Identification and differentiation of Panax species using ELISA, RAPD and eastern blotting

Immunochemical and genetic methods have been developed in order to distinguish Panax spp. With the aim of establishing immunochemical methods, two hybridomas (3H4 and 5H8), each secreting a monoclonal antibody (MAb) against proteins of Panax ginseng, were prepared by fusing splenocytes immunized with two kinds of ginseng water-soluble fractions and a hypoxanthine-thymidine-aminopterin-sensitive mouse myeloma cell line, P3-X63-Ag8-U1. MAb 3H4 cross-reacted with four Panax spp., whereas the MAb 5H8 cross-reacted with P. ginseng in the enzyme-linked immunosorbent assay (ELISA). ELISA and western blotting methods using a ginseng water-soluble fraction as the solid-phase antigen were developed for the unambiguous authentication of P. ginseng. A combination of random amplified polymorphic DNA (RAPD) and eastern blotting analyses using anti-ginsenoside Rb1 and Rgl monoclonal antibodies was used for the identification of P. notoginseng, P. quinquefolius and P. japonicus. RAPD can be used to differentiate the species of Panax from each other. An important parameter used for differentiating P. notoginseng is the absence of ginsenoside Rc in the extract of P. notoginseng with eastern blotting. The combination of these methods enabled a reliable identification of Panax spp.     

Extractable polysaccharides of Panax quinquefolius L. (North American ginseng) root stimulate TNFalpha production by alveolar macrophages.

We have investigated the immunostimulatory activity of the medicinal plant Panax quinquefolius L. (North American ginseng). Rat alveolar macrophages were treated with different extracts from 4-year old roots, and tumour necrosis factor alpha (TNF) production was used as a measure of immunostimulatory activity. Aqueous extracts of P. quinquefolius root (1-100 microg/ml) were found to significantly stimulate alveolar macrophage TNF release. Both a P. quinquefolius methanol extract containing ginsenosides (but no polysaccharides), and pure ginsenoside-Rb1, the major ginsenoside present in P. quinquefolius, were found to be inactive as TNF-stimulating agents. Significant TNF-stimulating activity was found in the extractable polysaccharide fraction, which was hydrolyzed and found to contain glucose, galactose, arabinose, rhamnose, and mannose. This represents the first evidence that North American ginseng exerts cytokine-stimulating activity on macrophages.     

Genetic identification of Panax ginseng and Panax quinquefolius by pyrosequencing methods

This study was performed to determine whether two ginseng species (Panax ginseng and Panax quinquefolius) can be identified by genetic analysis and to verify pyrosequencing analysis, which was used to assess genetic variation. The pyrosequencing results constituted clear data. Panax quinquefolius showed a very different pattern than Panax ginseng. Pyrosequencing analysis might be able to identify the Panax species.     

Molecular authentication of Panax species

Using conserved plant sequences as primers, the DNA sequences in the ribosomal ITS1-5.8S-ITS2 region have been amplified and determined for six Panax species, P. ginseng C. A. Mey. (Oriental ginseng), P. quinquefolius L. (American ginseng), P. notoginseng (Burkill) F. H. Chen (Sanchi), P. japonicus C. A. Mey. (Japanese ginseng), P. trifolius L. and P. major Ting, as well as two common adulterants of ginseng, Mirabilis jalapa L. and Phytolacca acinosa Roxb. An authentication procedure based upon the restriction fragment length polymorphism (RFLP) in the region is able to differentiate between P. ginseng and P. quinquefolius, and to discriminate the ginsengs from the two common poisonous adulterants. Broader application of this approach to authenticate other morphologically similar Chinese medicinal materials is rationalised.     

POLLEN MORPHOLOGY IN THE GENUS ERYTHRONIUM (LILIACEAE) AND ITS SYSTEMATIC IMPLICATIONS

Pollen morphology was examined in 40 samples from 19 species in the genus Erythronium (Liliaceae) using scanning (SEM) and transmission electron microscopy (TEM). Pollen grains are monosulcate monads, except for a plant of E. oregonum which has dyad grains. North American and European species are characterized by reticulate exine sculpture composed of various degrees of wavy muri and variably-sized lumina. The most distinctive feature of exine sculpture in the genus is exhibited by the Asian species E. japonicum, which has a unique reticulate pattern composed of striate muri and a distinctive exine structure without columellae. The distinctness of E. japonicum pollen suggests that it has specialized in isolation from species in North America and Europe.     

Decreased Hill reaction rates and slow turnover of transitory starch in the obligate shade plant Panax quinquefolius L. (American ginseng)

To identify physiological processes that might limit photosynthesis in Panax quinquefolius L. (American ginseng) a comparison has been made with Panax ginseng C.A. Meyer (Korean ginseng), Pisum sativum L. (pea) and Spinacia oleracea L. (spinach). The quantum yield of oxygen evolution in intact leaves and isolated thylakoid membranes was found to be smaller in ginseng than in pea or spinach. However, the number of photosystem II (PSII) centers on a chlorophyll basis was found to be similar in all species. This suggests that ginseng thylakoid membranes possess relatively more inactive PSII centers than thylakoids of pea and spinach when grown under similar conditions. Unexpectedly, whole-chain electron transport from water to methyl viologen, and partial photosystem I reactions, demonstrated that electron transport rates to methyl viologen were anomalously low in P. quinquefolius and P. ginseng. Additionally, at elevated light intensities, intact leaves of P. quinquefolius were more susceptible to lipid peroxidation than pea leaves. In plants grown at a light intensity of 80 micro mol photons m(-2) s(-1) the levels of fructose and starch were higher in both ginseng species than in pea or spinach. Significantly, the level of starch in P. quinquefolius was relatively constant throughout the entire 12 h/12 h light/dark cycle and remained high after an extended dark time of 48 h. In addition, P. quinquefolius had lower activities of alpha-amylase and beta-amylase than P. ginseng, pea and Arabidopsis thaliana (L.) Heynh. The significance of the elevated levels of leaf starch in P. quinquefolius remains to be determined. However, the susceptibility of P. quinquefolius to photoinhibition may arise as a consequence of a reduced fraction of active PSII centers. This may result in the normal dissipative mechanisms in these plants becoming saturated at elevated, but moderate, light intensities.     

Genetic diversity in harvested and protected populations of wild American ginseng, Panax quinquefolius L. (Araliaceae)

Genetic diversity was examined at 16 allozyme loci in 21 wild populations of the medicinal plant American ginseng, Panax quinquefolius L. (Araliaceae). This species has been harvested from forests in North America for more than 250 years. Average expected heterozygosity was significantly greater within protected populations (H(e) = 0.076) than within populations in which harvesting was permitted (H(e) = 0.070). More notably, genetic structure was greater among unprotected populations (G(ST) = 0.491) than among protected populations (G(ST) = 0.167). These differences in the level and distribution of genetic diversity in American ginseng populations indicate that harvesting may have significant evolutionary implications for this species. Age class structure also shifted toward smaller, nonreproductive plants in unprotected populations. Juvenile plants had lower genetic diversity (H(e) = 0.067) than reproductive plants (H(e) = 0.076) suggesting that conserving a proportion of the largest (oldest) plants in each population is important to protect reproductive fitness and the evolutionary potential of the species. Due to its high genetic structure, conservation recommendations include protecting populations throughout the range of P. quinquefolius.     

Ginseng modifies the diabetic phenotype and genes associated with diabetes in the male ZDF rat

Asian ginseng (Panax ginseng) and its close relative North American ginseng (Panax quinquefolius) are perennial aromatic herbs that are widely used in Oriental medicine and have been acclaimed to have various health benefits including diabetes treatment. In this study, we compared the effects of a diet containing rosiglitazone to a diet containing ginseng (Panax quinquefolius) in male Zucker diabetic fatty (ZDF) rats. Animals were assigned to one of three diets: control, rosiglitazone (0.1 g/1 kg diet), or ginseng (10 g/1 kg diet). During the 11-week study, body weight, food intake, organ weight, blood glucose, plasma cholesterol, and plasma triglyceride levels were evaluated. Animals treated with rosiglitazone or ginseng exhibited increased body weight (p<0.05) and decreased kidney weight (p<0.05) compared to control animals. The rosiglitazone group demonstrated decreased food intake and plasma triglyceride levels versus the other groups (p<0.05). The ginseng group revealed decreased cholesterol levels relative to the control group (p<0.05). Furthermore, ginseng and rosiglitazone had marked effects on the expression of genes involved in PPAR actions and triglyceride metabolism compared to controls. In conclusion, ginseng modified the diabetic phenotype and genes associated with diabetes in the male ZDF rat. These data are encouraging, and warrant further research to determine the therapeutic value of this medicinal herb in treating human diabetes.     

Phylogenetics and biogeography of eastern Asian-North American disjunct genus Pachysandra （Buxaceae） inferred from nucleotide sequences

Pachysandra is an eastern Asian-North American disjtunct genus with three species, two in eastern Asia （Pachysandra axillaris and Pachysandra terminalis） and one in eastern North America （Pachysandra procurnbens）. Although morphological and cytological studies suggest a close affinity of Pprocumbens with P axillaris, molecular data from nuclear and chloroplast DNA regions have provided conflicting signals. In this study, we tested previous phylogenetic hypotheses using sequences of nuclear ribosomal DNA internal transcribed spacers and chloroplast ndhF gene from multiple individuals of each of the three species. We also estimated the time of divergence between eastem Asia and eastern North America. Our results support the morphological and cytological conclusion that P procumbens is more closely related to P axillaris than to P terminalis. The estimated time of divergence of P axillaris and P procumbens was 14.6±5.5 mya, consistent with estimates from many other eastern Asian-North American disjunct genera. The migration of Pachysandra populations from eastern Asia to North America might have occurred by way of the North Atlantic land bridge.     

Mycotoxins in root extracts of American and Asian ginseng bind estrogen receptors alpha and beta

The estrogenic activity of ginseng has been the subject of conflicting reports. Cell proliferation, induction of estrogen-responsive genes, and isolated cases of adverse reactions such as postmenopausal vaginal bleeding and gynecomastia have been reported after ginseng treatment. Other studies report antiproliferative effects with no induction of estrogen-responsive genes. We developed estrogen receptor (ER) alpha and ER alpha competitive binding assays using recombinant receptors and [(3)H]-17 alpha-estradiol to detect phytoestrogens in extracts of Asian ginseng root (Panax ginseng C. A. Meyer) and American ginseng root (Panax quinquefolius L.). Root extracts contained substances that bound both receptor isoforms. These substances had a two to three times greater affinity for ER alpha. Significantly higher binding was found in methanol extracts than in hot water extracts. Subsequent analysis of the extracts revealed significant ER binding attributable to zearalenone, the estrogenic mycotoxin produced by several Fusarium species. The ER showed no binding affinity for Rb1 and Rg1, the major ginsenosides found in P. quinquefolius and P. ginseng, respectively. Thus, ginseng extraction methods, plant species tested, and mycotoxin contaminants may help to explain the disparate literature reports. The prevalence and health significance of fungal contamination in herbal products used for medicinal purposes should be further investigated.     

The cyto-taxonomic studies on some species of Panax L.

Abstract 1. It was observed that somatic chromosome numbers of four species of the genus Panax L. are as follows: Panax japonicus 2n=24, P. notoginseng 2n=24, P. ginseng 2n=44 and P. quinquefolius 2n=48. The somatic chromosome numbers of P. japonicus from Lushan and Jinggangshan (Jiangxi Province, China) is different from that of Japanese population (2n=48). The chromosome numbers (2n=24) of P. notoginseng is first reported. 2. The P. japonicus, one of the diploid species, which has the widest geographical distribution is perhaps the more primitive type among the living species of Panax. While, the P. ginseng and P. quinquefolius, the tetraploid species, are more advanced types than the diploid species. The conclusion from cytotaxonomy is thus different from that of chemical taxonomy. 3. The cytological analysis together with the geographic distribution of this four species shows that the Southwestern China is the modern distribution center, also the most variational center, and perhaps the center of origin for the genus Panax L.     

三七叶、人参叶和西洋参叶皂苷类成分的研究(英文)

三七叶、人参叶和西洋参叶其皂苷类成分相近,但专属性成分各异,皂苷类成分的分布比例也各不相同。本文建立了HPLC-UV法测定上述皂苷成分的方法,经过方法学考察,各种皂苷成分精密度好、加样回收率高,方法可靠。11种皂苷成分总含量顺序为:西洋参叶>人参叶>三七叶;二醇组皂苷成分含量:西洋参叶>三七叶>人参叶;三醇组皂苷成分含量:人参叶>西洋参叶>三七叶。西洋参叶中二醇组皂苷和人参叶中三醇组皂苷含量明显高于其他。西洋参叶中人参皂苷Rb3和Rd的含量之和占11种皂苷成分的60%以上。鉴于其中人参皂苷的高含量,三七叶、人参叶和西洋参叶应该作为皂苷来源得到充分利用;不同的皂苷成分有不同的药理活性,应基于它们的皂苷组成和比例选择性进行研究和开发。     

POLLEN MORPHOLOGY AND DETAILED STRUCTURE OF FAMILY COMPOSITAE,TRIBE CICHORIEAE. I. SUBTRIBE STEPHANOMERIINAE

A survey of pollen morphology in 20 species representing the 11 genera of the North American subtribe Stephanomeriinae by light, scanning electron, and transmission electron microscopy revealed 10 of the 11 genera to have echinate, tricolporate pollen grains, Lygodesmia being the only genus with echinolophate pollen. Sectioned exines of most of the species examined are similar, being composed of ektexine and endexine. The ektexine surface is composed of spines which typically have globose perforate bases. A cavus occurs as a separation between the basis (foot layer) and the columellae in all of the genera examined except Chaetadelpha. Pollen of the two species of Glyptopleura were found to be strikingly different in exomorphology. Pollen of the putatively self-fertile G. marginata has much shorter spines than the closely related G. setulosa. Atrichoseris, Anisocoma, Calycoseris, Glyptopleura, Pinaropappus, Prenanthella, and most species of Malacothrix have pollen which lack paraporal ridges. The remaining genera, Chaetadelpha, Lygodesmia, Rafinesquia, and Stephanomeria have well-developed ridges of fused spine bases around the apertures. Pollen characters, particularly those of the aperture region, have been found to be systematically useful in the subtribe, therefore acetolyzed material gives more useful information than untreated pollen.     

Proteome of Oriental ginseng Panax ginseng C. A. Meyer and the potential to use it as an identification tool

Oriental ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) are two widely used valuable traditional Chinese medicines (TCM). Previously, the identification of ginseng was mainly performed by analyzing the ginsengnosides using high performance liquid chromatography and amplification of polymorphic DNA using polymerase chain reaction. However, these methods cannot be used to distinguish TCM samples which are from different parts (main root, lateral roots, rhizome head and skin) of ginseng and ginseng culture cells from wild-grown ginseng. The present study aimed to identify different species of ginseng, different parts of the same ginseng and cultured cells of ginseng using a proteomic approach. Two-dimensional electrophoresis (2-DE) maps were established from the American ginseng main root, different parts (main root, lateral roots, rhizome head and skins) of Oriental ginseng and Oriental ginseng culture cells. Our results show that the 2-DE maps of different ginseng samples contain sufficient differences to permit easy discrimination. We have also identified common and specific protein spots in the 2-DE maps of different ginseng samples. The use of these "marker proteins" may help to speed up the identification process.     

Pollen morphology in thePortulacaceae (Centrospermae)

Pollen of 110 species from 18 genera in thePortulacaceae has been examined by light and scanning electron microscopy, and a representative number by transmission electron microscopy. Three basic pollen types were found: 3-colpate with thick tectum and foot layer with prominent unbranched columellae and an extremely thin endexine; pantoporate with thick tectum and foot layer with branched columellae enclosing pores and an endexine that is one to two layers thick; pantocolpate with thin tectum and foot layer with broad, short unbranched columellae and an inconspicuous endexine. All pollen types, however, have a spinulose and tubuliferous/punctate ektexine. Also, all the genera except three,Calandrinia H.B.K.,Montia L. andTalinum Adanson are stenopalynous. There is, however, no absolute correlation between pollen morphology and geographical distribution, although both the major centre of palynological diversity and the majority of all species with tricolpate grains occur in South America.     

RAPD- and allozyme analysis of genetic variability of Panax ginseng C.A. Meyer and P. quinquefolius L

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.     

Generation of ginsenosides Rg3 and Rh2 from North American ginseng

Rg3 and Rh2 ginsenosides are primarily found in Korean red ginseng root (Panax ginseng C.A. Meyer) and valued for their bioactive properties. We quantified both Rh2 and Rg3 ginseng leaf and Rg3 from root extracts derived from North American ginseng (Panax quinquefolius). Quantification was obtained by application of HPLC with ion fragments detected using ESI-MS. Ginseng leaf contained 11.3+/-0.5 mg/g Rh2 and 7.5+/-0.9 mg/g Rg3 in concentrated extracts compared to 10.6+/-0.4 mg/g Rg3 in ginseng root. No detectable Rh2 was found in root extracts by HPLC, although it was detectable by ESI-MS analysis. Ginsenosides Rg3 and Rh2 were detected following hot water reflux extraction, but not from tissues extracted with 80% aqueous ethanol at room temperature. Therefore ginsenosides Rg3 and Rh2 are not naturally present in North American ginseng, but are products of a thermal process. Using ESI-MS analysis, it was found that formation of Rg3 and Rh2, among other compounds, were a function of heating time and were breakdown products of the more abundant ginsenosides Rb1 and Rc. Our findings that heat processed North American ginseng leaf is an excellent source of Rh2 ginsenoside is an important discovery considering that ginseng leaf material is obtainable throughout the entire plant cycle for recovery of valuable ginsenosides for pharmaceutical use.     

POLLEN MORPHOLOGY OF THE DILLENIACEAE AND ACTINIDIACEAE

Pollen of 53 species of Dilleniaceae and Actinidiaceae was examined by light microscopy, scanning electron microscopy, and a selected group in transmission electron microscopy. Dilleniaceae pollen ranges from tricolpate, tricolporate, tetracolpate, and incipiently inaperturate. Tricolpate types occur only among the Old World subfamily Dillenioideae and the compound aperturate (3-colporate) condition is restricted to the subfamily Tetraceroideae. Within the Dilleniaceae the tricolpate pollen type with elongated apertures is considered primitive, having given rise to the 3-colporate and 4-colpate conditions. The striking pollen dimorphism in the Neotropical species of Tetracera, all of which are androdioecious, is documented; however, in contrast to previous reports, pollen from bisexual flowers appears to be incipiently inaperturate and not pantoporate. The inaperturate condition is interpretated as an early stage in the evolution of outcrossing. Pollen morphology does not support a close relationship between Dilleniaceae and Actinidiaceae. Pollen morphological differences that can be noted between these families are: tectum complete and predominantly psilate or psilate-granular in Actinidiaceae, tectum incomplete, punctate to reticulate in Dilleniaceae; an equatorial bridge of ektexine over the endoaperture usually present in Actinidiaceae, absent in Dilleniaceae; columellae reduced in Actinidiaceae, columellae usually well-developed in Dilleniaceae. Pollen morphology does not argue against a close relationship between Actinidiaceae and Theaceae.     

A phylogenetic analysis ofPanax (Araliaceae): Integrating cpDNA restriction site and nuclear rDNA ITS sequence data

A phylogenetic analysis ofPanax was performed using restriction site variations of eight PCR-amplified chloroplast regions. Twenty populations were examined, representing 13 of the 14 species ofPanax. Aralia cordata was used as the outgroup. The 11 restriction endonucleases produced a total of 105 restriction sites and length variations from the large single-copy region of cpDNA. Forty restriction variations are polymorphic. The cpDNA tree is largely congruent with the nuclear ribosomal ITS phylogeny. Similar to the ITS tree, the cpDNA dataset suggests the following relationships: (1)P. trifolius from eastern North America is sister to the clade consisting of all otherPanax species; (2)P. ginseng andP. japonicus from eastern Asia form a clade withP. quinquefolius from eastern North America; (3) the HimalayanP. pseudoginseng is most closely related toP. stipuleanatus of southwestern China; and (4) the medicinally importantP. notoginseng forms a clade with the closely relatedP. bipinnatifidus, P. ginseng, P. japonicus, P. major, P. quinquefolius, P. sinensis, P. wangianus, andP. zingiberensis. Two biogeographic disjunctions are detectable withinPanax. One is the connection of the eastern North AmericanP. trifolius with the rest ofPanax species. The other is the more recent disjunction between the North AmericanP. quinquefolius and the eastern AsianP. ginseng andP. japonicus. The active orogenies caused by the collision of the Indian Plate with Asia may have facilitated the diversification ofPanax taxa in Asia in the late Tertiary.     

Pollen characteristics of New Zealand Gnaphalieae (Compositae) and their taxonomic significance II. Tem

Pollen ultrastructure has been investigated in 35 taxa of Gnaphalieae (Compositae), predominantly from New Zealand. Pollen grains of all taxa examined are tectate‐columellate and caveate. The characters regarded as particularly distinctive include variability of columellar form and internal organisation of the columellae. The importance of pollen characters in the classification of the New Zealand Gnaphalieae is discussed. The species of Haastia, characterised by senecioid pollen and internal tecta, do not belong to the Gnaphalieae. In the species with helianthoid pollen and sub‐columellae five different columellae types can be distinguished.