Effects of population, age, and cultivation methods on ginsenoside content of wild American ginseng (Panax quinquefolium)

Genotype and environmental effects on ginsenoside content among eight wild populations of American ginseng (Panax quinquefolium) were investigated. Root concentrations of six ginsenosides were determined at the time of collection of plants from the wild (T0) and 2 years (T2) after transplanting roots from each of the eight populations to each of two different forest garden locations. Both location and population had significant effects on root and shoot growth. Overall, ginsenoside Rb1 was most abundant, followed by Rg1 and Re. Concentrations of Rg1 and Re were inversely related among and within populations. The relative ranking of populations differed depending upon the particular ginsenoside and sampling time. The relative importance of genotype and environment was not the same for all ginsenosides. Ginsenoside Re was influenced by population but not location, whereas Rb1, Rc, and Rb2 were influenced only by location (environment), while Rg1 and Rd were influenced by both. Ginsenoside levels were consistently lower, but growth was consistently higher at the more intensively managed garden location.     

Simultaneous determination of ginsenosides and polyacetylenes in American ginseng root (Panax quinquefolium L.) by high-performance liquid chromatography

A method for simultaneous determination of ginsenosides and polyacetylenes in Panax quinquefolium L. (American ginseng) roots was developed. The ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, Ro, malonyl-Rb1, malonyl-Rc, and malonyl-Rd and the polyacetylenes falcarinol and panaxydol were extracted from fresh ginseng roots in a sequential extraction process with 100% methanol followed by 80% aqueous methanol and quantified simultaneously in extracts by high-performance liquid chromatography using diode array detection. Separations were achieved with a phosphate buffer-acetonitrile gradient system using an RP-C18 column. Except for Rd, the present extraction method resulted in similar or significantly higher concentrations of both ginsenosides and polyacetylenes in comparison to commonly used extraction methods for these compounds. The contents of polyacetylenes and ginsenosides were determined in the root hairs, lateral roots, and main roots of 6 year old ginseng plants. The total mean concentrations of ginsenosides and polyacetylenes in root hairs were 31.0 g/kg fresh weight (FW) and 2.6 g/kg FW, respectively, whereas the concentrations of these bioactive compounds in the main roots were significantly lower with total mean concentrations of 17.8 g/kg FW for ginsenosides and 0.6 g/kg FW for polyacetylenes. The concentration of individual and total ginsenosides and polyacetylenes did not differ significantly between main roots of different sizes. Consequently, it is possible to do quantitative screening for ginsenosides and polyacetylenes to breed ginseng roots with higher levels of bioactive compounds.     

Simplified extraction of ginsenosides from American ginseng (Panax quinquefolius L.) for high-performance liquid chromatography-ultraviolet analysis

Four methods were tested for extraction and recovery of six major ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) found in roots of American ginseng (Panax quinquefolius): method A, sonication in 100% methanol (MeOH) at room temperature (rt); method B, sonication in 70% aqueous MeOH at rt; method C, water extraction (90 degrees C) with gentle agitation; and method D, refluxing (60 degrees C) in 100% MeOH. After 0.5-1 h, the samples were filtered and analyzed by high-performance liquid chromatography (HPLC)-UV. A second extraction by methods C and D was done, but 85-90% of ginsenosides were obtained during the first extraction. Lyophilization of extracts did not influence ginsenoside recovery. Method D resulted in the highest significant recoveries of all ginsenosides, except Rg1. Method C was the next most effective method, while method A resulted in the lowest ginsenoside recoveries. Method B led to similar recoveries as method C. All methods used one filtration step, omitted time-consuming cleanup, but maintained clear peak resolution by HPLC, and can be used for quantitative screening of ginsenosides from roots and commercial ginseng preparations.     

Saponins composition in American ginseng leaf and berry assayed by high-performance liquid chromatography

The root of American ginseng is a commonly used herbal medicine in the United States. However, the compositions of American ginseng leaves and berries are not clear to date. In this study, we improved a method for the analysis of 12 ginsenosides based on solid phase extraction and high-performance liquid chromatography-ultraviolet. Good resolution was obtained for all tested ginsenosides: Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, 20(R)-Rg2, Rg3, Rh1, and Rh2. Ginsenosides Rh1, Rg2, and 20(R)-Rg2 were easily separated with this column. The modified gradient elution program resulted in satisfactory linearity and precision. Solid phase extraction made the analysis accurate and efficient. Other investigators recently observed that ginsenoside Rb3 is a potent neuroprotective compound; it can promote learning and memory. In this report, we found that the major ginsenoside in American ginseng leaves and berries was ginsenoside Rb3, while Rb3 only had limited amounts in the root of American ginseng and other species of the Panax genus. Ginsenoside Rb3 was quantified as 4.71% in American ginseng leaves and 5.35% in berries, suggesting that American ginseng leaves and berries are new sources of ginsenoside Rb3.     

Simultaneous quantification of ginsenosides in American ginseng (Panax quinquefolium) root powder by visible/near-infrared reflectance spectroscopy.

Near-infrared reflectance spectroscopy (NIRS) was examined as a possible alternative to high-performance liquid chromatography (HPLC) for the analysis of ginsenosides from American ginseng (Panax quinquefolium) root powder (n = 26). NIR spectra were collected over 400-2500 nm. For each sample and individual ginsenoside quantified by HPLC, spectral data were regressed against the chemical data to develop prediction equations. The spectral prediction equations produced high correlation coefficient (1-VR) values and low standard errors of cross validation (SECV) values for the determination of individual and total ginsenosides. The contents of individual ginsenosides, Rb(1), Rb(2), Rc, Rd, Re, Rg(1), Ro, m-Rb(1), m-Rb(2), m-Rc, m-Rd, and total ginsenosides (X +/- SECV) were (1.29+/-0.18)%, (0.273+/-0.096)%, (0.298+/-0.052)%, (0.091+/-0.027)%, (1. 015+/-0.114)%, (0.116+/-0.018)%, (0.25+/-0.040)%, (0.776+/-0.116)%, (0.197+/-0.074)%, (0.239+/-0.083)%, (0.143+/-0.042)%, and (4.393+/-0.283)%, respectively. The (1-VR) values of cross validation were 0.877, 0.872, 0.955, 0.834, 0.899, 0.919, 0.325, 0.849, 0.902, 0.877, 0.871, and 0.963, respectively. Results indicated that the NIRS method could be used for the analysis of the major ginsenosides, Rb(1), Re, and m-Rb(1), as well as the total ginsenosides in American ginseng.     

Protopanaxatriol-type ginsenosides from the root of Panax ginseng

Ginseng, the root of Panax ginseng C. A. Meyer (Araliaceae), is one of the most important traditional medicines and functional foods. A detailed phytochemical investigation on the roots of P. ginseng led to the isolation of 6 new natural protopanaxatriol (PPT)-type ginsenosides, ginsenosides Re(1)-Re(6) (compounds 1-6), along with 10 known PPT-type ginsenosides. Their structures were elucidated on the basis of chemical and spectroscopic analyses, including high-resolution mass spectrometry (HRMS) and 1D and 2D nuclear magnetic resonance (NMR). The unusual α-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl sugar chain, as found in compounds 1 and 2, is reported in the genus Panax for the first time.     

Influence of zinc on the growth,distribution of elements,and metabolism of one‐year old American ginseng plants

Using a water culture technique, 0.05 ppm zinc (Zn) was found to be the critical deficiency concentraction for one‐year American ginseng (Panax quinquefolium L) plants, 0.3 ppm was optimum, 0.5 ppm the critical Zn‐toxicity concentration, and 10 ppm the concentration when severe toxicity occurs. Therefore, the optimum Zn concentration for the growth of American ginseng plants was between 0.1 ppm ‐ 0.3 ppm. Zinc deficiency symptoms of one‐year old American ginseng plants were indicated by the inhabition of root growth, with little fibrous root development, and smaller leaves compared to normal leaves. The symptoms of toxicity were also indicated by the inhibition of root growth, and when seedlings were suffering from an acute toxicity, no fibrous roots appeared, and eventually the roots yellowed and leaves grew slowly or even entirely ceased to grow, the final result being very small leaves which are also chlorotic. Zinc maintained within the 0.1 ppm to 0.3 ppm sufficiency range promoted the synthesis and accumulation of ginsenosides by American ginseng plants, and both low and high Zn concentrations restrained the synthesis and accumulation of ginsenosides. Both Zn deficiency and the optimum Zn concentration (0.3ppm) are beneficial to the accumulation of amino acids in the roots of American ginseng plants. Close to the optimum Zn concentration, the ratios of P/Zn and Fe/Zn in the shoot of American ginseng plants were maintained at 77 and 9.4, respectively.     

Determination of ginsenosides Rb1, Rc, and Re in different dosage forms of ginseng by negative ion electrospray liquid chromatography-mass spectrometry

A method based on high-performance liquid chromatography (HPLC) and negative ion electrospray mass spectrometry (LC-MS) has been used to determine ginsenosides Rb1, Rc, and Re in six different samples of ginseng. These included a liquid extract, capsules, tea bags, and an instant tea. It was found that four of the six samples had detectable levels of at least one of the ginsenosides. The liquid extract, capsules, instant tea, and tea bags labeled ginseng had ginsenosides. The labels on the two samples that did not have ginsenosides indicated that they were a mixture of green tea, licorice, and ginseng. Also, 13C NMR was used to identify the types of complex carbohydrates present in the samples. One of the samples of tea bags had none of the ginsenosides, but did have complex carbohydrates found in most of the other samples. The instant tea had all three ginsenosides, but had no complex carbohydrates, only sucrose. The amounts of ginsenosides found in standard doses from six different sources of ginseng varied considerably. It was found that steeping a tea bag for a longer time than that recommended on the label produced a larger recovery of ginsenosides and that reusing a tea bag produced even higher recoveries.     

Effects of harvester behavior on population growth rate affects sustainability of ginseng trade

When long-term, intense levels of harvest nearly extirpated Chinese ginseng (Panax ginseng C. Meyer) in the early 18th century, commercial harvest of American ginseng (P. quinquefolius L.) began and large quantities of the roots were exported to East Asia. Annual export figures have fallen over the past 200 years, but demand for wild American ginseng has not abated. Persistent harvest of long-lived, slow-maturing species can have negative impacts on population growth rates, yet those closest to wild resources are often in a position to be the best stewards of that resource. This study explored the consequences of different harvester behaviors on the population dynamics of American ginseng. Drawing on known behaviors, we developed three harvester ‘types’ and ran demographic simulations on wild ginseng populations, partitioning the sources of differences in population growth rates using a life table response experiment (LTRE). The simulations showed that ignoring size class limits and harvest season onset dates dramatically affected population growth rates. Existing laws in many states are not adequate to protect wild ginseng populations. A stewardship-oriented harvester, who delays harvest onset by two weeks, self-limits harvest intensity and plants ginseng seeds at the time of harvest can reverse declining population growth rates.     

DNA identification of commercial ginseng samples

An investigation was performed with the objective of developing a DNA-based protocol for the identification of commercial samples of the herbal compound ginseng. There are currently two major herbal products referred to as ginseng. They are Korean or Chinese ginseng (Panax ginseng) and American ginseng (Panax quinquefolius). The market for ginseng in the United States is estimated to be approximately $300 million annually. Current tests for ginseng species identification rely on expert botanical identification of fresh plant/root specimens or on biochemical characterization of active and marker compounds (e.g., ginsenosides). For the determination of the feasibility of ginseng identification by DNA analysis, a strategy based on the direct DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region was developed. Other genetic tests included sequence analysis of the chloroplast ribulose 1,5-bisphosphate carboxylase large subunit gene and DNA fingerprinting by the rapid amplification of polymorphic DNA technique. To confirm the results, each ginseng sample was identified using high-performance liquid chromatography. All methods were successful in distinguishing American from Korean ginseng. In addition, the protocol was improved for the isolation of genomic and plastid DNA from commercial ginseng preparations by incorporating an impact homogenization step into the standard column chromatography purification procedure.     

Steamed American ginseng berry: ginsenoside analyses and anticancer activities

This study was designed to determine the changes in saponin content in American ginseng berries after treatment by heating and to assess the anticancer effects of the extracts. After steaming treatment (100-120 degrees C for 1 h, and 120 degrees C for 0.5-4 h), the content of seven ginsenosides, Rg1, Re, Rb1, Rc, Rb2, Rb3, and Rd, decreased; the content of five ginsenosides, Rh1, Rg2, 20R-Rg2, Rg3, and Rh2, increased. Rg3, a previously identified anticancer ginsenoside, increased significantly. Two hours of steaming at 120 degrees C increased the content of ginsenoside Rg3 to a greater degree than other tested ginsenosides. When human colorectal cancer cells were treated with 0.5 mg/mL steamed berry extract (120 degrees C 2 h), the antiproliferation effects were 97.8% for HCT-116 and 99.6% for SW-480 cells. At the same treatment concentration, the effects of unsteamed berry extract were 34.1% for HCT-116 and 4.9% for SW-480 cells. After staining with Hoechst 33258, apoptotic cells increased significantly by treatment with steamed berry extract compared with unheated extracts. Induction of apoptosis activity was confirmed by flow cytometry after staining with annexin V/PI. The steaming of American ginseng berries augments ginsenoside Rg3 content and increases the antiproliferative effects on two human colorectal cancer cell lines.     

Degradation of ginsenosides in American ginseng (Panax quinquefolium) extracts during microwave and conventional heating

The degradation of ginsenosides in American ginseng (Panax quinquefolium) extracts during microwave and water (oil) bath heating (conventional heating) was investigated. Both the 50% ethanol-water extracts and the aqueous extracts were boiled in a modified laboratory microwave oven and in a water (or oil) bath, respectively. The neutral ginsenosides (Rb(1), Rc, Rd, and Re) and malonyl ginsenosides (m-Rb(1), m-Rc, and m-Rd) were determined by reverse-phase high-performance liquid chromatography. The results showed that the degradation of ginsenosides in 50% ethanol-water extracts was a first-order reaction. The malonyl ginsenosides were much less stable than the corresponding neutral ginsenosides, with the rate constant value of the malonyl ginsenosides being 3-60 times that of the neutral ginsenosides. At the same temperature, the effect of microwave heating on the degradation of ginsenosides was the same as that of conventional heating.     

影响人参发根皂苷含量基因rolC的克隆与序列分析

摘要：通过发根农杆菌?穴Agrobacterium rhizogenes ?雪与人参?穴Panax ginseng C. A. Mey?雪根外植体共培养，用直接接菌方法诱导出人参发根。培养4周后人参发根的总皂苷含量达到栽培3年生人参皂苷含量的水平，单体皂苷Rb1含量明显提高，说明TL-DNA具有影响人参皂苷生物合成的能力。根据Slightom等RiA4TL-DNA序列分析结果，利用PCR方法从人参发根中扩增并克隆了影响人参皂苷合成的基因rolC。与已发表序列相比较，核苷酸序列的同源性为99.9%。     

Phytochemical variation in echinacea from roots and flowerheads of wild and cultivated populations

Quantitative phytochemical variation was determined from roots and inflorescences of native plant populations in the genus Echinacea. Specimens were collected in situ throughout the natural range of each putative taxon and transplanted to greenhouse cultivation. Ethanolic extracts from individual plants were separated by reversed-phase HPLC to quantify the alkamides, polyenes/ynes, and phenolics, and then grouped by age and taxonomically, according to a recent morphometric taxonomic revision of the genus. Canonical discriminant analysis revealed that cichoric acid, the diene alkamides 1-3 and 7, and ketoalkene 24 were the best taxonomic markers. Mean content for each of 26 phytochemicals revealed useful agronomic information, such as those varieties and organs with the highest accumulations, as well as the optimal age and growth conditions for each variety. The highest amounts of cichoric acid were measured from the older, wild inflorescences of E. pallida var.sanguinea, whereas the highest quantities of the alkamides 1-3 and 7 were present in roots of wild and transplanted E. purpurea. Baseline phytochemical data and chromatographic profiles for all types of wild Echinacea may be used for protection of wild stands, germplasm identification, and crop improvement.     

Sanchi ginseng (<Emphasis Type="Italic">Panax notoginseng</Emphasis> (Burkill) F. H. Chen) in China: distribution,cultivation and variations

Sanchi ginseng (Panax notoginseng (Burkill) F. H. Chen) has been cultivated in China for more than 400 years, whose root is an important traditional Chinese medicine mainly used to stanch blood, to disperse gore and to reduce pain caused by injury due to falls. The cultivated populations of this crop are distributed in Wenshan mountain area of Yunnan with a narrow habitat, whose location is around N 23.5°, E 104° and altitude ranges from 1,200 to 2,000 m. Although its wild species has not been found, current cultivated populations show rich morphological variations in stem, leaf, root, flower and fruit. Recent studies exhibit that Sanchi root has more active compounds than the roots of P. ginseng and P. quinquefolius. Therefore, this crop needs further utilization.     

Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD)

AFLP profiles characteristic to Panax ginseng and Panax quinquefolius were generated using primers E-AGG/M-CAA. P. ginseng samples from different farms in China and Korea are homogeneous genetically [similarity index (SI) = 0.88-0.99], whereas samples of P. quinquefolius from different sources are much more heterogeneous (SI = 0.64-0.96). Detailed analysis of one of the polymorphic bands in P. ginseng led to the identification of a minisatellite Pg2, which contains eight repeats of 5'-AGGACTCATCACATTGTTACTC. The minisatellite DNA was consequently used in directed amplification minisatellite region DNA analysis to authenticate the two ginsengs.     

RAPD-based assessment of genetic relationships among and within American ginseng (Panax quinquefolius L.) populations and their implications for a future conservation strategy

American ginseng (Panax quinquefolius) is a native North American medicinal plant that is becoming increasingly vulnerable despite government harvest restrictions. To better understand the genetic diversity and gene flow of American ginseng, we studied RAPD variation in cultivated and wild populations. Classical and Bayesian analogues of genetic diversity statistics were estimated in seven wild and two cultivated populations. The wild populations were more highly structured (G _stβ  = 0.41) than the cultivated populations (G _stβ  = 0.24). The genetic diversity within populations ranged from H _eβ  = 0.05 to 0.38. Based on genetic pairwise distances, six of the wild populations clustered with the locally-derived cultivated population, while one wild population was more similar to the non-local cultivated population than the local populations. This wild population was highly diverse (P = 1.0; U = 1.0) suggesting that it was supplemented from exotic seed. A set of eight RAPD markers was identified that differentiated plants of local and non-local origin. As a conservation strategy, we recommend that regional gene banks be established based on molecular and geographic diversity to preserve the locally adapted germplasm. These regional gene banks would serve as a conservation tool and also provide a source of genes for genetic improvement of cultivated ginseng.     

In vitro study of the relationship between the structure of ginsenoside and its antioxidative or prooxidative activity in free radical induced hemolysis of human erythrocytes

Ginsenoside, the major active component in Panax ginseng, which has been used in traditional Chinese medicine, contains a series of derivatives of the triterpene dammarane being attached by some sugar moieties. To clarify the relationship between the structure of ginsenoside and its properties, 11 individual ginsenosides, along with the central structures of ginsenoside, protopanaxadiol and protopanaxatriol, are used in 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) induced hemolysis of human erythrocytes, a good experimental model to research free radical induced membrane damage and to evaluate the antioxidative or prooxidative activities of various antioxidants conveniently. It is found that the central structures of ginsenosides, either protopanaxadiol or protopanaxatriol, play a prooxidative role in AAPH-induced hemolysis of erythrocytes. As to the individual ginsenoside, if there are no sugar moieties attached to the 20-position of the triterpene dammarane, the ginsenoside acts as a prooxidant, that is, Rg3, Rh2, and Rg2. A glucose attached to the 6-position instead of the 20-position sugar moieties can make the ginsenoside an antioxidant, that is, Rh1. The antioxidants among ginsenosides follow two different mechanisms that can be expressed mathematically by the Boltzmann equation, that is, Rc and Rb1, and a polynomial equation, that is, Re, Rd, R1, Rg1, Rb3, and Rh1. The orders of antioxidative ability are Rc > Rb1 and Re > Rd > R1 > Rg1 > Rb3 > Rh1, respectively.     

Genetic diversity and variation of saponin contents in Panax notoginseng roots from a single farm

Radix notoginseng, the root of Panax notoginseng (Burk.) F. H. Chen, has been widely used in traditional Chinese medicine. Its main components, saponins, have been reported to have many pharmacological activities. To test the general assumption that herbs of a single species planted and harvested from a single location are uniform in chemical and genetic makeup, chemical analysis and DNA fingerprinting were carried out. High-performance TLC together with HPLC analysis were used to analyze 17 randomly sampled 3-year-old roots from a single farm for the presence of six saponins. Five roots showed distinct chemical profiles with changed ratios of ginsenosides Rd/Rg1, Re/Rg1, or Rb1/Rg1. The same samples, together with some 1- and 2-year-old samples, were also subjected to fluorescent amplified fragment length polymorphism (AFLP) analysis, and their internal transcribed spacer 2 (ITS 2) regions were sequenced. Fluorescent AFLP analysis was found to be much more polymorphic than the ITS 2 sequence and showed clear evidence of genetic diversity within the tested population. In conclusion, genetic diversity and variation of saponin contents between individual P. notoginseng roots have been detected. We suggest that genetic diversity affects the contents of the six saponins. The saponin contents variation and genetic diversity were also found among P. notoginseng root samples collected from China and Singapore markets. Since variable saponin contents may affect therapeutic efficacy, combining the use of genetic profiling with chemical profiling will help ensure greater uniformity in the quality of P. notoginseng roots. The genetic and chemical diversity within a population also provides the opportunity for breeding new cultivars with more desirable chemical constituents.