Extraction and Separation of Eight Ginsenosides from Flower Buds of Panax Ginseng Using Aqueous Ionic Liquid-Based Ultrasonic-Assisted Extraction Coupled with an Aqueous Biphasic System.

Ionic liquids (ILs) are recognized as a possible replacement of traditional organic solvents, and ILs have been widely applied to extract various compounds. The present work aims to extract ginsenosides from Panax ginseng flower buds using aqueous ionic liquid based ultrasonic assisted extraction (IL-UAE). The extraction yields of 1-alkyl-3-methylimidazolium ionic liquids with different anions and alkyl chains were evaluated. The extraction parameters of eight ginsenosides were optimized by utilizing response surface methodology (RSM). The model demonstrated that a high yield of total ginsenosides could be obtained using IL-UAE, and the optimum extraction parameters were 0.23 M [C4mim][BF4], ultrasonic time of 23 min, temperature of extraction set to 30 °C, and liquid-solid ratio of 31:1. After that, an aqueous biphasic system (ABS) was used to separate ginsenosides further. The nature and concentration of salt, as well as the value of pH in ionic liquid were evaluated, and the optimal conditions (6.0 mL IL extract, 3 g NaH2PO4, and pH 5.0) were obtained. The preconcentration factor was 2.58, and extraction efficiency reached 64.53%. The results indicate that as a simple and efficient method, an IL-UAE-ABS can be considered as a promising method for extracting and separating the natural active compounds from medicinal herbs.

Comprehensive Characterization for Ginsenosides Biosynthesis in Ginseng Root by Integration Analysis of Chemical and Transcriptome.

Herbgenomics provides a global platform to explore the genetics and biology of herbs on the genome level. Panax ginseng C.A. Meyer is an important medicinal plant with numerous pharmaceutical effects. Previous reports mainly discussed the transcriptome of ginseng at the organ level. However, based on mass spectrometry imaging analyses, the ginsenosides varied among different tissues. In this work, ginseng root was separated into three tissues-periderm, cortex and stele-each for five duplicates. The chemical analysis and transcriptome analysis were conducted simultaneously. Gene-encoding enzymes involved in ginsenosides biosynthesis and modification were studied based on gene and molecule data. Eight widely-used ginsenosides were distributed unevenly in ginseng roots. A total of 182,881 unigenes were assembled with an N50 contig size of 1374 bp. About 21,000 of these unigenes were positively correlated with the content of ginsenosides. Additionally, we identified 192 transcripts encoding enzymes involved in two triterpenoid biosynthesis pathways and 290 transcripts encoding UDP-glycosyltransferases (UGTs). Of these UGTs, 195 UGTs (67.2%) were more highly expressed in the periderm, and that seven UGTs and one UGT were specifically expressed in the periderm and stele, respectively. This genetic resource will help to improve the interpretation on complex mechanisms of ginsenosides biosynthesis, accumulation, and transportation.

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.

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, Rg2, Rb1, Rb2, Rc, Rd, Rg3, Rk1, Rg5, Rg6, and F4. Meanwhile, eight ginsenosides have been purified from white ginseng, including Rg1, Re, Rf, Rh1, 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.

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

The use of (1)H-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.

Effects of ginsenosides, active ingredients of Panax ginseng, on development, growth, and life span of Caenorhabditis elegans.

The backbone structure of ginsenosides, active ingredients of Panax ginseng, is similar with that of sterol, especially cholesterol. Caenorhabditis elegans (C. elegans) is one of free living nematodes and is well-established animal model for biochemical and genetic studies. C. elegans cannot synthesize de novo cholesterol, although cholesterol is essential requirement for its growth and development. In the present study, we investigated the effects of ginseng total saponins (GTS) on the average brood size, growth, development, worm size, and life span of C. elegans in cholesterol-deprived and -fed medium. Cholesterol deprivation caused damages on normal growth, reproduction, and life span of worms throughout F1 to F3 generations. GTS supplement to cholesterol-deprived medium restored the growth, reproduction, and life span of worms as much as cholesterol alone-fed medium. GTS co-supplement to cholesterol-fed medium not only promoted worm reproduction but also induced bigger worms and faster growth than cholesterol-fed medium. In study to identify which ginsenosides are responsible for life span restoring effects of GTS, we found that ginsenoside Rc supplement not only restored life span of worms grown in cholesterol-deprived medium but also prolonged life span of worms grown in cholesterol-fed medium. Worms grown in medium supplemented with ginsenoside Rb(1) or Rc to cholesterol-deprived medium exhibited strong filipin staining, in which filipin forms tight and specific complexes with 3beta-hydroxy sterols. These results show a possibility that ginsenosides could be utilized by C. elegans as a sterol substitute and further indicate that ginsenoside Rc is the component of Panax ginseng that prolongs the life span of C. elegans.

Embryotoxicity study of ginsenoside Rc and Re in in vitro rat whole embryo culture.

BACKGROUND: Pregnant women commonly consume ginseng. However, there is little data concerning the effects of ginseng on early pregnancy. METHODS: Rat embryos were exposed in vitro to different concentrations of Rc and Re from day 9.5 to day 11.5 after conception. Embryos were scored for growth and differentiation at the end of the culture period. RESULTS: Embryos exposed to 50.0 microg/ml Re had significantly lower median morphological score (29.0 versus 48.0), fewer number of somites (15.0 versus 21.0), and smaller yolk sac diameter (3.5 versus 4.1 mm) and crown-rump length (CRL) (2.9 versus 3.4 mm) compare to control embryos. There was no significant difference between embryos exposed to 5.0 microg/ml Re and control embryos. There was also no difference in the biometric and morphologic parameters among control and embryos exposed to 5.0 and 50.0 microg/ml Rc. CONCLUSION: There is a significant variability in embryotoxic effects of different ginsenosides. Further studies to evaluate the synergistic embryotoxic effects of ginsenosides are warranted.

Null and opposing effects of Asian ginseng (Panax ginseng C.A. Meyer) on acute glycemia: results of two acute dose escalation studies.

OBJECTIVE: We have repeatedly reported that a batch of American ginseng with a specific ginsenoside (glycosidal saponin) profile decreases acute postprandial glycemia. We investigated whether Asian ginseng is able to replicate this glycemia-lowering efficacy in two separate acute dose escalation studies. METHODS: Each study was conducted in a separate sample of 11 healthy subjects (gender: 8M:3F and 6M:5F, age: 29 +/- 2y and 27 +/- 3y, BMI: 28.5 +/- 2.1 kg/m(2) and 26.9 +/- 1.4 kg/m(2)) using a randomized, single-blind, placebo-controlled, multiple-crossover design. Treatments consisted of 0 (placebo), 1, 2, and 3 g of Asian ginseng for the first study and 0 (placebo), 3, 6, and 9 g Asian ginseng for the second study administered 40 minutes before a 75g-OGTT protocol with blood drawn at -40, 0, 15, 30, 45, 60, 90, and 120 minutes. Ginsenosides were analyzed by HPLC-UV. RESULTS: Neither the main effect of pooled-treatment, nor dose, nor either factors interaction with time was significant for incremental plasma glucose and insulin. But the diagnostically and therapeutically relevant two-hour plasma glucose (2h-PG) value was significantly higher for pooled Asian ginseng treatment than placebo (5.46 +/- 0.31 versus 4.99 +/- 0.30 mmol/L, p = 0.050). Ginsenoside analyses showed that the Asian ginseng contained up to 96% lower and sevenfold higher quantities of various ginsenosides and their ratios than our previous efficacious batch of American ginseng. CONCLUSIONS: Asian ginseng showed both null and opposing effects on indices of acute postprandial plasma glucose and insulin. This is in contrast to our findings with American ginseng. One explanation may be the marked ginsenoside differences. Practitioners and consumers should be aware of ginseng's variable effects.

The inhibitory effect of ginseng saponins on the stress-induced plasma interleukin-6 level in mice.

The effect of ginseng saponins on plasma interleukin-6 (IL-6) in non-stressed and immobilization-stressed mice were investigated. Ginseng total saponins, ginsenosides Rb2, Rg1 and Rd administered intraperitoneally attenuated the immobilization stress-induced increase in plasma IL-6 level. But, intracerebroventricular injection of each ginsenoside did not affect plasma IL-6 level induced by immobilization stress. Ginsenosides Rb2, Rd and Rg1 significantly decreased norepinephrine and/or epinephrine-induced increase of IL-6 level in macrophage cell line (RAW 264.7). Thus, it can be suggested that the inhibitory action of ginseng saponins against the immobilization stress-induced increase of plasma IL-6 level would be in periphery; at least in part, mediated by blocking norepinephrine- and/or epinephrine-induced increase of IL-6 level in macrophage rather than in the brain. Ginseng saponins might be proposed as a possible candidate in the research or therapeutic modulation of stress-related disorders.