yield of nevadensin was 0.87 mg/g. The average purity of nevadensin was higher than 98.0%. The chemical structure of collected fraction was identified by HPLC, ESI-MS and (1)H NMR. The results indicated that this on-line method was effective and fast for high-throughput isolation of nevadensin from L. pauciflorus Maxim.     

Application of analytical and preparative high-speed counter-current chromatography for the separation of Z-ligustilide from a crude extract of Angelica sinensis

Z-Ligustilide was separated and purified from the traditional Chinese medicinal plant Angelica sinensis by high-speed counter-current chromatography (HSCCC). Analytical HSCCC was first used for the systematic selection of the two-phase solvent system. Preparative HSCCC separation was performed with a two-phase solvent system composed of petroleum ether (60-90 degrees C)-ethanol-water at an optimum volume ratio of 10:17:10 (v/v). A total of 38 mg Z-ligustilide at 98.8% purity was obtained in one step from 200 mg crude extract as determined by HPLC analysis. The structure of the target compound was identified by electron impact ionisation mass spectrometry.