Application of preparative high-speed counter-current chromatography for separation of chlorogenic acid from Flos Lonicerae

Chlorogenic acid, an ester formed between caffeic acid and quinic acid, is a major phenolic compound in the traditional Chinese medicinal herb Flos Lonicerae. The separation and purification of chlorogenic acid from the crude extract of Flos Lonicerae was achieved by high-speed counter-current chromatography (HSCCC). A high acid, highly polar two-phase solvent system containing n-butanol-acetic acid-water (4:1:5) was run on a preparative scale. The upper phase was used as the mobile phase in the head to tail elution mode. A 300-mg quantity of the crude extract containing 5.97% chlorogenic acid was loaded on a 342-ml HSCCC column. Double separations were performed with the same solvent system yielding 16.9 mg chlorogenic acid at 94.8% purity with approximately 90% recovery.     

金银花的毛细管电泳指纹图谱研究

采用毛细管区带电泳法,以50 mmol/L硼砂(含20 mmol/L β-环糊精(CD),用磷酸调pH 8.0)为背景电解质,运行电压12 kV,紫外检测波长254 nm,重力进样15 s（高度8.5 cm）,建立了金银花药材水提取液的毛细管电泳指纹图谱(CEFP)。将13个不同产地的金银花药材供试液的CEFP进行比较,以电泳峰出现率100%计,确定金银花的共有指纹峰为18个。该CEFP具有较好的精密度和重现性,分离效能高且成本低廉。提出了指纹图谱宏观含量相似度R、投影含量相似度C和定量相似度P的概念,可从总体上评价药材化学组分的整体含量情况。从两个方面评价各产地药材与对照CEFP间的总体相似性,合格药材应具备以下两个条件：(1)代表化学成分分布相似性的定性相似度(S)≥0.90;(2)描述药材整体化学成分含量的定量相似度(R,C,P,Q)应在80%～120%。以此二类相似度指标控制金银花的质量,建立了指纹图谱评价的又一新方法。     

Study on phenolic acids of Lonicerae japonicae Flos based on ultrahigh performance liquid chromatography-tandem mass spectrometry combined with multivariate statistical analysis

Lonicerae japonicae Flos, a traditional Chinese medicine, has the function of evacuating heat and detoxifying. To promote the optimization of Lonicerae japonicae Flos germplasms and improve the quality of medicinal materials, 55 batches of five Lonicerae japonicae Flos germplasms with the same origin were collected during different periods, a UHPLC-TOF-MS method was established, and 22 kinds of phenolic acids were found and qualitatively analysed. Seventeen phenolic acids were selected for quantitative analysis by UHPLC-QqQ-MS/MS, and the quantitative results were analysed by principal component analysis, orthogonal partial least squares-discriminant analysis, and partial least squares discriminant analysis. The contents of phenolic acids in periods S1–S6 were found to be significantly different. There were also significant differences in the accumulation of phenolic acids in Lonicerae japonicae Flos during different growth periods. Ferulic acid, 5-O-caffeoylquinic acid, and caffeic acid were determined to be important components to distinguish the different growth periods of Lonicerae japonicae Flos. There were significant differences in the phenolic acid content of different germplasms of Lonicerae japonicae Flos, and the total amount of 17 phenolic acids and total acids (chlorogenic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) in “Hua Jin No. 6” was highest, so the quality of “Hua Jin No. 6” was better than that of the four other germplasms. In addition, chlorogenic acid methyl ester and caffeic acid were the smallest markers in combination to distinguish the five germplasms of Lonicerae japonicae Flos.     

Simultaneous quantification of Echinacea species, Flos Lonicerae, Radix Scutellaria and Fructus Forsythiae combinations by rapid resolution liquid chromatography

Echinacea angustifolia and E. purpurea are commonly used in North America for their anti-bacterial effects. Flos Lonicerae, Radix Scutellaria and Fructus Forsythiae are traditional Chinese medicinal herbs commonly used for the treatment of complaints such as pneumonia, acute upper respiratory tract infection, and acute bronchitis. A reproducible, simple, and reliable rapid resolution liquid chromatographic (RRLC) method has been developed to analyze extracts of products formulated containing E. angustifolia, E. purpurea, Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae simultaneously in one run in less than 6 minutes. The method uses a C18-HST column, a mobile phase consisting of 0.1% aqueous phosphoric acid solution and acetonitrile, and UV detection at 327 nm and 229 nm. A stability test was performed that revealed that chlorogenic acid is more stable in acidic pH, and hence it is best to keep the extract of E. augustifolia, E. purpurea, Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae in mild acidic conditions at approximately pH 5.     

Rapid quantitative analysis of adulterant Lonicera species in preparations of Lonicerae Japonicae Flos

Lonicerae Japonicae Flos is often adulterated with Lonicerae Flos, which is derived from the other four Lonicera species, in both the crude drug and Lonicerae Japonicae Flos preparations. We proposed a methodology for the quantitative analysis of adulterant Lonicerae Flos in Lonicerae Japonicae Flos preparations. Taking macranthoidins A, B, dipsacoside B (saponins), sweroside (iridoids), and luteolin‐7‐O‐d ‐glucoside (flavonoids) as markers, a method of ultra high performance liquid chromatography with triple quadrupole mass spectrometry was employed to determine their amounts in Lonicerae Flos, Lonicerae Japonicae Flos, and Lonicerae Japonicae Flos preparations. The proportion of adulterant Lonicerae Flos in Lonicerae Japonicae Flos preparations was estimated based on the saponin contents of Lonicerae Japonicae Flos and Lonicerae Flos. All analytes separated under isocratic elution in 12 min with acceptable linearity, precision, repeatability, and accuracy. Lonicerae Japonicae Flos was easily distinguished from Lonicerae Flos by the total amount of saponins (0.067 and > 45.8 mg/g for Lonicerae Japonicae Flos and Lonicerae Flos, respectively). Eighteen of twenty one Lonicerae Japonicae Flos preparation samples were adulterated with Lonicerae Flos in proportions of 11.3–100%. The developed ultra high performance liquid chromatography with triple quadrupole mass spectrometry method could be used for the identification of Lonicerae Japonicae Flos and the four species of Lonicerae Flos and for the analysis of Lonicerae Japonicae Flos preparations adulterated with Lonicerae Flos.     

HPLC determination and pharmacokinetics of chlorogenic acid in rabbit plasma after an oral dose of Flos Lonicerae extract

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of chlorogenic acid (3-O-caffeoyl-D-quinic acid) in plasma and applied to its pharmacokinetic study in rabbits after administration of Flos Lonicerae extract. Plasma samples are extracted with methanol. HPLC analysis of the extracts is performed on a C(18) reversed-phase column using acetonitrile-0.2% phosphate buffer (11:89, v/v) as the mobile phase. The UV detector is set at 327 nm. The standard curves are linear in the range 0.0500-1.00 microg/mL (r = 0.9987). The mean extraction recovery of 85.1% is obtained for chlorogenic acid. The interday precision (relative standard deviation) ranges from 5.0% to 7.5%, and the intraday precision is better than 9.0%. The limit of quantitation is 0.0500 microg/mL. The plasma concentration of chlorogenic acid shows a C(max) of 0.839 +/- 0.35 microg/mL at 34.7 +/- 1.1 min and a second one of 0.367 +/- 0.16 microg/mL at 273.4 +/- 39.6 min.     

胶束扫集毛细管电泳分离测定绿原酸和咖啡酸

采用胶束扫集毛细管电泳分离测定双黄连口服液中的绿原酸和咖啡酸.试验条件为:重力进样时间40 s;以20 mmol/L NaH_2PO_4,100 mmol/L 十二烷基磺酸钠(SDS)为电泳缓冲液(含体积分数15%甲醇,pH 2.20),分离电压-20 kV,检测波长214 nm,讨论了pH、SDS浓度、样品溶剂等对分离效果的影响.在优化条件下,绿原酸和咖啡酸的检出限分别达到1.02和0.168 μg/mL,线性范围分别为5.86～51.5 μg/mL和1.27～14.5 μg/mL.     

微波辅助萃取/高效液相色谱串联质谱法对山银花中活性成分含量的测定

建立了微波辅助萃取/高效液相色谱串联质谱法(MAE/HPLC-MS/MS)同时测定山银花中10种活性成分含量的方法。山银花药材采用MAE萃取,萃取溶剂为乙醇-水(7∶3),固液比1∶30,萃取温度70℃,萃取时间10 min。采用HPLC-MS/MS测定萃取液中活性成分的含量,色谱柱采用Agilent Poroshell120 SB-C18(100 mm×2.1 mm,2.7μm),以0.5%甲酸-乙腈为流动相进行梯度洗脱,负离子多重反应离子监测模式检测。在优化条件下,10种成分的定量分析在10 min内完成。结果表明,10种活性成分的线性范围为0.05～500 mg/L,相关系数(r)不低于0.996 9,检出限和定量下限分别在69～4 413μg/kg和231～14709μg/kg范围,回收率为94%～105%。采用该方法检测6个不同产地的山银花样品,10种活性成分的含量在3.98～14 356.31 mg/kg范围。该方法快速、准确,可有效地用于山银花药材的质量控制。     

高分辨采样二维液相色谱法同时测定金银花中绿原酸和木犀草苷含量

采用高分辨采样二维液相色谱法（HiRes 2D-LC）对金银花中绿原酸和木犀草苷进行准确定量分析。第一维液相色谱采用C18色谱柱，以乙腈和0.4%（v/v）磷酸水溶液为流动相进行梯度洗脱；第二维液相色谱采用SB-Phenyl色谱柱，以乙腈和0.5%（v/v）乙酸水溶液为流动相进行梯度洗脱；二维接口采用五位十通阀，并配置2个多中心切割阀，对绿原酸组分和木犀草苷组分进行多次连续切割。实验结果表明，二维液相色谱分析提高了绿原酸和木犀草苷色谱峰的确认能力，可揭示一维液相色谱分析中共洗脱或隐藏峰的信息；高分辨采样模式实现了一维目标组分的片段式整峰切割，提高了二维液相色谱分析的准确定量能力；通过线性关系、基质加标回收和重复性等考察结果，表明高分辨采样二维液相色谱具有优异的定量准确性和重复性，为中药等复杂基质组分样品的分离和准确定量提供了新方法。     

Simultaneous qualitation and quantification of thirteen bioactive compounds in Flos lonicerae by high-performance liquid chromatography with diode array detector and mass spectrometry

A high-performance liquid chromatography (HPLC) with diode array detector (DAD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of thirteen bioactive compounds in Flos Lonicerae. The optimal chromatographic conditions were obtained on a C(18) column (250x4.6 mm, 5.0 microm) with the column temperature at 30 degrees C. The mobile phase was composed of (A) acetic acid aqueous (0.4%, v/v) and (B) acetonitrile using a gradient elution, the flow rate was 1 ml/min. Detection wavelengths were set at 240 nm for iridoids (loganin, sweroside, secoxyloganin and centauroside), 330 nm for phenolic acids (chlorogenic acid, caffeic acid, 4,5-di-O-caffeoyl quinic acid and 3,4-di-O-caffeoyl quinic acid) and 360 nm for flavonoids (rutin, hyperoside, quercetin-3-O-glucoside, luteolin-7-O-glucoside and lonicerin). The identities of the peaks were accomplished by comparing retention times, UV and mass data with reference compounds under the same conditions. All calibration curves showed good linear regression (r(2)>0.9983) within test ranges. The developed method provided satisfactory precision and accuracy with overall intra-day and inter-day variations of 0.78--1.85% and 1.13--2.36%, respectively, and the overall recoveries of 91.3--104.2% for the thirteen compounds analyzed. The verified method was successfully applied to quantitative determination of the three types of bioactive compounds in ten commercial Flos Lonicerae samples from different markets in China. The analytical results demonstrated that the contents of the thirteen analytes were relatively variant.     

银黄冲剂中黄芩甙和绿原酸的毛细管电泳分离分析

摘要：用毛细管电泳法分离并测定了银黄冲剂中黄芩甙和绿原酸。以对硝基苯甲酸为内标,未涂层融硅毛细管（50μmi.d.,370μmo.d.,总长47cm,有效分离长度40cm）为分离通道,25mmol/L硼砂缓冲液（pH8.5）为电泳介质,17kPa·s压力进样,25kV恒压电泳,310nm检测。黄芩甙和绿原酸线性范围分别为160～960mg/L(r=0.9993,RSD=1.76％～2.33％）和80～960mg/L（r=0.9989,RSD=1.07％～2.51％）,加入回收率：黄芩甙为（102.09±1     

Binding study of Flos Lonicerae Japonicae with bovine serum albumin using centrifugal ultrafiltration and liquid chromatography

The screening and analysis of bioactive components in traditional Chinese medicines (TCMs) is very important not only for the quality control of Chinese herbs but also for elucidating the therapeutic principles. This study developed a new method for screening and analyzing bioactive compounds from TCMs using centrifugal ultrafiltration coupled with high-performance liquid chromatography. The method was successfully applied in the binding study of Flos Lonicerae Japonicae with bovine serum albumin (BSA), and 11 compounds were found to be bound with the BSA. Eight of them were positively identified as chlorogenic acid, caffeic acid, rutin, quercetin-3-O-glucoside, luteolin-7-O-glucoside, lonicerin, 3, 5-di-O-caffeoyl quinic acid and 3,4-di-O-caffeoyl quinic acid. Another three compounds were tentatively identified as two isomers of chlorogenic acid and one isomer of di-O-caffeoyl quinic acid by comparing the UV data and MS data with the previous reports. Based on modern pharmacological study, these compounds are the major bioactive components in Lonicera japonica. Therefore, the proposed method could be a good approach to predicting the potential bioactivities of multiple compounds in TCMs simultaneously.     

Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid-phase extraction

Flavonoids are an important bioactive group in the commonly used herbal medicine Flos Lonicerae. A new method of capillary zone electrophoresis (CZE) coupled with solid-phase extraction (SPE) was developed for simultaneous assay of flavonoid aglycones and glycosides in Flos Lonicerae. Optimum CZE separation was achieved with a background electrolyte (BGE) solution consisting of 80 mM boric acid and 20 mM phosphate acid, adjusted to pH 8.1, with 15% acetonitrile (v/v) added, and applying a separation voltage of 28 kV. The SPE method was used for pretreating the complex matrix of botanical materials and good reproducibility was obtained when avicularin was used as internal standard. Linearity of the method was excellent with correlation coefficients (r2) in the range of 0.9995-0.9999 and detection limits were lower than 0.6 microg/mL for the four flavonoids. The obtained recoveries varied between 93 to 104% while the relative standard deviations (RSDs) were below 4.4% (n=3). The developed CZE method was successfully used for the separation of eight flavonoids and the quantification of the four flavonoids in five species of Flos Lonicerae.     

Preparative separation of isomeric caffeoylquinic acids from Flos Lonicerae by pH-zone-refining counter-current chromatography

This work concentrates on pH-zone-refining counter-current chromatography of two isomeric dicaffeoylquinic acids, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid along with 3-caffeoylquinic acid, from crude extracts of Flos Lonicerae. The elution sequence of the isomeric dicaffeoylquinic acids, the mixing zone and mechanism of separation are discussed. The separation of 2.136g of the crude sample from Flos Lonicerae yielded two isomeric compounds: 0.289g 3,5-dicaffeoylquinic acid and 0.106g 3,4-dicaffeoylquinic acid plus 0.690g 3-caffeoylquinic acid at a high purity of over 92.9%, 94.2% and 97.5%, respectively.     

金银花中绿原酸的二阶导数差示脉冲极谱法定量研究

建立了中草药金银花中绿原酸的二阶导娄差示脉冲极谱定量分析方法,绿原酸在0.05mol/L硫酸－3mol/L亚硝酸钾－2.5mol/L乙酸钠－95％乙醇（1：1：1：17）的溶液中,于－0.276V(vs.Ag/AgCl)处出现一良好的二阶导数差示脉冲极谱峰,其峰幅值与绿原酸在0.1-0.6mmol/L范围内呈非常显著的线性关系（P＜0.01）,检出限为8.0nmol/L。本法简便、快速、灵敏,结果准确。     

Quality evaluation of Flos lonicerae through a simultaneous determination of seven saponins by HPLC with ELSD

A new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-beta-D-glucopyranosyl(6-->1)-O-beta-D-glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-alpha-L-arabinopyranosyl(2-->1)-O-alpha-L-rhamnopyranoside (7) in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb. Simultaneous separation of these seven saponins was achieved on a C18 analytical column. The mobile phase consisted of (A) acetonitrile-acetic acid (95:0.5) and (B) 0.5% aqueous acetic acid using a gradient elution of 29%A at 0-10 min, 29-46%A at 10-25 min and 46%A at 25-30 min. The drift tube temperature of ELSD was set at 106 degrees C, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2>0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0%, respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.     

Determination of five major iridoid glucosides in Flos Lonicerae by high-performance liquid chromatography coupled with evaporative light scattering detection

This study presents a new HPLC method using evaporative light scattering detection for the simultaneous determination of live major iridoid glucosides, namely 7-epi-loganin, sweroside, loganin, 7-epi-vogeloside, and secoxyloganin in Flos Lonicerae, an important traditional Chinese medicinal herb. The optimal conditions of separation and detection were achieved on a C18 analytical column with an isocratic mobile phase consisting of methanol-water (30:70, v/v) containing 0.5% acetic acid at the flow-rate of 1.0 ml/min, temperature for the detector drift tube set at 90 degrees C and the nitrogen flow-rate of 2.6 l/min. The limit of detection (S/N = 3) is less than 35.1 microg/ml and the limit of quantification (S/N = 10) is less than 140.1 microg/ml. All calibration curves show good linear regression (r2>0.996) within test ranges. This method provides good reproducibility for the quantification of the major iridoid glucosides in four Lonicera species with overall intra- and inter-day variation of less than 5% and 9%, respectively. The assay was successfully applied to quantify the main iridoid glucosides in the herb and to identify the botanical origin of Flos Lonicerae.     

Separation of chlorogenic acid and concentration of trace caffeic acid from natural products by pH‐zone‐refining countercurrent chromatography

Chlorogenic acid and caffeic acid were selected as test samples for separation by the pH‐zone‐refining countercurrent chromatography (CCC). The separation of these test samples was performed with a two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/water at a volume ratio of 4:1:5 v/v/v where trifluoroacetic acid (TFA; 8 mM) was added to the organic stationary phase as a retainer and NH₄OH (10 mM) to the aqueous mobile phase as an eluter. Chlorogenic acid was successfully separated from Flaveria bidentis (L.) Kuntze (F. bidentis) and Lonicerae Flos by pH‐zone‐refining CCC, a slightly polar two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/n‐butanol/water at a volume ratio of 4:1:1:5 v/v/v/v was selected where TFA (3 mM) was added to the organic stationary phase as a retainer and NH₄OH (3 mM) to the aqueous mobile phase as an eluter. A 16.2 mg amount of chlorogenic acid with the purity of 92% from 1.4 g of F. bidentis, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Lonicerae Flos have been obtained. These results suggest that pH‐zone‐refining CCC is suitable for the isolation of the chlorogenic acid from the crude extracts of F. bidentis and Lonicerae Flos.     

高效毛细管电泳法测定银翘解毒片中的氯原酸、甘草酸和甘草次酸

建立了同时测定银翘解每片中氯原酸、甘草酸和甘草次酸的高效毛细管电泳法,电解缓冲液为20mmol／L磷酸二氢钠和5mmol／L硼砂的混合溶液（pH7.0）。方法简便快速,具有良好的精密度、回收率和线性关系,并测定了很翘解毒片中3组分的含量。     

Capillary high-performance liquid chromatography with mass spectrometry for simultaneous determination of major flavonoids, iridoid glucosides and saponins in Flos Lonicerae

Flos Lonicerae, a traditional herbal medicine, has been used in China to treat some inflammatory disease. Several different classes of compounds have been separated from the herb to assess their pharmacological activities. Among these classes, flavonoids, iridoid glycosides and saponins have been well studied and may be responsible for its clinical application. Therefore, quality control of Flos Lonicerae is an important issue for drug safety and validity evaluations. A quantitative method consisting of solid phase extraction followed by capillary high-performance liquid chromatography-diode array detection/electrospray ionization mass spectrometry (capillary HPLC-DAD/ESI-MS) was developed for simultaneously assay of 24 compounds in Flos Lonicerae. Under optimized capillary HPLC-DAD/ESI-MS conditions, these compounds, including nine flavonoids, eight iridoid glucosides and seven saponins, were separated with high efficiency in the selected-ion monitoring (SIM) mode. Linearity of the method was good with correlation coefficients (r(2)) in the range of 0.9935-0.9998 and detection limits were lower than 2.57 ng/mL for most of analytes. The obtained recoveries varied between 91.0 and 108.7% with the relative standard deviations (RSDs) within 8.74% (n=3). The capillary HPLC-DAD/ESI-MS method was also successfully applied to the analysis of these compounds in five species of Flos Lonicerae. It was demonstrated to be a powerful tool for comprehensive analysis of herbal medicines, owing to its exclusive selectivity and excellent sensitivity.