Development of a quality evaluation system for Panax quinquefolium. L based on HPLC chromatographic fingerprinting of seven major ginsenosides

A HPLC fingerprinting method based on the distribution and contents of seven major ginsenosides has been developed for the quality evaluation of Panax quinquefolium. L roots cultivated in China, Singapore and Canada. The method is ideally suited for the fingerprinting of P. quinquefolium. L samples or its derived products for sample authentication or quality control, e.g. pharmaceutical stability studies. Different extraction methods have been evaluated and a protocol established to maximize the gensinosides yields in routine operations. Hierarchical clustering analysis of the fingerprints demonstrates that the distribution and contents of ginsenosides in P. quinquefolium. L root vary depending on geographic location; and the quality of Jilin-cultivated P. quinquefolium. L roots is similar in composition to Canada-cultivated ones, which are generally considered among the best quality in P. quinquefolium. L.     

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.     

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.     

Isolation and determination of ginsenosides in American ginseng leaves and root extracts by LC-MS

Ginseng saponins (ginsenosides) were extracted from the root and leaves of locally cultivated American ginseng (Panax quinquefolium L.). For the isolation of compounds from plant samples three different extraction methods were utilized: accelerated solvent extraction, the ultrasound-assisted solvent extraction and mechanical shaking assisted solvent extraction. The separation of compounds was achieved with a water–acetonitrile gradient system using a C18 reversed-phase column. Target compounds were identified in MS² and MS³ experiments. The relative distribution of these ginsenosides in each root and leaf extract was established. The limit of detection of the method was less than 30 ng/ml. Recovery of ginseng saponins in spiked samples exceeded 80%, while the relative standard deviation ranged from 7.1 to 9.1%. The total concentrations of ginsenosides were 41 and 13 mg/g in root and leaves.     

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.     

A novel strategy for rapid quantification of 20(S)-protopanaxatriol and 20(S)-protopanaxadiol saponins in Panax notoginsengP. ginseng and P. quinquefolium

A novel strategy for the qualitative and quantitative determination of 20(S)-protopanaxatriol saponins (PTS) and 20(S)-protopanaxadiol saponins (PDS) in Panax notoginseng, Panax ginseng and Panax quinquefolium, based on the overlapping peaks of main components of PTS (calibrated by ginsenoside Rg1) and PDS (calibrated by ginsenoside Rb1), was proposed. The analysis was performed by using high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD). Under specific chromatographic conditions, all samples showed two overlapping peaks containing several main ginsenosides belonging to PTS and PDS, respectively. The overlapping peaks were also identified by using HPLC–MS. Based on the sum and ratio of PTS and PDS, 60 tested Panax samples were divided into three main clusters according to their species. The findings suggested that this strategy provides a simple and rapid approach to quantify PTS and PDS in Panax herbs.     

Production of Minor Ginsenosides from Panax notoginseng Flowers by Cladosporium xylophilum

Panax notoginseng flowers have the highest content of saponins compared to the other parts of Panax notoginseng, but minor ginsenosides have higher pharmacological activity than the main natural ginsenosides. Therefore, this study focused on the transformation of the main ginsenosides in Panax notoginseng flowers to minor ginsenosides using the fungus of Cladosporium xylophilum isolated from soil. The main ginsenosides Rb₁, Rb₂, Rb₃, and Rc and the notoginsenoside Fa in Panax notoginseng flowers were transformed into the ginsenosides F₂ and Rd₂, the notoginsenosides Fd and Fe, and the ginsenoside R₇; the conversion rates were 100, 100, 100, 88.5, and 100%, respectively. The transformation products were studied by TLC, HPLC, and MS analyses, and the biotransformation pathways of the major ginsenosides were proposed. In addition, the purified enzyme of the fungus was prepared with the molecular weight of 66.4 kDa. The transformation of the monomer ginsenosides by the crude enzyme is consistent with that by the fungus. Additionally, three saponins were isolated from the transformation products and identified as the ginsenoside Rd₂ and the notoginsenosides Fe and Fd by NMR and MS analyses. This study provided a unique and powerful microbial strain for efficiently transformating major ginsenosides in P. notoginseng flowers to minor ginsenosides, which will help raise the functional and economic value of the P. notoginseng flower.     

A new triterpenoid saponin from the leaves and stems of Panax quinquefolium L.

One new triterpenoid saponin,quinquenoside L_(17)(1),was isolated from the leaves and stems of Panax quinquefolium L.,and its structure was elucidated as 20-O-[(β-D-xylopyranosyl-(1-6)-O-β-D-glucopyranosyl)]-6-O-β-D-glucopyranosyl-dammar-24-ene- 3,6,12,20-tetraol,by the combination analysis of one-dimensional NMR and two-dimensional NMR,mass spectrometry,CD spectrum and chemical evidences.     

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.     

Simultaneous determination of five active hydrolysis ingredients from Panax quinquefolium L. by HPLC‐ELSD

An effective method for simultaneous determination of five hydrolysis products of 20 (R)‐dammarane‐3β,6α,12β,20,25‐pentol, 24(R)‐ocotillol, 20(R)‐protopanaxatriol, 20(S)‐panaxatriol and 20(R)‐dammarane‐3β,12β,20,25‐tetrol was developed using high‐performance liquid chromatography with evaporative light scattering detection (HPLC‐ELSD). The hydrolysis products from Panax quinquefolium L. in the stems and leaves, berries, flower buds and roots components were successfully separated on a Kromasil C₁₈ column using methanol and water (83:17, v/v) as mobile phase in 18 min. The parameter for the ELSD was set to a probe temperature of 40°C and the nebulizer for nitrogen gas was adjusted to 3 L/min. All calibration curves showed good linear regression (r > 0.9975) within test ranges. The validation of the method included recovery, linearity, accuracy and precision (intra‐ and inter‐day variation). The accuracy and precision were satisfactory, with the overall intra‐ and inter‐day variation being less than 3.11%, and recoveries of this method were greater than 95.0%. This study developed an effective and rapid method for simultaneous determination of multiple hydrolysis components from Panax quinquefolium L. Copyright © 2010 John Wiley & Sons, Ltd.     

液相色谱-大气压化学电离质谱法分析人参中的人参皂甙

研究了用反相高效液相色谱-大气压化学电离质谱(HPLC/APCI-MS)分析人参皂甙的方法。液相色谱采用乙腈-水流动相进行梯度洗脱,质谱采用正负离子同时扫描并结合二级质谱进行定性,用选择反应离子模式(SRM)测定检测限。实验发现虽然人参皂甙是热不稳定物质,但在大气压化学电离质谱的高温汽化过程中仍能检测到很强的负离子分子离子峰,而且随着汽化温度的升高,人参皂甙的负离子分子离子峰的强度增加。该方法对人参皂甙Rb1和Rg1的检测限分别为1.2×10-13 g和3.0×10-14 g,并检测出白参中包括丙二酰人参皂甙在内的29种人参皂甙。该法灵敏度高,重复性好,结果准确,能有效地对药材提取物中的多种人参皂甙进行检测和结构分析。     

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.

Biotransformation of Paeonol and Emodin by Transgenic Crown Galls of <Emphasis Type="Italic">Panax quinquefolium</Emphasis>

Two aromatic substrates, paeonol (1) and emodin (2), were biotransformed by using transgenic crown galls of Panax quinquefolium. Four biotransformed products (3–6) were isolated and identified by physicochemical and spectral methods. A β-glucoside (3, 73.2% of biotransformation yield) and a 1-(2,4-dimethoxyphenyl)- ethanone (4, 8.03%) were isolated from the suspension cultures after 7-day incubation of substrate 1. Upon administration of substrate 2, another β-glucoside [emodin-6-O-β-d-glucopyranoside (5), 19.2%] and a hydroxylated derivative, citreorosein (6, 54.6%), were also obtained. The results demonstrate that transgenic crown galls of P. quinquefolium have the capacities to catalyze glycosylation, hydroxylation, and methylation reactions in the plant cells on those aromatic compounds.     

Biotransformation of the saponins in Panax notoginseng leaves mediated by gut microbiota from insomniac patients

Saponins extracted from Panax notoginseng leaves by methanol or water could be orally administrated for insomnia with very low bioavailability, which might be bio-converted by gut microbiota to generate potential bioactive products. Moreover, gut microbiota profiles from insomniac patients are very different from healthy subjects. We aimed to compare the metabolic characteristics and profiles of the two saponins extract by incubation with gut microbiota from insomniac patients. The ginsenosides, notoginsenosides, and metabolites were identified and relatively quantified by high-performance liquid chromatography-tandem mass spectrometry. Gut microbiota was profiled by 16S ribosomal RNA gene sequencing. The results showed that saponins were very different between methanol or water extract groups, which were metabolized by gut microbiota to generate similar yields. The main metabolites included ginsenoside Rd, ginsenoside F₂, ginsenoside C-Mc or ginsenoside C-Y, ginsenoside C-Mx, ginsenoside compound K, and protopanaxadiol in both groups, while gypenoside XVII, notoginsenoside Fe, ginsenoside Rd₂, and notoginsenoside Fd were the intermediates in the methanol group. Moreover, the microbial, Faecalibacterium prausnitzi, could bio-convert the saponins to obtain the corresponding metabolites. Our study implied that saponins extracted from P. notoginseng leaves by methanol or water could be used for insomniac patients due to gut microbiota biotransformation.     

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.     

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.     

High‐performance anion‐exchange chromatography coupled with diode array detection for the determination of dencichine in Panax notoginseng and related species

A high‐performance anion‐exchange chromatography coupled with diode array detection method was developed for the determination of dencichine in Panax notoginseng and related species. The analysis was performed on an Eprogen Synchropak WAX column (4.6 × 250 mm, 6 μm) with 50 mM NaH₂PO₄ aqueous solution isocratic elution. The method was validated in terms of linearity, sensitivity, precision, stability, and accuracy. It was found that the calibration curve for dencichine showed good linearity (R² = 0.9999) within the test range. The LOD and LOQ were 0.77 and 3.06 ng, respectively. The RSD for intra‐ and interday repeatability was 0.2 and 0.5%, respectively. The test solution of dencichine is stable at least for three days at room temperature and for seven days at 4°C. The mean recovery of dencichine was 102.0%. The established method was successfully applied to determine dencichine in the raw root of P. nogoginseng, P. ginseng, and P. quinquefolium as well as the steamed root of P. notoginseng. Compared with previous reports, this method is sensitive, selective, and accurate, which is helpful to evaluate the quality of P. notoginseng and related species.     

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.     

Differentiation of <Emphasis Type="BoldItalic">Panax quinquefolius</Emphasis> grown in the USA and China using LC/MS-based chromatographic fingerprinting and chemometric approaches

American ginseng (Panax quinquefolius) is one of the most commonly used herbal medicines in the world. Discriminating between P. quinquefolius grown in different countries is difficult using traditional quantitation methods. In this study, a liquid chromatographic mass spectrometry fingerprint combined with chemometric analysis was established to discriminate between American ginseng grown in the USA and China. Fifteen American ginseng samples grown in Wisconsin and 25 samples grown in China were used. The chromatographic fingerprints, representing the chemical compositions of the samples, made it possible to distinguish samples from the two locations. In addition, it was found that some ginsenosides varied widely from P. quinquefolius cultivated in these two countries. P. quinquefolius grown in the USA is higher in ginsenoside R_c, ginsenoside R_d, quinquenoside III/pseudo-ginsenoside RC₁, malonyl ginsenoside R_b1, and ginsenoside R_b2, but lower in ginsenoside R_b1 compared with P. quinquefolius grown in China. These ginsenosides may be responsible for the class separation seen using fingerprinting and chemometric approaches.