Growth pattern and ginsenoside production of Agrobacterium-transformed Panax ginseng roots

Panax ginseng roots transformed by Agrobacterium rhizogenes grew rapidly in a hormone-free medium. The transformed roots showed biphasic growth: rapid during the first two weeks and slower thereafter. Sucrose in the medium was almost all converted to glucose and fructose during the first two weeks, and the root growth slowed down after the depletion of sucrose in the medium. Periodic changes of the medium maintained the high growth rate, and the dry weight increased by 31 times in 32 days, which is the highest growth rate so far reported for cultured tissues of ginseng. The medium exchange also increased the ginsenoside content in the roots. Effective scale-up of the root culture was achieved in a turbine-blade type bioreactor.     

Cryopreservation of Panax ginseng cells

The impacts of cryoprotectants (CP) and cell status during the growth cycle on Panax ginseng cell viability during cryopreservation were investigated. The ginseng cells used had a 5–7 times proliferation rate (compared with inoculum) in 2–3 weeks and were subcultured at 2- and 4-week intervals in liquid and on solid media, respectively. After testing various CP solutions of glycerol, dimethylsulphoxide, ethylene glycol and sucrose, a combination of 10% (v/v) glycerol and 4% (w/v) sucrose was selected for its least cytotoxicity and highest cell viability after thawing. With this CP solution, cells throughout the growth cycle exhibited a ‘U’-shaped fluctuation of post-thaw cell viability. The highest viability (86.5%) occurred during the lag phase from cells already maintained in suspension culture and then in the late exponential phase (61.7%); the lowest level of 15.4% was in the mid-exponential phase. Callus freshly transferred to liquid medium showed a less obvious fluctuation pattern. The recovered cells were brown-to-reddish at first and gradually returned to a light yellow colour after several subcultures. Received: 1 October 1998 / Revision received: 6 January 2000 / Accepted: 11 January 2000     

Anti-neuroinflammatory of Chloroform Extract of Panax ginseng Root Culture on Lipopolysaccharide-stimulated BV2 Microglia Cells

Background:It is believed that activation of microglia in the central nervous system upon detection of stimulus like lipopolysaccharides provokes neuroinflammation via the production of pro-inflammatory mediators and cytokines. The cytoprotective and anti-inflammatory properties of various folk medicine has been gaining attention as a strategy to combat various disease. This study aimed to assess the anti-neuroinflammatory properties of chloroform extract of in vitro Panax ginseng root culture based on nitric oxide and cytokines production.Methods:The study was initiated with the determination of maximum non-toxic dose (MNTD) of P. ginseng root culture chloroform extract using the MTT assay. The lipopolysaccharides-stimulated BV2 microglia cells were treated with MNTD and ½MNTD of the extract and its anti-neuroinflammatory properties were assessed by measuring the production of nitric oxide (NO) via Griess assay, as well as TNF-α, IL-6 and IL-10 using Quantikine ELISA.Results:It was found that the MNTD and ½MNTD of the extract did not play a significant role in the production of pro-inflammatory cytokines such as NO, TNF-α and IL-6. However, the MNTD and ½MNTD of chloroform extract significantly increased the anti-inflammatory IL-10 compared to the untreated cells.Conclusion:With this, the chloroform extract of P. ginseng root culture potentially exerts anti-neuroinflammatory properties.Key Words: Anti-inflammatory cytokines, Ginsenosides, Microglia cells, Neuroinflammation, Pro-inflammatory cytokines     

三七环二肽成分和人参内酰胺成分

From the roots of Panax notoginseng fourteen cyclodipeptides 1-14 were isolated including one new compound (1),seven new natural compounds (4-10) and six known compounds (2-3,11-14) together with one known other compound 15.The chemical structure of 1 was elucidated as cyclo-(Leu-Thr) based on spectral methods.From the roots of Panax ginseng five known lactams (16-20) includingpyrng lutamic acid were isolated together with butyric diacid,daucosterol and sucrose.The primary binactivity test showed that pyroglutamic acid and its n-butyl derivative have weak Ca^2 antagonistic activity.     

Studies on the Triterpenoid in Panax ginseng

Pure saponins were extrated from the roots of Panax ginseng. By means of thin-layer chromatography, twelve kinds of single saponins in ginsenosides were de- termined. According to S. Shibata's nomenclature, they are named Ginsenoside -Ro, -Ra, -Rb1, -Rb2, -Rc, -Rd, -Re, -Rf, -Rg1, -Rg2, and -Rh respectively. Sapogenin was obtained from the hydrolysis of ginsenoside. And three kinds of artificial single saponins were separated from sapogenin through Silica gel .G. According to the results of thin-layer chromatographic analysis, determination of melting point and identification of IR and NMR, the separated these three artificial single sapogenins are referred to as panaxadiol, panaxatriol and oleanolic acid respectively.     

A ribonuclease from Chinese ginseng (Panax ginseng) flowers

A ribonuclease, with a molecular mass of 23kDa, and much higher activity toward poly(U) than poly(C) and only negligible activity toward poly(A) and poly(G), was isolated from the aqueous extract of Chinese ginseng (Panax ginseng) flowers. The ribonuclease was unadsorbed on diethylaminoethyl-cellulose and adsorbed on Affi-gel blue gel and carboxymethyl-cellulose. High activity of the ribonuclease was maintained at pH 6-7. On either side of this pH range, there was a precipitous drop in enzyme activity. The activity of the enzyme peaked at 50 degrees C and fell to about 20% of the maximal activity when the temperature was lowered to 20 degrees C or raised to 80 degrees C. The characteristics of this ribonuclease were different from those of ribonuclease previously purified from ginseng roots.     

Identification of new microsatellite markers in Panax ginseng

Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The (AG)n motif was most common (23.1%), followed by the (AAC)n motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species.     

RAPD analysis of genome variability of planted ginseng,Panax ginseng

Genome variability of 23 ginseng plants (Panax ginseng) grown in culture in Primorskii Krai was studied by RAPD method. Eleven arbitrary chosen primers were used to analyze 138 loci of DNA samples, 17 of which appeared to be polymorphic. The OPD-11-1000 fragment was found to be a RAPD marker allowing plants to be differentiated according to their morphotype. Using five primers, it was demonstrated that the genetic polymorphism of the cultivated plants is lower than that in nature (7.6% and 10.6%, respectively). Dendrograms of genetic relatedness are in accord with genetic differences between individuals of plantedP. ginseng belonging to different morphotypes, and demonstrate close relatedness of one of the morphotypes to wild plants. This morphotype could be recommended for reintroduction into natural habitats.     

肝素结合性表皮生长因子在心肌重塑中的作用

肝素结合性表皮生长因子（HB-EGF）是表皮生长因子家族成员之一。HB-EGF是多种细胞的有丝分裂原,参与一系列生理和病理过程,包括心肌细胞肥大,成纤维细胞增生,胶原纤维表达增多,是心肌重塑发生发展过程中的一个重要生长因子。本文综述了HB-EGF在心肌重塑过程中的研究进展。     

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.     

云南丽江人参、西洋参的氨基酸营养

本试验对云南丽江人参、西洋参的氨基酸含量进行了分析,并与吉林人参及西洋参的氨基酸含量进行比较。结果表明,两地人参、西洋参的总氨基酸含量稍有差异,但碱性、中性、酸性氨基酸在总氨基酸中所占百分比的高低基本一致。     

Diversity of yeasts associated with Panax ginseng

Biodiversity of yeasts was investigated in the ginseng cultivation field. Among 34 isolates tested in this study, 26 isolates belonged to the hymenomycetous yeast group. These 26 strains were classified into 12 species including four new-species candidates that did not have clear affiliation to any established species. Seven isolates among the remaining strains were classified into three ascomycetous yeast species, and one isolate was identified as a urediniomycetous yeast species.     

Acetylenes from the callus of Panax ginseng

Two new polyacetylenes were isolated from dried callus of Panax ginseng. The structures of the polyacetylenes were confirmed as heptadeca-3-oxo-4,6-diyne-9,10-diol and its dihydro derivative by their IR, ¹H NMR, ¹³C NMR and mass spectra, and some chemical reactions. The new acetylenes exhibited growth inhibition against Yoshida sarcoma cells in tissue culture.     

A polyacetylenic compound from Panax ginseng roots

A new polyacetylenic compound was isolated from Korean ginseng roots. The structure was determined to be heptadeca-1,8-dien-4,6-diyn-3,10-diol by spectral methods.     

Release of Calcitonin Gene-Related Peptide-Like Immunoreactive Substance from Neuromuscular Junction by Nerve Excitation and Its Action on Striated Muscle

In a rat phrenic nerve-hemidiaphragm preparation, calcitonin gene-related peptide (CGRP) increased the twitch contraction induced by nerve or transmural stimulation dose dependently. Either electrical or high K+ stimulation of the phrenic nerve caused release of a CGRP-like immunoreactive substance (CGRP-LIS) in a Ca2(+)-dependent manner. Electrical stimulation of the phrenic nerve also increased the cyclic AMP content in diaphragm. This increase was not observed in Ca2(+)-free medium and was blocked by antiserum against CGRP. These results indicate that excitation of the motor nerve causes release of CGRP-LIS at nerve terminals and that the released CGRP-LIS increases the cyclic AMP content of skeletal muscles and potentiates twitch contraction.     

Mutation of Panax ginseng genes during long-term cultivation of ginseng cell cultures

It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during the long-term cultivation of transgenic cell cultures of Panax ginseng. In the present report, we analyzed the nucleotide sequences of selected plant gene families in the 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We sequenced the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL) and dammarenediol synthase genes (DDS), which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK) genes, which control plant development. We demonstrate that the plant genes also developed mutations during long-term cultivation. The highest level of nucleotide substitution was detected in the sequences of the SERK genes (2.00 ± 0.11 nt per 1000 nt), and the level was significantly higher when compared with the cultivated P. ginseng plant. Interestingly, while the diversity of Actin genes was similar in the P. ginseng cell culture and the cultivated plants, the diversity of the DDS and SERK genes was less in the 20-year-old cell culture than in the cultivated plants. In this work, we detail the level of nucleotide substitutions in different plant genes during the long-term culture of plant cells.     

Explant preparation affects culture initiation and regeneration of Panax ginseng and Panax quinquefolius

The influence of explant preparation on culture initiation and regeneration was investigated. Explant preparation was defined as the application of surface disinfection and homogenization to ginseng roots and embryos. Surface disinfection significantly affected culture initiation and subsequent embryogenesis. A high success rate (80–97%) in culture initiation was associated with explants from non-surface disinfected root tissue. The cumulative contamination rate after 8 months was below 4%; in contrast, a contamination rate of 85% was observed in disinfected root explants. For non-disinfected explants, visible callus was induced in 1 week, adventitious root and somatic embryos were formed in 2 and 6 months, respectively. Explants from disinfected roots required 1 month to induce visible callus, 5–7 months for adventitious roots and 10 months for somatic embryogenesis. For germinating embryos, disinfected embryos required double the time for embryogenesis than non-disinfected ones. It was considered that surface disinfection imposed a stress and subsequently a carry-over effect on explants.