Separation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi (Huang-qin in Chinese) was successfully established by using ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) as the two-phase solvent system. The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) was used as the stationary phase of HSCCC. Baicalin (58.1 mg) and wogonoside (17.0mg) with the purity of 99.2 and 99.0%, respectively, were separated successfully in one-step separation from 120 mg of crude sample from S. baicalensi, Georgi. The structures of baicalin and wogonoside were identified by 1H NMR and 13C NMR.     

Isolation and purification of baicalein, wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high-speed counter-current chromatography

The medicinal plant Scutellaria baicalensis Georgi has been used widely in traditional Chinese medicine for anti-inflammation, anticancer, antiviral and antibacterial infections, reducing the total cholesterol level and decreasing blood pressures. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of three bioactive flavonoids, namely, baicalein, wogonin and oroxylin A, from S. baicalensis Georgi. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-water (1:1:8:10, v/v/v/v) was successfully performed by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-1) after 4 h. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 144.8 mg of baicalein at 95.7% purity, 50.2 mg of wogonin at 98.5% purity, and 12.4 mg of oroxylin A at 93.2% purity from 500 mg of the crude extract in a one-step separation. The recoveries of baicalein, wogonin and oroxylin A were 92.7%, 91.6% and 92.5%, respectively.     

高效毛细管电泳法分离测定黄芩复方制剂中的黄芩甙

 建立了清开灵口服液和双黄连口服液中黄芩甙定量分析的高效毛细管电泳方法。选择毛细管区带电泳分离模式,以40mmol/L硼砂缓冲液(pH9.0)为电泳介质,未涂层石英毛细管(50μmi.d.×39.5cm,有效分离长度34.8cm)为分离通道,对硝基苯甲酸为内标,压力进样(68.95kPa×s),17kV恒压电泳(25℃),于285nm下分离分析两种黄芩复方制剂中的黄芩甙。实验结果表明,黄芩甙可在15min内与其它成分得到很好的分离,在10～640mg/L范围内定量分析。加样回收率分别为:清开灵口服液(100.31±1.98)%,双黄连口服液(100.60±2.36)%。方法简便、快速、准确,重现性好,可用于黄芩复方制剂。     

A simple isocratic HPLC method for the simultaneous determination of bioactive components of Scutellariae Radix extract

Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi, has been widely used in Asian countries for the treatment of dermatitis, diarrhoea, inflammatory disease and hepatic disease. A simple, sensitive and precise reversed-phase liquid chromatographic method with isocratic elution was developed to simultaneously determine four bioactive compounds in Scutellariae Radix: baicalein, baicalin, wogonin and wogonoside. Chromatographic analysis was performed on a YMC Pack Pro C(8) column (150?×?4.6?mm(2), 3?μm), with a mobile phase of 0.1% formic acid?:?acetonitrile (70?:?30, v/v) at a flow rate of 1.0?mL?min(-1), and UV detection at 280?nm. Linear behaviour was observed over the investigated concentration range (0.25-10?μg?mL(-1)) for all analytes, with a correlation coefficient of >0.997. The intra- and inter-day precisions were <8.07%, and accuracies were 92.3-102.9%. This method was successfully applied for the analysis of marker compounds for the quality control of Scutellariae Radix extract.     

Simple preparation of baicalin from Scutellariae Radix

A simple, rapid and reproducible method was developed to isolate high-purity baicalin from Scutellariae Radix, the dried roots of Scutellaria baicalensis Georgi. The method involves partition/recrystallization steps without repeated column chromatography or special instruments. Isolated baicalin was characterized by comparisons of TLC, HPLC, IR, MS, and NMR data with an authentic sample. Moreover, beta-glucuronidase hydrolysis of baicalin sequentially yielded glucuronic acid and baicalein as confirmed by co-TLC with authentic samples. The purity of baicalin was more than 97% with yield ca. 8.7% (w/w). The method presented here appears suitable for commercial application.     

不同产地黄芩中黄芩甙及元素的含量比较

用高效液相色谱法和原子吸收光谱法分别对１０个不同产地正品黄芩中黄芩甙及元素的含量进行了分析比较。     

6,7-二乙酰氧基黄芩素的一步合成、选择性水解及甲醚化

以中药黄芩中的主要活性成分黄芩苷为原料,在醋酸钾、吡啶催化回流条件下,用醋酐为乙酰化试剂,以67.6%的收率一步实现了6,7-二乙酰氧基黄芩素的合成,6,7-二乙酰氧基黄芩素经K2CO3/丙酮-水条件下的水解反应或Me2SO4/K2CO3/丙酮中的甲醚化反应以良好收率及高选择性得到6-乙酰氧基黄芩素或7-甲氧基-6-乙酰氧基黄芩素.     

A chromatographic method for baicalin quantification in rat thalamus

A rapid reversed-phase high-performance liquid chromatographic (rp-HPLC) assay for the determination of baicalin in rat thalamus was developed. This was carried out on a Hypersil -C(18) column using 4-nitro-benzoic acid as the internal standard with a mobile phase of methanol-water-H(3)PO(4) (45:55:0.2, v/v/v). Detection was by UV at 277 nm. The calibration curve for baicalin was linear (r=0.9992) over the concentration range of 0.05--4.0 microg/mL and the limit of detection was 10 ng/mL. The coefficients of variation of intra- and inter-day assays were 2.64, 5.19 and 3.19% and 3.46, 6.21 and 5.58% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The recoveries of baicalin from rat thalamus were 85.4+/- 5.62, 90.7+/- 2.43 and 89.1+/- 4.75% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The method was applied to determine the time course of baicalin in rat thalamus, following a single dosage of intravenous administration of Scutellariae radix extract at 90 mg/kg of baicalin to male Wistar rats.     

Orthogonal array design for the optimization of supercritical fluid extraction of baicalin from roots of Scutellaria baicalensis Georgi

Supercritical fluid was used to extract baicalin from the roots of Scutellaria baicalensis Georgi. An orthogonal array design (OAD), OA(9)(3(4)), was employed as a chemometric method for the optimization of the supercritical fluid extraction of baicalin from the herbal medicine. Four parameters, namely, modifiers, temperature and pressure of supercritical fluid, and the dynamic extraction time, were studied and optimized by a three-level OAD in which the interactions between the parameters were neglected. Determinations of the extracts were performed by high-performance liquid chromatography. The effects of parameters were studied using analysis of variance. The results showed that selection of the modifier was the main factor in attaining higher yields of baicalin. While the other three factors had some effect on the extraction of the compound, the effect was much less than that of the modifiers. 1,2-Propanediol-modified supercritical fluid was more effective than 95% ethanol-modified supercritical carbon dioxide or methanol-modified supercritical carbon dioxide for the extraction of baicalin from the solid matrix. Finally, experimental conditions were proposed which can provide the highest extraction yield with respect to the considered factors.     

Microwave‐assisted extraction coupled with countercurrent chromatography for the rapid preparation of flavonoids from Scutellaria barbata D. Don

As a famous Chinese herb having good inhibitory effects on numerous human cancers both in vitro and in vivo, Scutellaria barbata D. Don attracts extensive attention worldwide. In this work, four flavonoids named scutellarin, baicalin, luteolin, and apigenin were simply and rapidly prepared from S. barbata by microwave‐assisted extraction coupled to countercurrent chromatography. Extraction conditions including irradiation time, extraction temperature, liquid/solid ratio, and microwave power were optimized using an orthogonal array design method. The extract of S. barbata was separated and purified with a two‐phase solvent system composed of hexane/ethyl acetate/methanol/acetic acid/water (1:5:1.5:1:4, v/v/v/v/v) and 4.5 mg of scutellarin, 4.6 mg of baicalin, 1.1 mg of luteolin, 2.1 mg of apigenin were obtained from 2.0 g original sample in a single run. The purities of scutellarin, baicalin, luteolin, and apigenin determined by HPLC were 93.6, 97.3, 97.6, and 98.4%, respectively. The targeted compounds were identified by LC with MS and ¹H NMR spectroscopy. The total time including extraction, separation, and purification was <300 min. Compared to traditional methods, microwave‐assisted extraction coupled to countercurrent chromatography method is more simple and rapid for the extraction, separation, and purification of flavonoid compounds from natural products.     

Solid-state NMR studies and DFT calculations of flavonoids: baicalein, baicalin and wogonoside

Three flavonoids of pharmaceutical importance-baicalein, baicalin, and wogonoside-were isolated from a Chinese medicinal plant Scutellaria baicalensis Georgi and studied by 13C NMR in solution and solid state. Two-dimensional (2D) NMR spectroscopy in the liquid phase and dipolar dephasing (DD) experiments in magic-angle spinning (MAS) spectra enabled the assignment of 13C resonances. The cross-polarization (CP) time constants T(CH) and relaxation times T(H) (1rho) were obtained from the variable-contact time experiments. The principal elements of the 13C chemical shift tensor were determined in the spectra recorded under slow sample spinning (2 kHz) using phase-adjusted spinning sideband (PASS)-2D NMR technique, and were verified by density functional theory gauge-independent atomic orbital (DFT GIAO) calculations of shielding constants. Analysis of the 13C delta(ii) and comparison with shielding parameters calculated for different conformers of compounds 1-3 enabled the selection of the most reliable geometry in the solid phase. In all three compounds, an intramolecular hydrogen bond C5--OH...=C4 is formed; the existence of baicalein and baicalin with 'anticlockwise' orientation of OH groups is more probable.     

高效液相色谱-二极管阵列检测-电化学检测联用技术同时测定三精双黄连口服液中的4种化合物

建立了应用高效液相色谱-二极管阵列检测-电化学检测(HPLC-DAD-ECD)联用技术同时测定三精双黄连口服液中的绿原酸、咖啡酸、黄芩甙和木樨草素的方法。以Zorbax SB-C18柱(150 mm×4.6 mm i.d., 5.0 μm)为色谱柱,柱温为30 ℃，流动相为(A)甲醇和(B)甲醇-水-冰醋酸(体积比为50∶50∶1),其梯度洗脱程序为2%A3 min3%A12 min25%A5 min80%A。流速为0.8 mL/min。二极管阵列检测波长为275 nm。电化学单安培检测器的工作电压为0.7 V。在上述条件下实现了绿原酸、咖啡酸、黄芩甙和木樨草素的分离检测。上述4种化合物的回收率为96.6%～99.6%,相对标准偏差(RSD)为2.5%～4.1%,检测限依次为1,0.2,9和7 mg/L。该方法简便、快速,重现性和准确度较好,可作为测定双黄连口服液中绿原酸、咖啡酸、黄芩甙和木樨草素的有效方法。     

HPLC Determination of Flavonoids in Hairy-Root Cultures of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

A simple and sensitive liquid chromatographic method has been established for determination of the main flavonoid components of Scutellaria baicalensis Georgi (Lamiaceae) roots and hairy-root cultures. Wogon, the dried root of the plant, is used in traditional Chinese medicine for treatment of bronchitis, hepatitis, tumors, and inflammatory diseases. Lyophilized hairy roots were extracted with methanol. The crude extracts were purified by SPE on Supelco LC-8 cartridges. HPLC separations were performed on a Eurospher 100-C₈ reversed-phase column. The mobile phase was a gradient prepared from mixtures of acetonitrile and 0.1% trifluoroacetic acid. Peaks were identified by addition of standards and/or by diode-array detection. Baicalein 7-O-glucuronide (baicalin), wogonin 7-O-glucuronide (wogonoside), baicalein, wogonin, and acteoside were determined by the external standard method at 280 nm. We found that the aglycon (baicalein and wogonin) content of the transformed roots was consistently higher than that of the intact root from Siberia.     

Determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San, which contains Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix, was established. The samples were separated with a Cosmosil 5C₁₈-AR column by gradient elution with 0.03% (v/v) phosphoric acid- acetonitrile (0 min 96:4, 5 min 84:16, 7 min 82:18, 14–30 min 78:22) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 245 nm. Methylparaben was used as the internal standard and three equations were derived showing linear relationships between the peak- area ratios of marker components (paeoniflorin, ferulic acid and baicalin) to methylparaben and concentration. The recoveries of paeoniflorin, ferulic acid and baicalin were 27.86, 33.89 and 49.31%, respectively. The repeatability (relative standard deviation) was generally less than 5% (n = 5). The effects of various processes such as concentration by reduced-pressure evaporation, freeze-drying and spray-drying were studied and commercial concentrated herbal preparations containing Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix were also analysed.     

D241树脂分离纯化黄芩总黄酮的研究

本文研究了用241树脂分离纯化黄芩总黄酮的方法和工艺。实验结果表明：D241树脂对黄芩总黄酮的静态交换容量是77mg/ml树脂。在pH11.0、流速2.0BV/h、提取液中总黄酮浓度17.5mg/ml条件下，D241树脂对黄芩总黄酮的动态交换容量为43.8mg/ml。用60％甲醇作为黄芩总黄酮洗脱剂，在PH4.0、洗脱流速1.5BV/h条件下，4．5BV洗脱剂即可完全洗脱被D241树脂交换的黄芩总黄酮。与酸沉淀法相比较，经过除去果胶，总黄酮纯度由33．34％提高到74．9％，粗品收得率由11.52%降低至4.83%；经过D241树脂分离纯化，其纯度达到91.5%，产品收得率3.54%。     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

Preparative isolation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. The crude extracts from P. cuspidatum Sieb. et Zucc were treated with light petroleum-ethyl acetate-methanol-water (2:5:4:6, v/v). Sample 1 was obtained from the lower phase and sample 2 from the upper phase. The sample 1 was separated with light petroleum-ethyl acetate-water (1:5:5, v/v) and yielded 19.3mg of piceid, 17.6 mg of anthraglycoside B from 200mg of sample 1. The sample 2 was separated with light petroleum-ethyl acetate-methanol-water (3:5:4:6, v/v) and light petroleum-ethyl acetate-methanol-water (3:5:7:3, v/v) in a gradient elution and yielded 18.5mg of resveratrol, 35.3mg of emodin and 8.2mg of physcion from 220 mg of sample 2. The purity of each compound is over 95% as determined by HPLC. The chemical structures of these components were identified by (1)H NMR and (13)C NMR.     

生附子中C19型二萜生物碱的高速逆流色谱分离及其结构鉴定

应用高速逆流色谱法分离制备了生附子中的3个C19型二萜生物碱类化合物。以正己烷-乙酸乙酯-甲醇-水(3:5:4:5, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速850 r/min、流动相流速2.0 mL/min、检测波长235 nm条件下进行分离制备;一次性从90 mg附子总碱粗提物中分离制备得到15.3 mg北草乌碱,35.1 mg中乌头碱和22.7 mg次乌头碱,经高效液相色谱分析,测得它们的纯度分别为97.9%、96.2%和99.2%。并应用波谱(电喷雾离子质谱、核磁共振氢谱和核磁共振13C谱)解析法确定了它们的结构。利用该方法可以对生附子中的二萜类生物碱成分进行快速的分离和纯化     

青藤碱和黄芩甙的极谱特性

用单扫示小极谱法研究了青藤碱和黄芩甙的电化学行为，青藤碱在０．１ｍｏｌ／Ｌ Ｎａ２Ｂ４Ｏ７中有两个还原峰，峰Ｐ１和Ｐ２的电位分别为－１．４５Ｖ和－１．６７Ｖ，峰Ｐ１的峰电流与浓度在０．０６－１．８ｍｇ／Ｌ和２．０－３４．６ｍｇ／Ｌ范围内呈线性关系，检测限为０．０２ｍｇ／Ｌ，在０．０５ｍｏｌ／Ｌ棕檬酸钠＋０．２ｍｏｌ／Ｌ ＨＣｌ底液中，黄芩甙于－１．１４Ｖ（Ｐ３０和－１．３２Ｖ（Ｐ４）处产生两还原峰     

柴胡舒肝丸的毛细管电泳指纹图谱及其黄芩苷含量的测定

建立了柴胡舒肝丸(Chaihu Shugan Pill, CHSGP)的毛细管区带电泳指纹图谱(capillary electrophoresis fingerprint, CEFP),并采用内标法测定了黄芩苷的含量。以50 mmol/L硼砂-150 mmol/L磷酸二氢钠-50 mmol/L磷酸氢二钠(1:1:1, v/v/v)(含5 mmol/L庚烷磺酸钠)为背景电解质(BGE)溶液,采用未涂层石英毛细管(总长度75 cm,有效分离长度63 cm,内径75 μm),以色谱指纹图谱分离量指数(RF)为目标函数优化实验条件,在紫外检测波长265 nm、运行电压11 kV条件下,以黄芩苷峰为参照物峰,确定了22个共有指纹峰,建立了CHSGP的CEFP,通过对20批样品聚类分析确定用其中13批生成对照CEFP(RCEFP),以此RCEFP为标准用系统指纹定量法鉴别20批柴胡舒肝丸质量。结果其中的4批化学成分数量和分布比例不合格,4批含量明显偏低,其他12批完全合格。采用内标法测定黄芩苷的含量,在5～200 mg/L范围内线性良好(r=0.9999),平均回收率(n=9)为98.2%。该法具有较好的精密度和重现性,为柴胡舒肝丸的质量控制提供了一种新的参考。