Performance Evaluation of Charged Aerosol and Evaporative Light Scattering Detection for the Determination of Ginsenosides by LC

A sensitive LC-CAD method was developed for simultaneous determination of seven major triterpenoid saponins, namely ginsenosides Rg₁, Re, Rb₁, Rc, Rb₂, Rb₃ and Rd in Panax ginseng C. A. Meyer, a commonly used traditional Chinese medicine. This CAD method was evaluated in sensitivity, linearity and reproducibility compared to ELSD and UV. It was found the developed method has improved sensitivity, linearity and reproducibility compared to ELSD. This method was successfully applied to analyze the ginsenosides in ten samples of Panax ginseng. The validation results indicated that the improved method can be utilized as another approach for quality control of P. ginseng.     

177 Saponins,Including 11 New Compounds in Wild Ginseng Tentatively Identified via HPLC-IT-TOF-MSn,and Differences among Wild Ginseng,Ginseng under Forest,and Cultivated Ginseng

Wild ginseng (W-GS), ginseng under forest (F-GS, planted in mountain forest and growing in natural environment), and cultivated ginseng (C-GS) were compared via HPLC-DAD and HPLC-IT-TOF-MSⁿ. A total of 199 saponins, including 16 potential new compounds, were tentatively identified from 100 mg W-GS (177 saponins in W-GS with 11 new compounds), F-GS (56 saponins with 1 new compound), and C-GS (60 saponins with 6 new compounds). There were 21 saponins detected from all the W-GS, F-GS, and C-GS. Fifty saponins were only detected from W-GS, including 23 saponins found in ginseng for the first time. Contents of ginsenosides Re (12.36–13.91 mg/g), Rh₁ (7.46–7.65 mg/g), Rd (12.94–12.98 mg/g), and the total contents (50.52–55.51 mg/g) of Rg₁, Re, Rf, Rb₁, Rg₂, Rh₁, and Rd in W-GS were remarkably higher than those in F-GS (Re 1.22–3.50 mg/g, Rh₁ 0.15–1.49 mg/g, Rd 0.19–1.49 mg/g, total 5.69–18.74 mg/g), and C-GS (Re 0.30–3.45 mg/g, Rh₁ 0.05–3.42 mg/g, Rd 0.17–1.68 mg/g, total 2.99–19.55 mg/g). Contents of Re and Rf were significantly higher in F-GS than those in C-GS (p < 0.05). Using the contents of Re, Rf, or Rb₁, approximately a half number of cultivated ginseng samples could be identified from ginseng under forest. Contents of Rg₁, Re, Rg₂, Rh₁, as well as the total contents of the seven ginsenosides were highest in ginseng older than 15 years, middle–high in ginseng between 10 to 15 years old, and lowest in ginseng younger than 10 years. Contents of Rg₁, Re, Rf, Rb₁, Rg₂, and the total of seven ginsenosides were significantly related to the growing ages of ginseng (p < 0.10). Similarities of chromatographic fingerprints to W-GS were significantly higher (p < 0.05) for F-GS (median: 0.824) than C-GS (median: 0.745). A characteristic peak pattern in fingerprint was also discovered for distinguishing three types of ginseng. Conclusively, wild ginseng was remarkably superior to ginseng under forest and cultivated ginseng, with ginseng under forest slightly closer to wild ginseng than cultivated ginseng. The differences among wild ginseng, ginseng under forest, and cultivated ginseng in saponin compositions and contents of ginsenosides were mainly attributed to their growing ages.     

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 Rg₁ 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.     

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.     

Mechanism of Incompatible Herb Pairs,Panax ginseng and Veratrum nigrum L.: Material Basis and Metabolic Profiles of Ginsenosides in Rat Intestinal Bacteria

In traditional Chinese medicine theory, Panax ginseng and Veratrum nigrum L. is an important incompatible herb pair. Studies on the content variation of main components and the influences on the metabolism in rat intestinal bacteria are useful to understand the mechanism of incompatibility of this herb pairs. In this study, the content variation of ginsenosides and their metaboltic profiles in the extracts of P. ginseng and compatibility of P. ginseng with V. nigrum L. (G‐V) were investigated using relative quantitative method of electrospray ionization mass spectrometry (ESI‐MS) and UPLC‐MSⁿ, respectively. The relative contents of most ginsenosides were reduced in the extract of G‐V. Furthermore, ginsenosides Rb₁, Rb₂, Rc and Rd could be metabolized to Rd, F2 and C‐K in rat intestinal bacteria. The metabolic speeds of Rb₁, Rb₂ and Rc in the G‐V extracts at ratios of 10:5, 10:7 and 10:10 and the metabolic rates of ginsenosides Rb₁, Rb₂ and Rc to Rd, Rd to F2 in all compatibility extracts were lower than that in the P. ginseng extract. In conclusion, this study illustrated the mechanism of effect‐reducing by comparison of the relative contents and metabolic profiles of ginsenosides after compatibility of P. ginseng and V. nigrum L.     

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.     

Simplified ultrasonically- and microwave-assisted solvent extractions for the determination of ginsenosides in powdered Panax ginseng rhizomes using liquid chromatography with UV absorbance or electrospray mass spectrometric detection

New approaches for the recovery of ginsenosides are presented that greatly simplify the liquid chromatographic (LC) determination of the total content of eight ginsenosides (Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁ and Rg₂) in powdered Panax ginseng rhizomes. The extraction protocols not only recover the neutral ginsenosides, but also simultaneously incorporate base-catalyzed hydrolysis of the malonyl-ginsenosides using dilute potassium hydroxide added to the methanol–water extractant. This eliminates the need for an independent extraction step followed by acid- or base-catalyzed hydrolysis. Both ultrasonically-assisted and microwave-assisted extraction methods are developed. The optimization of these simplified methods to remove pendant malonate esters, while retaining the glycosidic linkages, was determined by LC through variation of the extraction/hydrolysis time, order of hydrolysis reagent addition, and evaluation of multiple extractions. A comparison of the ginsenoside profiles obtained with and without addition of base to the extractant solution was made using LCMS with positive-mode electrospray ionization (ESI⁺) detection. A number of malonyl-ginsenosides were tentatively identified by their mass spectral fragmentation spectra and indicating that they were converted to the free ginsenosides by the new extraction/hydrolysis procedure.

Comprehensive Investigation on Ginsenosides in Different Parts of a Garden-Cultivated Ginseng Root and Rhizome

Background: Ginseng is widely used as herb or food. Different parts of ginseng have diverse usages. However, the comprehensive analysis on the ginsenosides in different parts of ginseng root is scarce. Methods: An ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) combined with UNIFI informatics platform and ultra-high-performance liquid chromatography-charged aerosol detection (UHPLC-CAD) were employed to evaluate the different parts of cultivated ginseng root. Results: 105 ginsenosides including 16 new compounds were identified or tentatively characterized. 22 potential chemical markers were identified, 20, 17, and 19 for main root (MR) and fibrous root (FR), main root (MR) and branch root (BR), and main root (MR) and rhizome (RH), respectively. The relative contents of Re, Rb₁, 20(R)-Rh₁, Rd, and Rf were highest in FR. The relative content of Rg₁ was highest in RH. The total relative content of pharmacopoeia indicators Rg₁, Re, and Rb₁ was highest in FR. Conclusion: The differences among these parts were the compositions and relative contents of ginsenosides. Under our research conditions, the peak area ratio of Rg₁ and Re could distinguish the MR and FR samples. Fibrous roots showed rich ingredients and high ginsenosides contents which should be further utilized.     

An LC‐MS method for simultaneous determination of nine ginsenosides in rat plasma and its application in pharmacokinetic study

A sensitive and reliable LC‐ESI‐MS method for simultaneous determination of nine ginsenosides (Rh₁, Rg₂, Rg₁, Rf, Re, Rd, Rc, Rb₂ and Rb₁) in rat plasma was developed and validated using saikosaponin A as an internal standard. The samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Hypersil Gold C₁₈ column (100 × 2.1 mm, 5 µm) by stepwise gradient elution with water (0.1% formic acid, v/v) and acetonitrile as the mobile phase. Detection was determined by selective ion monitoring mode using electrospray ionization in the negative ion mode. Good linearity over the investigated concentration ranges was observed with the values of r higher than 0.9900. The intra‐ and inter‐day precisions were all no more than 15% and the average recoveries varied from 71.8 to 91.7%. This quantitative measurement was successfully applied to a pharmacokinetic study of Yi‐Qi‐Fu‐Mai injection. Copyright © 2010 John Wiley & Sons, Ltd.     

Microwave-assisted extraction of ginsenosides from ginseng root

The extractions of ginsenosides Rg₁ and Rb₁ from ginseng root under atmospheric pressure by focused microwave-assisted technique have been investigated. The parameters used for the optimization were solvent composition, extraction time, and applied microwave power. The ginsenosides were quantified by high-performance liquid chromatography equipped with UV/Vis detector. The results of the 15-min microwave-assisted extraction (0.28% of Rg₁ obtained in 70% water-ethanol and 1.31% of Rb₁ obtained in 30% water-ethanol under 150 W of microwave power) were better than that from 10-h conventional solvent extraction (0.22% of Rg₁ and 0.87% of Rb₁ obtained in 70% water-ethanol).     

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.     

Simultaneous quantification of 10 saponins in Chinese Shizhu Panax by UPLC-ESI-MS

A simple and rapid method was established and validated for the simultaneous quantification of 10 saponins, namely ginsenosides-Rb₁, Rb₂, Rb₃, Rc, Rd, Rg₁, Rg₂, Re, Rf and Notoginsenside R₁, in Chinese Shizhu Panax by ultra performance liquid chromatography coupled with an electrospray mass spectrometry (UPLC-ESI-MS). In addition, the contents of the analytes in different parts of Chinese Shizhu Panax were also analysed. The results showed that the concentration of saponins had a reference to the different parts of Chinese Shizhu Panax. The established method could be used as a new analytical approach for assessment of the quantity of Chinese Shizhu Panax.     

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.     

Isolation and Purification of Ginsenosides from Plant Extract of Panax quinquefolium L. by High Performance Centrifugal Partition Chromatography Coupled with ELSD

A high performance centrifugal partition chromatography (HPCPC) combined with evaporative light scattering detection (ELSD) was developed for the separation and purification of ginsenosides from Panax quinquefolium. Three compounds, ginsenosides Rc, Rb₁, and Re were isolated and purified by HPCPC using an optimized two-phase solvent system composed of ethyl acetate–n-butanol–water (1:1:2, v/v/v). The purities of the three ginsenosides were 96.5, 97.6, and 98.5%, respectively as determined by liquid chromatography (LC–ELSD). The CPC fractions were analyzed by LC–ELSD and electrospray ion source mass spectroscopy (ESI-MSⁿ) in negative ion mode. The identification of the ginsenosides Rc, Rb₁, and Re in the extract of P. quinquefolium was based on matching their retention times, the detection of the molecular ions, and the fragment ions of the molecular ion obtained in the CID experiments with those of the authentic standards and data reported in the literature. The results demonstrate that HPCPC coupled with ELSD is a feasible and efficient technique for systematic isolation of non-chromophoric components from traditional medicinal herbs.     

An efficient isolation and purification of broad partition coefficient range ginsenosides from roots of Panax quinquefolium L. by linear gradient counter-current chromatography coupled with preparative high-performance liquid chromatography

As a famous health food, roots of Panax quinquefolium L. possessed immune regulation and enhancement of the central nervous system, in which ginsenosides are the main active component with different numbers and positions of sugars, causing different chemical polarities with a challenge for the separation and isolation. In this study, a fast and effective bilinear gradient counter-current chromatography was proposed for preparative isolation ginsenosides with a broad partition coefficient range from roots of Panax quinquefolium L. In terms of the established method, the mobile phases comprising n-butanol and ethyl acetate were achieved by adjusting the proportion. Coupled with the preparative HPLC, eleven main ginsenosides were successfully separated, including ginsenoside Rg₁ ( 1 ), Re ( 2 ), acetyl ginsenoside Rg₁ ( 3 ), Rb₁ ( 4 ), Rc ( 5 ), Rg₂ ( 6 ), Rb₃ ( 7 ), quinquefolium R₁ ( 8 ), Rd ( 9 ), gypenoside X VII ( 10 ) and notoginsenoside Fd ( 11 ), with purities exceeding 95% according to the HPLC results. Tandem mass spectrometry and electrospray ionization mass spectrometry were adopted for recognizing the isolated compound architectures. Our study suggests that linear gradient counter-current chromatography effectively separates the broad partition coefficient range of ginsenosides compounds from the roots of Panax quinquefolium L. In addition, it can apply to active compound isolation from other complicated natural products.     

Tartaric acid induced conversion of protopanaxadiol to ginsenosides Rg3 and Rg5 and their in situ recoveries by integrated expanded bed adsorption chromatography

Panax ginseng has been applied in traditional Chinese medicine for over 2000 years. It is still one of the most popular herbs in recent decades. The prescribed ginseng‐containing medicines consist of protopanaxadiol and protopanaxatriol ginsenosides, which are the major constituents of the herb. Minor ginsenosides at low levels in the herb, such as Rg₃ and Rg₅, have attracted more rising attention than the major ones. The existing approaches to prepare Rg₃ and Rg₅ usually rely on either steamed red ginseng as the source or chemical/enzymatic conversion of protopanaxadiol to the targets. It is still highly desirable to effectively achieve such minor components. In this paper, a method integrated extraction of protopanaxadiol and conversion of it to Rg₃ and Rg₅ has been proposed. Protopanaxadiol was extracted and simultaneously converted to Rg₃ and Rg₅ by d ,l ‐tartaric acid. The targets were absorbed by resins on expanded bed adsorption chromatography and were then separated from other ginsenosides in different stages. Compared with conventional methods, the developed process has advantages in shortening time consumption and improving the conversion ratio of protopanaxadiol, which is promising in directly achieving Rg₃ and Rg₅ from P. ginseng.     

Quantitative Determination of Eight Major Constituents in the Traditional Chinese Medicinal Yi-Qi-Fu-Mai Preparation by LC

A high-performance liquid chromatography-diode array detection method was developed and validated for the simultaneous determination of eight marker components, ginsenoside Rf, Rb₁, Rb₂, Rb₃, Rd, Rg₃, 20(S)-ginsenoside F₂ and schizandrin, present in traditional Chinese medicinal Yi-Qi-Fu-Mai preparations. Samples were prepared using a solid phase extraction procedure. Chromatographic separation was carried out on a Waters Symmetry C₁₈ column (4.6 × 250 mm, 5 μm) by stepwise gradient elution with water (0.05% phosphoric acid, v/v) and acetonitrile as the mobile phase. Good linear relationships between were observed between peak areas and concentrations with r ² values above 0.9990 for all the analytes. Average recoveries for the eight markers ranged from 97.7 to 100.4%. This method was found to be rapid, sensitive, accurate, and could be readily applied to the quality assessment of Yi-Qi-Fu-Mai preparations.     

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.     

Simultaneous Quantitative Analysis of Ginsenosides Isolated from the Fruit of Panax ginseng C.A. Meyer and Regulation of HO-1 Expression through EGFR Signaling Has Anti-Inflammatory and Osteogenic Induction Effects in HPDL Cells

Periodontitis is a set of chronic inflammatory diseases caused by the accumulation of Gram-negative bacteria on teeth, resulting in gingivitis, pocket formation, alveolar bone loss, tissue destruction, and tooth loss. In this study, the contents of ginsenosides isolated from Panax ginseng fruit extract were quantitatively analyzed, and the anti-inflammatory effects were evaluated in human periodontal ligament cells. The major ginsenosides, Re, Ra8, and Rf, present in ginseng fruit were simultaneously analyzed by a validated method using high-performance liquid chromatography with a diode-array detector; Re, Ra8, and Rf content per 1 g of P. ginseng fruit extract was 1.01 ± 0.03, 0.33 ± 0.01, and 0.55 ± 0.04 mg, respectively. Ginsenosides-Re, -Ra8, and -Rf inhibited the production of pro-inflammatory factors and the expression of important cytokines in periodontitis by inducing the expression of heme oxygenase 1 (HO-1), promoting osteoblast differentiation of periodontal ligament cells, suppressing alveolar bone loss, and promoting the expression of osteoblast-specific genes, such as alp, opn, and runx2. An inhibitory effect of these ginsenosides on periodontitis and alveolar bone loss was observed via the regulation of HO-1 and subsequent epidermal growth factor receptor (EGFR) signaling. Silencing EGFR with EGFR siRNA confirmed that the effect of ginsenosides on HO-1 is mediated by EGFR. In conclusion, this study evaluated the contents of ginsenosides-Re, -Ra8, and -Rf isolated from P. ginseng fruit extract. Therefore, these results provide important basic data for future P. ginseng fruit component studies and suggest that ginsenosides Re, Ra8, and Rf have potential as future treatment options for periodontitis.     

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.