Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS

A high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry( HPLC-ESI-MS/MS) method was developed for the analysis and identification of ginsenosides in the extracts of raw Panax ginseng(RPG) and steamed Panax ginseng at high temperatures(SPGHT). A total of 25 ginsenosides were extracted include of which 10 low-polar ginsenosides, such as ginsenosides F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3 and so on, were identified according to their HPLC retention time and MS/MS data. The results indicated that the low polar ginsenosides were seldom found in RPG. For the exploration of the transformation pattern of the ginsenosides in steam processing, the standards of ginsenosides Re, Rg1, Rb1, Rc, Rb2, Rb3 and Rd were selected and hydrolyzed at a temperature of 120 ℃. The results show that these polar ginsenosides can be converted to low-polar ginsenosides such as Rg2, Rg6, F4, Rk3 and Rg5 by hydrolyzing the sugar chains.     

人参中人参皂苷的直接高压微波辅助降解

采用高效液相色谱-电喷雾质谱联用法测定了人参提取液中的人参皂苷. 考察了天然人参皂苷发生降解的条件, 同时研究了单体人参皂苷Rg1, Re, Rb1, Rc, Rb2和Rd的降解, 并对降解产物进行了分析. 结果表明, 随着提取压力的升高, 提取液中天然人参皂苷的含量逐渐减少, 同时产生多种次级人参皂苷. 当微波提取压力达到600 kPa, 提取时间为10 min时, 提取液中的主要天然人参皂苷达到完全降解, 次级人参皂苷Rg3含量达到最高. 在单体人参皂苷Rb1, Rc, Rb2和Rd的降解产物中均得到人参皂苷Rg3.     

Determination of ginsenosides (ginseng saponins) in dry root powder from Panax ginseng, Panax quinquefolius, and selected commercial products by liquid chromatography: interlaboratory study

Twelve collaborating laboratories assayed 4 products, namely, Panax ginseng, Panax quinquefolius, and 2 ginseng products, for 6 ginsenosides: Rb1, Rb2, Rc, Rd, Re, and Rg1. Collaborators also received a negative control for the recovery study. Pure ginsenosides were provided as reference standards for the liquid chromatography (LC) analysis and the system suitability tests. The LC analyses were performed on the methanol extract using UV detection at 203 nm. For P. ginseng, individual ginsenosides were consistent in their means; repeatability standard deviations (RSDr) ranged from 4.17 to 5.09% and reproducibility standard deviations (RSDR) ranged from 7.27 to 11.3%. For P. quinquefolius, the Rb1 and Rb2 ginsenosides were higher and lower in concentration than P. ginseng, with RSDr values of 3.44 and 6.60% and RSDR values of 5.91 and 12.6% respectively, and other analytes at intermediate precisions. For ginseng commercial products, RSDr values ranged from 3.39 to 8.12%, and RSDR values ranged from 7.65 to 16.5%. A recovery study was also conducted for 3 ginsenosides: Rg1, Re, and Rb1. The average recoveries were 99.9, 96.2, and 92.3%, respectively. The method is not applicable for the determination of Rg1 and Re in ginseng product at levels <300 mg/kg.     

Simultaneous determination of triterpene saponins in ginseng drugs by high-performance liquid chromatography

A HPLC method for the simultaneous determination of 11 triterpene saponins with four-type aglycones (protopanaxadiol, protopanaxatriol, ocotillol and oleanolic acid types) in Ginseng drugs was developed and validated. Using a gradient of acetonitrile and 10 mM K-phosphate buffer (pH 5.80) as the mobile phase and UV detection at 196 nm, more than 18 ginsenosides with different aglycones were separated satisfactorily within 60 min. The detection limits (signal/noise> or =3) were 0.1 microg for ginsenosides Rb1, Rc, Rd, Re and Rg1, chikusetsusaponin III, and notoginsenoside R2, 0.2 microg for gisenoside Ro and chikusetsusaponin IVa, 0.3 microg for chikusetsusaponin IV, and 3 microg for majonoside R2. The calibration curve of each saponin had a correlation coefficient close to 1. Intra- and interday precisions were less than 2.1% (n=5) and 3.3% (n=15), respectively. The recovery rates of extraction were in the range of 96.4-102.7% for all ginsenosides. By adopting this method, the determinations of 11 ginsenosides in three Ginseng drugs derived from Panax ginseng, Panax vietnamensis var. fuscidiscus and Panax japonicus (Japan) were achieved.     

动态泡沫浮选法分离富集人参提取液中的二醇型人参皂苷

采用动态泡沫浮选法分离富集人参提取液中的二醇型人参皂苷, 用高效液相色谱法测定6种人参皂苷Rg₁, Re, Rb₁, Rc, Rb₂和Rd的含量. 考察了浮选液pH值、电解质NaCl浓度、载气流量、料液浓度及料液流速对人参皂苷浮选率的影响, 确定了动态泡沫浮选的最佳条件, 并与溶剂提取法、溶剂浮选法以及静态泡沫浮选法进行了比较. 结果表明, 动态泡沫浮选法对二醇型人参皂苷Rb₁, Rc, Rb₂和Rd具有高富集效率, 回收率分别为93.3%, 98.6%, 96.9%和98.3%, 而对三醇型人参皂苷Rg1和Re的富集效率却很低, 回收率分别为4.8%和4.2%. 该法是分离纯化二醇型人参皂苷的一种简便有效的方法.     

Major ginsenoside contents in rhizomes of Panax sokpayensis and Panax bipinnatifidus

This study compared eight major ginsenosides (Rg1, Rg2, Rf, Re, Rd, Rc, Rb1 and Rb2) between Panax sokpayensis and Panax bipinnatifidus collected from Sikkim Himalaya, India. High-performance liquid chromatographic analysis revealed that all major ginsenosides were present in the rhizomes of P. sokpayensis except ginsenoside Rc, whereas ginsenoside Rf, Rc and Rb2 were not detected in P. bipinnatifidus.     

Studies on Intestinal Transport of Ginsenoside Compatibility with Veratrum nigrum via Caco-2 Cell Monolayer Model Coupled with UPLC-ESI-MS Method

The Caco-2 cells have been recognized as effective tools to be applied to imitating the drug absorption in human intestine for the transport of drug. Herein, Caco-2 cell monolayer model was used to study the transport of the ginsenoside compatibility with Veratrum nigrum in different proportions. A specific high performance liquid chromatography-electrospray ionization-mass spectrometry(HPLC-ESI-MS) method was developed for the semiquantitative determination of ginsenoside in intestinal transport with Dioscin as an internal standard. For the Caco-2 model constructed, two influencing factors were investigated, including time and concentration. The results suggest that the absorption of ginsenoside Re, Rg1, Rb1, Rc, Rb2 and Rd are time- and concentration-dependent and the excretions of Rb1, Rc, Rb2 and Rd have a relatronship with some transport proteins. The bioavailability of the ginsenosides has reduced compared to the single Panax ginseng extract when compatibility with a certain amount of Veratrum nigrum.     

Chemical characteristics of three medicinal plants of the Panax genus determined by HPLC-ELSD

The morphological appearance and main ingredients of three Chinese medicines (CMs), P. ginseng, P. quinquefolius, and P. notoginseng of the Panax genus, are similar. However, their pharmacological activities are obviously different. To ensure their safety and efficacy, chemical characteristics of the three CMs were determined using pressurized liquid extraction and HPLC-evaporative light scattering detection. Twelve major saponins, namely notoginsenoside R1, pseudo-ginsenoside F11, ginsenosides Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, Rb3, Rd, and Rg3 were also quantitatively compared among the three CMs. The contents of total investigated saponins varied considerably, by up to 4-14-fold, between the highest (P. notoginseng, 82.8-136.5 mg/g) and the lowest values (P. ginseng, 10.0-21.1 mg/g). Hierarchical clustering analysis based on the characteristics of 11 investigated saponins (except ginsenoside Rb3) and notoginsenoside R1, pseudo-ginsenoside F11, and the ratio of ginsenoside Rg1/Rb1 and Rg1/Re showed that 56 tested samples were divided into three main clusters in accordance with the three Panax species. Similarity evaluation of chromatograms was also performed using "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A)". The results showed that a high degree of similarity existed within individual clusters, but a low degree between the clusters, which could be used for quality control of the three CMs.     

Determination of Seven Major Ginsenosides in Different Parts of Panax quinquefolius L.(American Ginseng) with Different Ages

Ginsenosides Rg1,Re,Rb1,Rc,Rb2,Rb3,and Rd in different parts of the American ginseng plant were investigated.The extraction process was a pressurized microwave-assisted extraction(PMAE).The seven ginsenosides were separated and determined by high-performance liquid chromatography(HPLC) with a ultraviolet(UV) detector,at 203 nm.The experiment results showed significant variations in the individual ginsenoside contents of the American ginseng in different parts and ages of the plant.The results demonstrated that the leaves,root hairs,and rhizomes of Panax quinquefolius L.contained higher ginsenoside contents,followed by the main roots and stems.The leaves contained dramatically higher levels of ginsenoside Rg1,Rb3,and Rd than the other four parts.Higher contents of Rb1 and Re were present in the main roots,root hairs,and rhizomes.The amount of ginsenoside content in the stems was the lowest.The total content of the seven ginsenosides in main roots,root hairs and rhizomes increased with the age of the plant.In contrast,the ginsenoside contents in the leaves and stems decreased with a year of growth.     

高效液相色谱-飞行时间质谱联用法分析人参及人参皂苷与山楂配伍过程中的水解行为

利用高效液相色谱-飞行时间质谱联用的方法,分别对人参配伍山楂前后人参皂苷的变化进行分析,同时对人参皂苷Re、Rg1、Rb1、Rd与山楂配伍的水解规律进行系统研究,并与单独煎煮液、仿山楂配伍pH值煎煮液的水解产物进行比较,结果发现人参与山楂配伍后人参皂苷Rg1、Rb1含量明显减少,而人参皂苷Re、Rd、Rg2、Rg3、F2、Rh1含量明显增加,其中人参皂苷Re与山楂配伍后水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2,仿山楂配伍pH值水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2、Rg4、Rg6;人参皂苷Rg1与山楂配伍后水解产物为20(S)-Rh1、20(R)-Rh1,仿山楂pH值水解产物为20(S)-Rh1、20(R)-Rh1、Rh4、Rk3;人参皂苷Rb1与山楂配伍后水解产物为Rd、20(S)-Rg3,仿山楂pH值水解产物为F2、20(S)-Rg3;人参皂苷Rd与山楂配伍后水解产物为F2、20(S)-Rg3、20(R)-Rg3,仿山楂pH值水解产物为20(S)-Rg3、20(R)-Rg3。研究表明,不同人参皂苷和山楂配伍后与仿山楂pH值的水解产物并不相同,人参与山楂配伍改变了人参皂苷成分的种类及含量。本研究为临床方剂中人参与山楂配伍后成分的变化提供物质基础数据。     

Analysis of saponins in raw and steamed Panax notoginseng using high-performance liquid chromatography with diode array detection

A reversed-phase high-performance liquid chromatography-diode array detection method was developed and validated for the simultaneous determination of six saponins (notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rc, Rd) in raw and steamed Panax notoginseng. Linearity (r2 > 0.9988), intra- and inter-day precision (RSD < 4%), limit of detection (0.008-0.013 mg/ml), limit of quantification (0.027-0.042 mg/ml) of the saponins were determined. The method was successfully applied to 11 pairs of raw and steamed P. notoginseng products. Three products showed discrepancies between theirlabelled claims (raw or steamed) and the results of analysis. This new, simple and reliable method could be used in the quality control of raw and steamed P. notoginseng.     

Simultaneous quantification of Panax and Epimedium species using Rapid Resolution Liquid Chromatography (RRLC)

Two Rapid Resolution Liquid Chromatography (RRLC) methods have been developed and validated for simultaneous quantification of eight major ginsenosides from Panax species, namely, R1, Rg1, Re, Rf, Rb1, Rb2, Rc, and Rd, and flavonoids from Epimedium species, namely, epimedins A, B, and C and icariin. The analyses were performed using an Agilent 1200 series RRLC system with Phenomenex Luna C18-HST and Zorbax Eclipse XDB columns. The separation was performed with a gradient mobile phase of A (pure water) and B (acetonitrile) at a flow rate of 1.0 mL/min and 2.5 mL/min, respectively. Both columns were kept at 40 degrees C with the detection wavelength set at 203 nm. Specific eluted compounds were identified by using reference samples of ginsenosides R1, Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd, and epimedins A, B, C and icariin. Baseline separation was achieved in less than 15 minutes for the Phenomenex Luna column and 4 minutes for the Zorbax Eclipse column. Characteristic RRLC profiles were established for complex mixtures of ginsenosides from Panax species and flavonoids from Epimedium species. Both methods developed here are effective for the quality control of formulated products containing both Panax and Epimedium varieties.     

Development of a rapid and convenient method to separate eight ginsenosides from Panax ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detection

Ginsenosides exhibit diverse biological activities and are major well-known components isolated from the radix of Panax ginseng C.A. Meyer. In the present work, a rapid and facile method for the separation and purification of eight ginsenosides from P. ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detector (HSCCC-ELSD) was successfully developed. The crude samples for HSCCC separation were first purified from ginseng extract using a macroporous resin; the extract was loaded onto a Diaion-HP20 column and fractionated by methanol and water gradient elution. The ginsenosides-protopanaxadiol (PPD) and protopanaxatriol (PPT) fractions were subsequently eluted with 65 and 80% methanol and water gradient elution, respectively. Furthermore, these two fractions were separated by HSCCC-ELSD. The two-phase solvent system used for separation was composed of chloroform/methanol/water/isopropanol at a volume ratio of 4:3:2:1. Each fraction obtained was collected and dried, yielding the following eight ginsenosides: Rg(1), Re, Rf, Rh(1), Rb(1), Rc Rb(2) and Rd. The purity of these ginsenosides was greater than 97% as assessed by HPLC-ELSD, and their structures were characterized by electrospray-ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. This is the first report regarding the separation of the ginsenosides Rh(1), Rb(2) and Rc from P. ginseng by HSCCC.     

Rational development of a selection model for solvent gradients in single-step separation of ginsenosides from Panax ginseng using high-speed counter-current chromatography

A new model of solvent gradients selection was rationally developed for the preparative separation of target compounds. The solvent gradients were selected based on a three-stage screening process where stationary phase retention was ensured by introducing a new parameter termed as the phase ratio. The phase ratio was calculated after mixing the upper phase of a solvent system with the lower phase of a different solvent system (1:1, v/v). The developed model was applied to the one-step separation of eight ginsenosides from Panax ginseng. Three gradients were selected on the basis of new model and eight ginsenosides, Rb(1), Rb(2), Rc, Rd, Re, Rg(1), Rf, and Rh(1), were efficiently separated by high-speed counter-current chromatography coupled with evaporative light scattering detector. The structures of all compounds were characterized by electrospray-ionization mass spectrometry and nuclear magnetic resonance spectroscopy.     

Determination of marker constituents in radix Glycyrrhizae and radix Notoginseng by near infrared spectroscopy

High-performance liquid chromatographic (HPLC) methods were developed for the determination of glycyrrhizin in radix Glycyrrhizae and ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf and Rg, in radix Notoginseng. These methods were used as reference methods for near-infrared (NIR) spectroscopy. Spectroscopic calibrations were developed for the determination of glycyrrhizin, the total content of ginsenosides and the individual major ginsenosides Rb1, Rd, Re and Rg1. Standard errors of cross validation (SECV) were 1.22 mg g(-1) for glycyrrhizin (concentration range 21.3-34.1 mg g(-1)) and 0.99 mg g(-1) for the sum of ginsenosides (concentration range 55.3-71.1 mg g(-1)). The corresponding coefficients of determination (R2) were 0.94 and 0.98, respectively. The SECVs were generally less than a factor of 2.5 of the repeatability standard deviation of the HPLC methods.     

Simultaneous determination of 11 saponins in Panax notoginseng using HPLC-ELSD and pressurized liquid extraction

A new HPLC coupled with evaporative light scattering detection (ELSD) method was developed for simultaneous determination of 11 major triterpene saponins, namely notoginsenoside R1 (1), ginsenosides Rg1 (2), Re (3), Rf(4), Rb1 (5), Rg2 (6), Rc (7), Rb2 (8), Rb3 (9), Rd (10), and Rg3 (11) in Panax notoginseng, a commonly used traditional Chinese medicine (TCM). Pressurized liquid extraction (PLE) was employed for sample preparation, and the analysis was achieved using a Zorbax ODS C18 column eluted with gradient water-ACN in 60 min. The drift tube temperature of ELSD was set at 60 degrees C, and nitrogen flowrate was at 1.4 L/min. The method provided good repeatability and sensitivity for quantification of 11 saponins with overall precision (including intra- and interday) and LOD of less than 2.9% (RSD) and 98 ng, respectively. The validated method was successfully applied to quantify 11 saponins in 28 samples of P. notoginseng collected in different places, which is helpful to control the quality of P. notoginseng and its related products.     

Simultaneous determination of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 in rat plasma by HPLC/ESI/MS: platform for the pharmacokinetic evaluation of total panax notoginsenoside, a typical kind of multiple constituent traditional Chinese medicine

A platform for the pharmacokinetic study of multiple constituent traditional Chinese medicine was developed and validated. An HPLC/ESI/MS method was employed for the simultaneous determination of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 in rat plasma. After the addition of digoxin as an internal standard (IS), rat plasmas were extracted with n-butanol saturated with pure water and all analytes were separated on a reversed-phased C(18) column with a mobile phase of acetonitrile-water (0.5 mM ammonium chloride) and pumped at a flow rate of 0.2 mL/min. Analytes were determined in a single quadrupole mass spectrometer using an electrospray ionization source. HPLC/ESI/MS was performed in the selected-ion monitoring mode with the chlorinated adducts of molecular ions [M + Cl]( -) at m/z 967.75, 835.80, 981.80, 981.80, 1143.65 and 815.40 for R1, Rg1, Rd, Re, Rb1 and digoxin, respectively. The method showed excellent linearity over the concentration range 3.03-775.00 ng/mL (r(2) = 0.9994) for R1, 4.00-1025.00 ng/mL (r(2) = 0.9991, 0.9988, 0.9991) for Rg1, Rd and Re, respectively, and 2.77-710.00 ng/mL for Rb1 (r(2) = 0.9990). The low limit of quantification was 3.03, 4.00, 4.00, 4.00 and 2.77 ng/mL for R1, Rg1, Rd, Re and Rb1, respectively, with S/N > 10. The intra- and inter-day precisions were below 12.00% and the accuracy was between -2.31 and +4.43% for all analytes. The extract recoveries of analytes were from 67.47 to 94.18%. All analytes were stable in rat plasma after storage for 12 h at ambient temperature, at 4 degrees C for 12 h in the sample pool, at -20 degrees C for 4 weeks and at -20 degrees C for three thaw-freeze cycles. The HPLC/ESI/MS technique provided an excellent method for the simultaneous quantification of R1, Rg1, Rd, Re and Rb1 in rat plasma and was successfully applied to the pharmacokinetic study of a multiple-constituent traditional Chinese medicine, total panax notoginsenoside (Xuesaitong injection).     

Identification and Evaluation of a Panel of Ginsenosides from Different Red Ginseng Extracts with Nootropic Effect

Red ginseng has been gradually discovered to have pharmacological and physiological effects. It is well known that the most important bioactive components of ginseng are ginsenosides. The nootropic effect of ginsenosides from nine different red ginseng extracts was evaluated here. Nine groups of mice were perfused with different concentrations of nine red ginseng extracts, respectively, and two groups of mice with distilled water. The nootropic effect of ginsenosides on mice was evaluated with behavior tests and a biochemical indicator study. The extracts were identified by rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(RRLC-Q-TOF-MS). Furthermore, principal component analysis(PCA) was used to analyze the contribution of chemical components from different ginseng groups. The extracts with the most and the weakest effective nootropic were found. It is notable that extract processing is a very important factor to decide pharmacological functions of ginseng extracts. As a conclusion, the most effective extract method for ginsenosides has been found. A panel of 13 ginsenosides has been screened out as chemical markers with nootropic effect, which include high level ginsenosides Ra0, Rb1, Rc, Rb2, Rb3, Re, Rd, and Rg1 and low level ginsenosides mRb1, mRc, mRb2, mRd, and F2. Low level ginsenosides were first time to be discovered as possible nootropic compounds. This method may shed light on fast discovery of bioactive compounds of medicinal plants with low level compounds.     

重组内切纤维素酶Fpendo5A转化三七总皂苷

从嗜热细菌基因组中克隆到1个新的纤维素酶基因,并在大肠杆菌中进行了高效可溶性表达,粗酶液经镍柱亲和层析进行分离纯化.利用快速分离液相色谱-四极杆飞行时间质谱联用仪(RRLC/Q-TOF-MS)对重组内切纤维素酶Fpendo5A转化三七总皂苷的产物结构进行了鉴定,并进一步阐明其转化机制.结果表明,该酶的最适反应温度和pH值分别为80℃和5.5.Fpendo5A能够催化三七总皂苷中的主要皂苷成分,即Ra_1,Rb_1,Rc,Rd和Rg_3的侧链糖基的水解反应,但对于不同的皂苷底物,Fpendo5A选择性催化的侧链糖基类型不同.经鉴定,Fpendo5A转化Ra_1,Rb_1,Rc,Rd和Rg_3的转化产物分别为Rb_2,GypⅩⅦ,CMC_1,F_2和Rh_2.由此可见,Fpendo5A通过水解Rb_1,Rc,Rd和Rg3的C3位的β-(1,2)糖苷键分别生成GypⅩⅦ,CMC1,F2和Rh2.在转化Ra_1时,Fpendo5A通过水解Ra_1的C20位的α-(1,4)木糖苷键生成Rb2.     

Simultaneous LC Determination of Ginsenosides Using a Modified Extraction Procedure and an Improved Step Gradient Program

A novel, accurate and precise high performance liquid chromatographic method has been developed for simultaneous determination of seven important ginsenosides (Rg1, Re, Rf, Rb1, Rc, Rb2 and Rd) in ginseng products. The separation was performed on a Shim-pack VP-ODS column (5 μm, 150 ×2 mm i.d) with ultraviolet detection at 200 nm by using the improved step gradient elution program. The LODs (S/N = 3) were in the range 0.29 to 1.33 ng μL⁻¹. All calibration curves showed a good linearity (R² > 0.998) over the ranges tested. The recoveries obtained from spiked sample were between 95.1% and 98.7%. The proposed method was successfully applied to several ginseng pharmaceutical samples. For the sample preparation, a modified extraction method was made to improve the extraction efficiency by evaluation of five solvent systems. The results demonstrated that the extraction with methanol-water (80:20, v/v) is suitable method preferably for the extraction of the ginsenosides. On leave from Department of Pharmacy and Applied Chemistry, Jilin Institute of Chemical Technology, Jilin 132022, P. R. China