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.     

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.     

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.     

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.     

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.     

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.     

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.

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.     

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.     

Two New Dammarane‐Type Bisdesmosides from the Fruit Pedicels of Panax notoginseng

Two new dammarane‐type triterpenoidal saponins, notoginsenosides FP₁ ( 1 ) and FP₂ ( 2 ), were isolated from the fruit pedicels of Panax notoginseng, along with 22 known compounds. Their structures were elucidated on the basis of spectroscopic evidences and chemical methods. The known compounds were identified as ginsenosides Rg₁ ( 3 ), Re ( 4 ), Rb₃ ( 5 ), Rc ( 6 ), Rd ( 7 ), Rb₂ ( 8 ), Rb₁ ( 9 ), F₂ ( 10 ), and F₁ ( 11 ); as notoginsenosides R₁ ( 12 ), Fa ( 13 ), and Fc ( 14 ); as vina‐ginsenoside R₇ ( 15 ); as gypenosides IX ( 16 ), XVII ( 17 ), and XIII ( 18 ), and as chikusetsusaponin‐L₅ ( 19 ), quercetin 3‐O‐β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐galactopyranoside ( 20 ), kaempferol 3‐O‐β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐galactopyranoside ( 21 ), benzyl‐β‐primeveroside ( 22 ), (S)‐tryptophan ( 23 ), and icariside B₆ ( 24 ). Compounds 15, 19 and 22 – 24 are reported for the first time from the title plant.     

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.     

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.     

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.     

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.     

The use of linear ion trap for qualitative analysis of phytochemicals in Korean ginseng tea

A new approach to qualitative analysis of ginsenosides in challenging matrices was developed on the basis of high‐performance liquid chromatography/tandem mass spectrometry. Using the extracts from samples of ginseng tea, the approach was validated. Analysis of extracts was carried out using a reversed‐phase chromatography with SB‐C₁₈ sorbent. For compound identification, electrospray ionization and a quadrupole/linear ion trap mass‐spectrometer in different modes were used. A meticulous study of the fragmentation of ginsenosides in the linear ion trap and its application for analysis of these compounds was performed in this work. The accuracy of the identification was proven with standards of ginsenosides Rb1, Rg1, Re, Rf, Rd, Rb2, Rb3 and Rc. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

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.     

Microcalorimetric investigation of water vapor adsorption on silica gel

In this study, the activities of four ginsenosides Rc, Re, Rd, and Rf on splenic lymphocytes growth were studied by microcalorimetry. Some qualitative and quantitative information, such as the metabolic power–time curves, growth rate constant k, maximum heat-output power of the exponential phase P _max and the corresponding appearance peak time t _max, total heat output Q _t, and promotion rate R _p of splenic lymphocytes growth affected by the four ginsenosides were calculated. In accordance with thermo-kinetic model, the corresponding quantitative relationships of k, P _max, t _max, Q _t, R _p, and c were established, . Also, the median effective concentration (EC₅₀) was obtained by quantitative analysis. Based on both the quantitative quantity–activity relationships (QQAR) and EC₅₀, the sequence of promotion activity was Rc > Re > Rd > Rf. The analysis of structure–activity relationships showed that the number, type, and position of sugar moieties on the gonane steroid nucleus had important influences on the promotion activity of Rc, Re, Rd, and Rf on splenic lymphocytes growth. Microcalorimetry can be used as a useful tool for determining the activity and studying the quantity–activity relationship of drugs on cell.     

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze‐dried powder

A simple and rapid liquid chromatography–mass spectrometry (LC‐MS) method was developed and validated for analysis of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze‐dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C₁₈ column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion‐pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra‐ and inter‐day RSDs were all less than 10% with the relative error (RE) within ±9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze‐dried powder. Copyright © 2011 John Wiley & Sons, Ltd.     

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.