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

Ginsenosides Rgl, 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.     

Distinguishing Ontario ginseng landraces and ginseng species using NMR-based metabolomics

The use of ¹H-NMR-based metabolomics to distinguish and identify unique markers of five Ontario ginseng (Panax quinquefolius L.) landraces and two ginseng species (P. quinquefolius and P. ginseng) was evaluated. Three landraces (2, 3, and 5) were distinguished from one another in the principal component analysis (PCA) scores plot. Further analysis was conducted and specific discriminating metabolites from the PCA loadings were determined. Landraces 3 and 5 were distinguishable on the basis of a decreased NMR intensity in the methyl ginsenoside region, indicating decreased overall ginsenoside levels. In addition, landrace 5 was separated by an increased amount of sucrose relative to the rest of the landraces. Landrace 2 was separated from the rest of the landraces by the increased level of ginsenoside R_b1. The Ontario P. quinquefolius was also compared with Asian P. ginseng by PCA, and clear separation between the two groups was detected in the PCA scores plot. The PCA loadings plot and a t-test NMR difference plot were able to identify an increased level of maltose and a decreased level of sucrose in the Asian ginseng compared with the Ontario ginseng. An overall decrease of ginsenoside content, especially ginsenoside R_b1, was also detected in the Asian ginseng’s metabolic profile. This study demonstrates the potential of NMR-based metabolomics as a powerful high-throughput technique in distinguishing various closely related ginseng landraces and its ability to identify metabolic differences from Ontario and Asian ginseng. The results from this study will allow better understanding for quality assessment, species authentication, and the potential for developing a fully automated method for quality control.

Region-Specific Biomarkers and Their Mechanisms in the Treatment of Lung Adenocarcinoma: A Study of Panax quinquefolius from Wendeng,China

Panax quinquefolius, a popular medicinal herb, has been cultivated in China for many years. In this work, the region-specific profiles of metabolites in P. quinquefolius from Wendeng was investigated using liquid-chromatography–quadrupole–time-of-flight-(LC–Q–TOF)-based metabolomics analysis. The three most abundant biomarkers, identified as ginsenoside Rb₃, notoginsenoside R₁, and ginsenoside Rc, were the representative chemical components employed in the network pharmacology analysis. In addition, molecular docking and western blotting analyses revealed that the three compounds were effective binding ligands with Hsp90α, resulting in the inactivation of SRC and PI3K kinase, which eventually led to the inactivation of the Akt and ERK pathways and lung cancer suppression. The outcomes obtained herein demonstrated the intriguing chemical characteristics and potential functional activities of P. quinquefolius from Wendeng.     

Dose‐dependent exposure profile and metabolic characterization of notoginsenoside R1 in rat plasma by ultra‐fast liquid chromatography–electrospray ionization–tandem mass spectrometry

Notoginsenoside R₁ (NGR₁), a diagnostic protopanaxatriol‐type (ppt‐type) saponin in Panax notoginseng, possesses potent biological activities including antithrombotic, anti‐inflammatory, neuron protection and improvement of microcirculation, yet its pharmacokinetics and metabolic characterization as an individual compound remain unclear. The aim of this study was to investigate the exposure profile of NGR₁ in rats after oral and intravenous administration and to explore the metabolic characterization of NGR₁. A simple and sensitive ultra‐fast liquid chromatographic–tandem mass spectrometric method was developed and validated for the quantitative determination of NGR₁ and its major metabolites, and for characterization of its metabolic profile in rat plasma. The blood samples were precipitated with methanol, quantified in a negative multiple reaction monitoring mode and analyzed within 6.0 min. Validation parameters (linearity, precision and accuracy, recovery and matrix effect, stability) were within acceptable ranges. After oral administration, NGR₁ exhibited dose‐independent exposure behaviors with t_1/2 over 8.0 h and oral bioavailability of 0.25–0.29%. A total of seven metabolites were characterized, including two pairs of epimers, 20(R)‐notoginsenoside R₂/20(S)‐notoginsenoside R₂ and 20(R)‐ginsenoside Rh₁/20(S)‐ginsenoside Rh₁, with the 20(R) form of saponins identified for the first time in rat plasma. Five deglycometabolites were quantitatively determined, among which 20(S)‐notoginsenoside R₂, ginsenoside Rg₁, ginsenoside F₁ and protopanaxatriol displayed relatively high exploration, which may partly explain the pharmacodynamic diversity of ginsenosides after oral dose.     

Simultaneous determination of ginsenoside Rg1, Re and notoginsenoside R1 in human plasma by LC‐MS/MS and its application in a pharmacokinetic study in Chinese volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for simultaneous quantification of ginsenosides Rg₁, Re and notoginsenoside R₁ in human plasma. Chromatography was performed on Capcell Pak C₁₈ MG II column using a binary gradient using mobile phase A (5 mm ammonium formate solution) and B (methanol, containing 5 mm ammonium formate) at a flow rate of 0.3 mL/min. The entire chromatographic run time was 3.2 min. Quantification was achieved using multiple reaction monitoring in positive mode using API 3000. This method was validated in terms of specificity, linearity, precision, accuracy, matrix effect and stability. The calibration curves were linear in the concentration range of 0.020–5.00 ng/mL for ginsenosides Rg₁, Re and notoginsenoside R₁. The lower limit of quantification (LLOQ) of this method was 0.020 ng/mL. The intra‐run and inter‐run precision values were within 12.31% for ginsenoside Rg₁, 14.13% for ginsenoside Re and 11.46% for notoginsenoside R₁ at their LLOQ levels. The samples were stable under all tested conditions. This method was successfully applied to study the pharmacokinetics of ginsenoside Rg₁ and notoginsenoside R₁ in 24 healthy volunteers following oral administration of 200 mg Sanqi Tongshu Enteric‐Pellets Capsule.     

Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines

The present study aims at developing an extraction protocol for efficient ginsenoside recovery from cell suspensions of Panax quinquefolius and P. sikkimensis. Methanol (100%, 70% and 30%), water (40°C, 90°C), water-saturated butanol and butanol-saturated water were compared for their ultrasonication-assisted ginsenoside retrieval efficacy. HPLC and HP-TLC analysis revealed 100% methanol as the best solvent for maximum retrieval of Rb (diol) and Rg (triol) ginsenosides (P. quinquefolius: Rb: 0.189, Rg: 3.163 mg/g DW; P. sikkimensis: Rb: 0.245, Rg: 4.073 mg/g DW), followed by water (90°C). Methanolic solutions, especially 70%, proved to be significant retrievers of Rg1 (1.812 and 1.327 mg/g DW in P. quinquefolius and P. sikkimensis), with poor Re recovery (0.328 and 0.342 mg/g DW). Water-saturated butanol also led to significant ginsenoside extraction (72.4% of content extracted by methanol), selectively in P. quinquefolius, with a less than 50% of total content extracted by methanol, in P. sikkimensis.     

State-of-the-art separation of ginsenosides from Korean white and red ginseng by countercurrent chromatography

Ginseng (Panax ginseng C. A. Meyer) has been one of the most popular herbs used for nutritional and medicinal purposes by the people of eastern Asia for thousands of years. Ginsenosides, the mostly widely studied chemical components of ginseng, are quite different depending on the processing method used. A number of studies demonstrate the countercurrent chromatography (CCC) separation of ginsenosides from several sources; however, there is no single report demonstrating a one-step separation of all of these ginsenosides from different sources. In the present study, we have successfully developed an efficient CCC separation methodology in which the flow-rate gradient technique was coupled with a new solvent gradient dilution strategy for the isolation of ginsenosides from Korean white (peeled off dried P. ginseng) and red ginseng (steam-treated P. ginseng). The crude samples were initially prepared by extraction with butanol and were further purified with CCC using solvent gradients composed of methylene chloride–methanol–isopropanol–water (different ratios, v/v). Gas chromatography coupled with flame ionization detector was used to analyze the components of the two-phase solvent mixture. Each phase solvent mixture was prepared without presaturation, which saves time and reduces the solvent consumption. Finally, 13 ginsenosides have been purified from red ginseng with the new technique, including Rg₁, Re, Rf, Rg_2, Rb₁, Rb₂, Rc, Rd, Rg₃, Rk₁, Rg₅, Rg₆, and F₄. Meanwhile, eight ginsenosides have been purified from white ginseng, including Rg₁, Re, Rf, Rh_1, Rb₁, Rb₂, Rc, and Rd by using a single-solvent system. Thus, the present technique could be used for the purification of ginsenosides from all types’ ginseng sources. To our knowledge, this is the first report involving the separation of ginsenoside Rg₂ and Rg₆ and the one-step separation of thirteen ginsenosides from red ginseng by CCC.     

A method for the analysis of ginsenosides, malonyl ginsenosides, and hydrolyzed ginsenosides using high-performance liquid chromatography with ultraviolet and positive mode electrospray ionization mass spectrometric detection

A high-performance liquid chromatographic separation coupled to diode array absorbance and positive mode electrospray mass spectrometric detection has been developed for the analysis of ginsenosides, malonyl ginsenosides, and hydrolyzed ginsenosides in extracts of Asian ginseng (Panax ginseng) and American ginseng (P. quinquefolius). The method is capable of separating, identifying, and quantifying the predominant ginsenosides found in heated alcoholic extracts of Asian and American ginseng roots routinely sold as nutraceuticals. It also separates and identifies the malonyl ginsenosides often found in cold alcoholic extracts of ginseng root and has the potential to quantify these compounds if pure standards are available. Furthermore, it can separate and identify ginsenoside hydrolysis products such as those readily produced in situations mimicking gastric situations, including those used for dissolution studies (i.e., 0.1 N HCl, 37 degrees C).     

Identification of Ginsenosides in Panax quinquefolium by LC-MS

An HPLC-APCI-MS method for the identification of ginsenosides in Panax quinquefolium has been developed. HPLC-APCI-MS could effectively identify ocotillol, protopanaxadiol, protopanaxatriol and oleanane-type ginsenosides in a single MS experiment since [M-H]⁻ ions and characteristic thermal degradation ions of ginsenosides could be simultaneously observed under negative and positive ionization conditions. Nine ocotillol-type ginsenosides including 24(R)-pseudoginsenoside F₁₁ were firstly identified and a total of 30 ginsenosides were identified in Panax quinquefolium. The ginsenoside profile differences between Chinese and American P. quinquefolium were investigated by HPLC-APCI-MS.     

Glycosides of the epigeal part of Panax ginseng

The qualitative and quantitative compositions of ginsensides in the epigeal part of ginseng cultivated in the Maritime Territory have been determined. The concentration of ginsenosides in the epigeal part of this plant is fairly high and depends on the growth site and seasonal conditions. A method of alkaline hydrolysis for obtaining ginsenoside R_g2 from R_e is described. Under these conditions for cleaving the glycosidic bond in the ginsenosides no epimerization at C₂₀ is observed.     

Foam Floatation-SPE for Separation and Concentration of Trace Ginsenosides

A foam floatation (FF) process and a solid phase extraction (SPE) process were synchronously applied to the separation and concentration of ginsenosides from extracts of Panax quinquefolius L. The selectivity and sensitivity for the determination of the ginsenosides were improved. The experimental conditions, including volumes of the sample solutions, pH value of sample solution, the flow rate of nitrogen gas and floatation time for FF and elution conditions for SPE were examined and optimized. Average recoveries for protopanaxadiol (PPD) ginsenosides Rc, Rb₂, Rb₃, Rd, and Rb₁ were between 84.5 and 98.8%. The relative standard deviations were lower than 6.73% for the PPD ginsenosides. The results were satisfactory since both FF and SPE were synchronously applied to both the separation and concentration. The proposed method is not only of importance for the concentration and separation of ginsenosides in extracts from P. quinquefolius L., but also of great potential in the separation and concentration of trace compounds in the other solution samples.     

Fast determination of ginsenosides in ginseng by high‐performance liquid chromatography with chemometric resolution

Ginseng is a well‐known traditional Chinese medicinal herb, and ginsenosides are its major active components. A method for the fast determination of ginsenosides in ginseng samples by high‐performance liquid chromatography was developed and used for the quantitative analysis of four ginsenosides in three different ginseng samples. In this method, instead of time‐consuming gradient elution, isocratic elution was used to speed up the analysis. Under strong isocratic elution, all the ginsenosides are eluted in 2.3 min. Although the measured signal is composed of overlapped peaks with the interferences and background, the signal of ginsenosides can be extracted by chemometric resolution. A non‐negative immune algorithm was employed to obtain the chromatographic information of the target components from the data. Compared with conventional chemometric approaches, the method can perform the extraction for one‐dimensional overlapping signals. The method was validated by the determination of four ginsenosides in three different ginseng samples. The recoveries of the spiked samples were in the range of 94.08–107.3%.     

Rapid differentiation of Panax ginseng and Panax quinquefolius by matrix-assisted laser desorption/ionization mass spectrometry

A matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based method has been developed for rapid differentiation between Panax ginseng and Panax quinquefolius, two herbal medicines with similar chemical and physical properties but different therapeutic effects. This method required only a small quantity of samples, and the herbal medicines were analyzed by MALDI-MS either after a brief extraction step, or directly on the powder form or small pieces of raw samples. The acquired MALDI-MS spectra showed different patterns of ginsenosides and small chemical molecules between P. ginseng and P. quinquefolius, thus allowing unambiguous differentiation between the two Panax species based on the specific ions, intensity ratios of characteristic ions or principal component analysis. The approach could also be used to differentiate red ginseng or P. quinquefolius adulterated with P. ginseng from pure P. ginseng and pure Panax quinquefolium. The intensity ratios of characteristic ions in the MALDI-MS spectra showed high reproducibility and enabled quantitative determination of ginsenosides in the herbal samples and percentage of P. quinquefolius in the adulterated binary mixture. The method is simple, rapid, robust, and can be extended for analysis of other herbal medicines.     

Orthogonal strategy development using reversed macroporous resin coupled with hydrophilic interaction liquid chromatography for the separation of ginsenosides from ginseng root extract

Ginsenosides have been widely conceded as having various biological activities and are considered to be the active ingredient of ginseng. Nowadays, preparative high‐performance liquid chromatography is considered to be a highly efficient method for ginseng saponins purification and preparation. However, in the process of practical application, due to the complex and varied composition of natural products and relatively simple pretreatment process, it is likely to block the chromatographic column and affect the separation efficiency and its service life. In this work, an orthogonal strategy was developed; in the first‐dimension separation, reverse‐phase macroporous resin was applied to remove impurities in ginseng crude extracts and classified ginseng extracts into protopanaxatriol and protopanaxadiol fractions. In the second‐dimension separation, the obtained fractions were further separated by a preparative hydrophilic column, and finally yielded 11 pure compounds. Eight of them identified as ginsenoside Rh₁, Rg₂, Rd, Rc, Rb₂, Rb₁, Rg₁, and Re by standards comparison and electrospray ionization mass spectrometry. The purity of these ginsenosides was assessed by high‐performance liquid chromatography with UV detection.     

Silver (Ι)‐assisted enantiomeric analysis of ginsenosides using electrospray ionization tandem mass spectrometry

For identification of ginsenoside enantiomers, electrospray ionization mass spectrometry (ESI‐MS) was used to generate silver complexes of the type [ginsenoside + Ag]⁺. Collision induced dissociation of the silver‐ginsenoside complexes produced fragment ions by dehydration, allowing differentiation of ginsenoside enantiomers by the intensity of [M + Ag ? H₂O]⁺ ion. In the meanwhile, an approach based on the distinct profiles of enantiomer‐selective fragment ion intensity varied with collision energy was introduced to refine the identification and quantitation of ginsenoside enantiomers. Five pairs of enantiomeric ginsenosides were distinguished and quantified on the basis of the distribution of fragment ion [M + Ag ? H₂O]⁺. This method was also extended to the identification of other type of ginsenoside isomers such as ginsenoside Rb2 and Rb3. For demonstrating the practicability of this novel approach, it was utilized to analyze the molar ratio of 20‐(S) and 20‐(R) type enantiomeric ginsenosides in enantiomer mixture in red ginseng extract. The generation of characteristic fragment ion [M + Ag ? H₂O]⁺ likely results from the reduction of potential energy barrier of dehydration because of the catalysis of silver ion. The mechanism of enantiomer identification of ginsenosides was discussed from the aspects of computational modeling and internal energy. Copyright © 2012 John Wiley & Sons, Ltd.     

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     

Simultaneous determination of seven ginsenosides in rat plasma by high‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry: application to pharmacokinetics of Shenfu injection

A high‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry (HPLC‐TOF MS) method was successfully developed and validated for the identification and determination of seven ginsenosides, R_e, R_f, R_b1, R_c, R_b2, R_o and R_d, in a Chinese herbal preparation, Shenfu injection, and rat plasma. Based on the method, the pharmacokinetic profiles of the seven ginsenosides were investigated following intravenous administration of single dose of Shenfu injection to six rats. The established method had high linearity, selectivity, sensitivity, accuracy and precision. The pharmacokinetic results showed that R_b1, R_c and R_b2 had similar pharmacokinetic profiles and relatively long half‐life values (19.29 ± 6.36, 29.54 ± 22.91 and 35.60 ± 30.66 h). The half‐lives of R_f and R_d were 4.21 ± 3.68 and 8.49 ± 5.20 h, respectively, indicating that they could be metabolized more rapidly than R_b1, R_c and R_b2. Copyright © 2014 John Wiley & Sons, Ltd.     

HPLC-MS结合多元统计分析区分人参产地及筛选皂苷类标志物

利用高效液相色谱-质谱联用(HPLC-MS)技术结合多元统计分析方法, 区分中国人参主产区5个不同产地的45个人参样本, 筛选出差异性皂苷类标志物. 根据人参总皂苷在反相C₁₈色谱柱中的洗脱顺序, 结合串联质谱分析和标准品比对, 在提取的人参总皂苷中鉴定出15种原人参三醇型、 24种原人参二醇型和2种齐墩果酸型共41种皂苷. 对人参总皂苷的HPLC-MS全扫描数据进行了多元统计分析. 正交偏最小二乘-判别分析(OPLS-DA)结果表明, 所建立的分析模型具有良好的数据描述能力和预测能力. 所有人参样本能够根据产地被区分, 并筛选得到同时区分5个产地的差异性皂苷类组分18种; 能够区分任意2个产地人参样本的差异性组分主要为在人参中含量较高的人参皂苷Rb1, Rg1, Re, Rc, Rd, Ro和m-Rb1等. 分层聚类分析(HCA)结果显示, 黑龙江和吉林两省的样本能够独自聚类, 但是绥化市的样本更接近于吉林省. 初步推断原因为绥化市地理位置较接近吉林省, 两地人参生长环境相似并可能存在种质资源交换.     

Transformation of Ginsenosides Rg1 and Rb1, and Crude Sanchi Saponins by Human Intestinal Microflora

Fractions major in ginsenosides Rg₁ and Rb₁ from Sanchi saponins were transformed by human fecal flora. This study yielded the corresponding aglycone, protopanaxatriol, in 49.4% from Rg₁, protopanaxadiol 20‐O‐glucoside in 54.8% from Rb₁, and dihydroprotopanaxadiol 20‐O‐glucoside in 87.6% from dihydro Rb₁, by incubation with healthy feces for 70 h in subgram level. Never the less large‐scale incubation of crude Sanchi saponins revealed the complete biotransformation of Rb₁ and the almost unchanged Rg₁. A small amount of Rg₁ was found to be converted into 20 R‐ginsenoside Rh₁ and its dehydration product, 20(22) Z‐ginsenoside Rh₄.     

Isocratic separation of ginsenosides by high-performance liquid chromatography on a diol column at subambient temperatures

An improved high-performance liquid chromatographic method for separation of a number of ginsenosides has been developed. The influence of temperature (from 0 to 25°C) on the retention and separation of the ginsenosides was studied by applying a binary mobile phase (acetonitrile/water, 82:18 v/v) and a diol column (LiChrospher 100 Diol). The column temperature is one of the more important parameters for the retention and separation of the components investigated. Selected thermodynamic parameters, including changes of enthalpy (ΔH°) and entropy (ΔS°), were estimated from linear van’t Hoff plots, and possible retention mechanisms were discussed. Moreover, the best separation conditions were selected based on optimization criteria including maximum retention time (t _{R max}), minimum resolution (R _{s min}), and relative resolution product (r). Temperature regions close to 14°C offered the highest selectivity and almost equal distribution of the ginsenosides peaks across the chromatogram. Under such isocratic conditions, excellent separation of chromatographic standards and selected ginseng samples was achieved in less than 16 min.