Ellagic acid formation from galloylglucoses by a crude enzyme of Cornus capitata adventitious roots

The aqueous extract of acetone powder, which had been prepared from Cornus capitata 'Mountain Moon' adventitious roots, cultured in MS medium with a high concentration of Cu2+(10 microM), showed strong oxidative activity toward galloylglucoses. A compound formed from galloyglucoses, such as 1,2,3,4,6-penta-O-galloyl-beta-D-glucose and tannic acid, by the reaction with the crude enzyme solution of the adventitious roots was isolated and characterized as ellagic acid by spectrometric analyses.     

Preparative isolation and purification of geniposide from gardenia fruits by centrifugal partition chromatography

The iridoid glycoside, geniposide was purified by centrifugal partition chromatography (CPC) with a two-phase solvent system composed of ethyl acetate:isopropanol:water (3:2:5, v/v) from an 80% methanolic extract of fruits of Gardenia jasminoides. Preparative CPC yielded 56.2 mg of geniposide in a one-step separation of 500 mg of extract, with a purity of 95% as determined by HPLC. Isolated geniposide was identified from its 1H-NMR, 13C-NMR and MS spectra.     

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。     

微生物发酵产辅酶Q10的高速逆流色谱法分离纯化

本文首次将高速逆流色谱法应用于微生物发酵液提取物中辅酶Q10的分离纯化,建立了一套可用于其制备分离的逆流色谱溶剂体系正庚烷－乙睛－二氯甲烷(12：7：3.5, v/v/v)。500mg发酵液粗提物经一步制备分离,可得到绝对纯度在98％以上辅酶Q10130mg。比较表明,该方法较传统的硅胶柱层析和结晶相结合的纯化方法在产物纯度、回收率及产率等方面都有一定的优势。     

Combinative application of pH-zone-refining and conventional high-speed counter-current chromatography for preparative separation of alkaloids from Stephania kwangsiensis

A method which involves the combination of pH-zone-refining counter-current chromatography (pH-zone-refining CCC) and conventional high-speed counter-current chromatography (HSCCC) was established for the preparative separation of alkaloids from the crude extracts of Stephania kwangsiensis. pH-zone-refining CCC was first performed with the solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v), where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 2.0 g of crude extract, 370 mg of sinoacutine and 600 mg of a mixture of three other alkaloids were obtained. Then, the mixture was further separated by conventional HSCCC with the solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v), yielding 42 mg of (-)-crebanine, 50 mg of (-)-stephanine and 30 mg of l-romerine from 150 mg mixture of three other alkaloids, respectively. The purities of the four compounds were all over 98% as determined by HPLC, and the chemical structures of the four compounds were confirmed by positive ESI-MS and (1)H NMR data. Results of the present study successfully indicated that this method was efficient for the preparative separation of alkaloids from natural plants.     

Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were successfully applied to the separation of alkaloids from crude extract of Nelumbo nucifera leaves. The conventional HSCCC separations were performed with a two-phase solvent system composed of tetrachloromethane–CHCl₃–methanol–0.1 M HCl at a volume ratio of 1:3:3:2 (v/v/v/v), and 120 mg crude extract could be successfully separated. pH-Zone-refining CCC was performed with a two-phase solvent system composed of petroleum ether (60–90 °C)–ethyl acetate–methanol–water (5:5:2:8, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluent. From 4.0 g of the crude extract, 120 mg N-nornuciferine, 1020 mg nuciferine and 96 mg roemerine were obtained in a single run each with a purity of over 98% as determined by HPLC. The structures of the isolated compounds were identified by ESI-MS, ¹H NMR and ¹³C NMR.     

Separation and purification of four chromones from radix saposhnikoviae by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for the isolation and purification of 1'-O-glucosylcimifugin (1), 4'-O-beta-d-glucosyl-5-O-methylvisamminol (2), cimifugin (3) and 3'-O-glucosylhamaudol (4) from the Chinese medicinal herb radix saposhnikoviae has been successfully developed. A sample of 300 mg of crude extract was separated using ethyl acetate:n-butanol:1% aqueous acetic acid (1:4:5, v/v) as the two-phase solvent system and yielded 102.4 mg of 1 and 81.6 mg of 2. During this separation 3 and 4 remained in the stationary phase, which was collected, evaporated to dryness and separated with another two-phase solvent system involving ethyl acetate:n-butanol:1% aqueous acetic acid (5:0.5:5, v/v) to yield 31.4 mg of 3 and 12.7 mg of 4. The purities of compounds 1-4 were 98.4, 98.7, 99.3 and 98.2%, respectively, as determined by HPLC. The chemical structures of these components were established by (1)H-NMR and (13)C-NMR.     

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。     

高速逆流色谱法同时分离制备头花蓼中没食子酸和原儿茶酸

对蓼科蓼属头状蓼组植物头花蓼进行化学成分的研究。本研究建立了HPLC测定中药头花蓼水提喷雾干燥粉末中化学成分含量的方法,然后应用高速逆流色谱法对头花蓼水提喷雾干燥粉末的乙酸乙酯粗提物的化学成分进行了半制备性分离研究,通过对分离方法和溶剂系统的筛选,寻找到最佳的溶剂系统(正己烷∶乙酸乙酯∶甲醇∶水=1∶5∶1∶5),上相为固定相,转速840 r/min,流速2.0 mL/min,进样量756 mg,检测波长272 nm。结果显示,在该条件下经一步分离可同时得到质量为148.5 mg和9.2 mg的两种产物,纯度为99.8%和97.4%,经紫外、红外、质谱及核磁共振等方法进行结构分析,确定分别为没食子酸和原儿茶酸。     

Halothane anaesthesia of normal and dystrophic hamsters.

Induction, carried out in a small clear-plastic box with 3-5% (v/v) halothane in 30:70 (v/v) oxygen: nitrous oxide, was quiet and rapid. Recovery was almost instantaneous. 2% halothane in the oxygen-nitrous oxide mixture was sufficient for maintenance anaesthesia. The anaesthetic mixture was given by face mask in an open circuit specially designed to function at low gas-flow rates. The halothane content of the muscle and blood after 25 min anaesthesia was estimated by gas chromatography of n-heptane extracts. The mean level (+/- s.e.m.) in blood was 22-8 +/- 2-7 mg/100 ml (n=4), and in dystrophic muscle 226 +/- 36-8 mg/100 g wet weight of tissue (n=4): there was a positive correlation (r=0-94) between them (p less than 0-02).     

Biosynthesis of gallotannins: beta-glucogallin-dependent formation of 1,2,3,4,6-pentagalloylglucose by enzymatic galloylation of 1,2,3,6-tetragalloylglucose

An acyltransferase was detected in young leaves of pedunculate oak (Quercus robur) that catalyzed the formation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, the common precursor of gallotannins and the related ellagitannins. This enzyme depended on beta-glucogallin (1-O-galloyl-beta-D-glucose) as acyl donor; 1,2,3,6-tetra-O-galloyl-beta-D-glucose was specifically required as acceptor molecule, whereas no reaction occurred with the 1,2,4,6-isomer of this substrate. The partially purified enzyme (Mr 260,000) was stable between pH 5.0 and 6.5; highest activities were observed at pH 6.3 and 40 degrees C. Km values of 2.3 and 1.0 mM, respectively, were determined for the substrates beta-glucogallin and tetragalloylglucose. In accordance with stoichiometric studies, the systematic name "beta-glucogallin: 1,2,3,6-tetra-O-galloylglucose 4-O-galloyltransferase" is proposed for this new enzyme.     

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.     

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.     

高速逆流色谱分离制备甘草中的甘草苷和芒柄花苷

应用高速逆流色谱分离制备甘草中的甘草苷和芒柄花苷。将甘草乙酸乙酯提取物经聚酰胺柱粗分后,30%乙醇洗脱物用高速逆流色谱进一步分离,所用两相溶剂系统为乙酸乙酯-水(5∶5,v/v),转速850 rpm,流速2.0 mL/min,检测波长254 nm,从50 mg30%乙醇洗脱物中得到甘草苷8.7 mg、芒柄花苷4.2 mg,纯度分别为99.5%和97.3%。所得产物的结构经核磁共振谱(NMR)鉴定。利用该方法可以对甘草中的甘草苷和芒柄花苷进行快速的分离和纯化。     

Combined microwave-assisted extraction and high-speed counter-current chromatography for separation and purification of xanthones from Garcinia mangostana

A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5 g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75 mg of α-mangostin at 98.5% purity, and 16 mg of γ-mangostin at 98.1% purity from 360 mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, (1)H NMR and (13)C NMR.     

Preparative separation of helvolic acid from the endophytic fungus <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> Ppf9 by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane-ethyl acetate-methanol-water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min(-1) and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, (1)H-NMR and (13)C-NMR spectra.     

Accelerated solvent extraction of monacolin K from red yeast rice and purification by high-speed counter-current chromatography

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L₉ (3)³. The optimum extraction conditions were determined as follows: extraction temperature 120 °C, static extraction time 7 min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26 mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300 mg of crude extract, 51.2 mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and ¹H NMR.     

Preparative separation of isoquinoline alkaloids from Stephania yunnanensis by pH-zone-refining counter-current chromatography

In this paper, five isoquinoline alkaloids were successfully separated from a crude extract of Stephania yunnanensis using pH-zone-refining counter-current chromatography in single-step. With a two-phase solvent system composed of methyl-tert-butyl ether (MtBE)–acetonitrile–water (2:2:3, v/v) where triethylamine (10 mM) was added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.4 g crude extract, 68.7 mg isocorydine, 78.2 mg corydine, 583.4 mg tetrahydropalmatine, 36.3 mg N-methylasimilobine, and 47.3 mg anonaine were separated with purities over 90%. Their structures were identified by ¹H NMR, ¹³C NMR, ESI-MS data.     

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.