Transcriptome-Wide Identification and Functional Analysis of PgSQE08-01 Gene in Ginsenoside Biosynthesis in Panax ginseng C.A.Mey.

Panax ginseng C.A.Mey.is an important plant species used in traditional Chinese medicine,whose primary active ingredient is a ginsenoside.Ginsenoside biosynthesis is not only regulated by transcription factors but also controlled by a variety of structural genes.Nonetheless,the molecular mechanism underlying ginsenoside biosynthesis has always been a topic in the discussion of ginseng secondary metabolites.Squalene epoxidase(SQE)is a key enzyme in the mevalonic acid pathway,which affects the biosynthesis of secondary metabolites such as terpenoid.Using ginseng transcriptome,expression,and ginsenoside content databases,this study employed bioinformatic methods to systematically analyze the genes encoding SQE in ginseng.We first selected six PgSQE candidates that were closely involved in ginsenoside biosynthesis and then identified PgSQE08-01 to be highly associated with ginsenoside biosynthesis.Next,we constructed the overexpression vector pCAMBIA3301-PgSQE08-01 and the RNAi vector pART27-PgSQE08-01 and transformed ginseng adventitious roots using Agrobacterium rhizogenes,to obtain positive hairy-root clones.Thereafter,quantitative reverse transcriptionpolymerase chain reaction and high-performance liquid chromatography were used to determine the expression of relevant genes and ginsenoside content,respectively.Then,we focused on the function of PgSQE08-01 gene,which was noted to be involved in ginsenoside biosynthesis.Thus,these findings not only provided a molecular basis for the identification of important functional genes in ginseng but also enriched genetic resources for the biosynthesis of ginsenosides using synthetic biology.

High-throughput transcriptional profiling of perturbations by Panax ginseng saponins and Panax notoginseng saponins using TCM-seq

Panax ginseng(PG)and Panax notoginseng(PN)are highly valuable Chinese medicines(CM).Although both CMs have similar active constituents,their clinical applications are clearly different.Over the past decade,RNA sequencing(RNA-seq)analysis has been employed to investigate the molecular mechanisms of extracts or monomers.However,owing to the limited number of samples in standard RNA-seq,few studies have systematically compared the effects of PG and PN spanning multiple conditions at the transcriptomic level.Here,we developed an approach that simultaneously profiles transcriptome changes for multiplexed samples using RNA-seq(TCM-seq),a high-throughput,low-cost workflow to molecularly evaluate CM perturbations.A species-mixing experiment was conducted to illustrate the accuracy of sample multiplexing in TCM-seq.Transcriptomes from repeated samples were used to verify the robustness of TCM-seq.We then focused on the primary active components,Panax notoginseng saponins(PNS)and Panax ginseng saponins(PGS)extracted from PN and PG,respectively.We also characterized the transcriptome changes of 10 cell lines,treated with four different doses of PNS and PGS,using TCM-seq to compare the differences in their perturbing effects on genes,functional pathways,gene modules,and molecular networks.The results of transcriptional data analysis showed that the transcriptional patterns of various cell lines were significantly distinct.PGS exhibited a stronger regulatory effect on genes involved in cardiovascular disease,whereas PNS resulted in a greater coagulation effect on vascular endothelial cells.This study proposes a paradigm to comprehensively explore the differences in mechanisms of action between CMs based on transcriptome readouts.

Activation of Plasma Membrane NADPH Oxidase and Generation of H_2O_2 Mediate the Induction of PAL Activity and Saponin Synthesis byEndogenous Elicitor in Suspension-Cultured Cells of Panax ginseng

Endogenous elicitor, termed cellulase-degraded cell wall (CDW), was prepared from the cell wall of suspension-cultured ginseng (Panax ginseng C.A. Meyer) cells via cellulase degradation. CDW activated the NADPH oxidase activity of isolated plasma membranes and stimulated in vivo H2O2 generation in ginseng cell suspensions. CDW also increased the activity of phenylalanine ammonia lyase (PAL), expression of a P. ginseng squalene epoxidase (sqe) gene and saponin synthesis. NADPH oxidase inhibitors inhibited both in vitro NADPH oxidase activity and in vivo H2O2 generation. Induction of PAL activity, saponin synthesis and sqe gene expression were all inhibited by such inhibitor treatments and reduced by incubation with catalase and HA scavengers. These data indicate that activation of NADPH oxidase and generation of H2O2 are essential signalling events mediating defence responses induced by the endogenous elicitor(s) present in CDW.

Ocotillone-type Ginsenoside from Leaves of Panax ginseng

An ocotillone type ginsenoside, together with 2 known ginsenosides was isolated from leaves of Panax ginseng and identified as pseudoginsenoside RT 5 on the basis of chemical and physicochemical evidences. It has been so far the first example of ocotillone type ginsenoside discovered in Panax ginseng and its plausible biotransformation pathway also discussed.

High-Performance Liquid Chromatographical Analysis of Ginsenosides in Panax ginseng,P.quinquef(?)lium and P.notoginseng

The compositions and contents of ginsenbsides in Panax ginseng,P.quinquefolium and P.notoginseng were determined and compared by reversed-phase High-Performance Liquid Chro- matography(HPLC).The method was performed on an Alltech Adsorbosphere HS C_(18) column,using 5×10^(-3)M NaH_2PO_4-H_3PO_4 buffer solution(pH 3.0)and acetonitrile-water(50:50)as gradient eluents. The baseline separation of ginsenosides Rb_1,Rb_2,Rb_1,Rc,Rd,Rf,Ro,and Re+Rg_1 was obtained in one analytical run.The ginsenosides are directly detected at 203 nm.The detection limit is 40μg at a signal to noise ratio of 3:1.The improved sample preparation and clean-up prior to injection with SEP-PAK C_(18)cartridge strongly reduced the front peaks caused by the impurities in the methanolic extracts of samples to afford a smooth baseline and clear background.The HPLC patterns of methanolic extracts mainly including the ginsenosides were found capable of serving as chemical fingerprints to differentiate the three species from each other.It was also found that there are no significant diffe- rences of the HPLC patterns between the wild Panax ginseng and the cultivated,the white and the red ginsengs,Chinese and Korean red ginsengs,and the tap roots of Panax ginseng collected in four consecutive months,only certain differences in contents of ginsenosides do exist.The contents of the nine major ginsenosides present in the rhizome,tap root and rootlet as well as the leaf of Panax quinquefolium were also determined and compared.

人参(Panax ginseng)根系分泌物成分对人参致病菌的化感效应

采用室内培养结合生物学测定的试验方法,研究了不同浓度人参(Panax ginseng)根系分泌物成分苯甲酸、邻苯二甲酸二异丁酯、十六酸和2,2二-(4羟-苯基)丙烷对人参立枯丝核菌(Rhizoctonia solani)、黑斑菌(Alternaria panax)、疫病菌(Phytophthora cactorum)、菌核菌(Sclerotinia schinseng)、锈腐菌(Cylindrocarpon destructans)和绿色木霉菌(Trichoderma viride)菌落生长及孢子萌发的化感效应。结果显示,不同浓度人参根系分泌物成分对人参致病菌及绿色木霉菌的化感效应存在显著差异。苯甲酸浓度与人参立枯丝核菌、菌核菌和锈腐菌菌落生长以及人参黑斑菌、锈腐菌孢子萌发呈负相关,与人参黑斑菌、绿色木霉菌菌落生长呈正相关;对人参疫病菌菌落生长的化感效应表现为低浓度和高浓度抑制,中浓度促进。邻苯二甲酸二异丁酯浓度与人参立枯丝核菌、黑斑菌、菌核菌和绿色木霉菌菌落生长以及人参黑斑菌孢子萌发呈负相关;对人参锈腐菌菌落生长和孢子萌发表现为低浓度和高浓度抑制,中浓度促进;对人参疫病菌菌落生长表现为低浓度和中浓度抑制,高浓度促进。2,2二-(4羟-苯基)丙烷浓度与人参立枯丝核菌、黑斑菌、疫病菌、绿色木霉菌菌落生长以及人参黑斑菌、锈腐菌孢子萌发呈负相关;对人参菌核菌、锈腐菌菌落生长表现中浓度促进,高浓度抑制。十六酸浓度与人参锈腐菌、疫病菌和绿色木霉菌菌落生长呈正相关,与人参锈腐菌孢子萌发呈负相关,对黑斑菌孢子萌发表现为中浓度抑制。4种根系分泌物的等量混合物浓度与人参致病菌及拮抗木霉菌菌落生长速率呈负相关。

人参(Panax ginseng)的氮过剩现象──人参氮毒原因初探

1985-1990年间，我们在长白县及公主岭进行的四次NPK三要素试验中，发现氮肥抑制了人参生长发育，降低了产量（5％显著水准），而倍量氮肥，减产达1％显著水平。继续研究证明，人参硝酸还原酶活力（NRA）甚低，追施氮肥使组织，积累，促进了呼吸作用，植株干物中N、Si及Cu、Zn、Fe、Mn含量增加，Ca、Mg、K、Na等阳离子减少，表现出氮毒征象。本文称氮过剩症，它是指氮素供应超过了由极低NRA催化的转化代谢速率，而引起的一种毒害现象。

Panax ginseng extract attenuates neuronal injury and cognitive deficits in rats with vascular dementia induced by chronic cerebral hypoperfusion

Panax ginseng is a slow-growing perennial plant.Panax ginseng extract has numerous biological activities,including antitumor,anti-inflammatory and antistress activities.Panax ginseng extract also has a cognition-enhancing effect in rats with alcohol-induced memory impairment.In this study,we partially occluded the bilateral carotid arteries in the rat to induce chronic cerebral hypoperfusion,a wellknown model of vascular dementia.The rats were then intragastrically administered 50 or 100 mg/kg Panax ginseng extract.Morris water maze and balance beam tests were used to evaluate memory deficits and motor function,respectively.Protein quantity was used to evaluate cholinergic neurons.Immunofluorescence staining was used to assess the number of glial fibrillary acidic protein-positive cells.Western blot assay was used to evaluate protein levels of vascular endothelial growth factor,basic fibroblast growth factor,Bcl-2 and Bax.Treatment with Panax ginseng extract for 8 weeks significantly improved behavioral function and increased neuronal density and VEGF and b FGF protein expression in the hippocampal CA3 area.Furthermore,Panax ginseng extract reduced the number of glial fibrillary acidic protein-immunoreactive cells,and it decreased apoptosis by upregulating Bcl-2 and downregulating Bax protein expression.The effect of Panax ginseng extract was dose-dependent and similar to that of nimodipine,a commonly used drug for the treatment of vascular dementia.These findings suggest that Panax ginseng extract is neuroprotective against vascular dementia induced by chronic cerebral hypoperfusion,and therefore might have therapeutic potential for preventing and treating the disease.

Ginsenoside-Rg_6, a Novel Triterpenoid Saponin from the Stem-Leaves of Panax ginseng C. A. Mey.

A novel dammarane-type triterpene oligoglycoside, named ginsenoside-Rg6 3, was isolated from the stem-leaves of Panax ginseng C. A. Mey., together with two known ones, 20(S)-ginsenoside-Rg2 1 and 20(R)-ginsenoside-Rg2 2. On the basis of chemical and physicochemical evidence , the structure of ginsenoside-Rg6 have been elucidated as 6-O-(-L-rhamnosyl-(1?2)-(-D-glucopyranosyl-dammarane-(E)-20(22), 24-diene-3(, 6(, 12(-triol.

Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS

A high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrome try（HPLC-ESI-MS/MS） method was developed for the analysis and identification of ginsenosides in the extracts of raw Panax ginseng（RPG） and steamed Panax ginseng at high temperatures（SPGHT）. A total of 25 ginsenosides were extracted include of which 10 low-polar ginsenosides, such as ginsenosides F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3 and so on, were identified according to their HPLC retention time and MS/MS data. The results indicated that the low polar ginsenosides were seldom found in RPG. For the exploration of the transformation pattern of the ginsenosides in steam processing, the standards of ginsenosides Re, Rg1, Rb1, Rc, Rb2, Rb3 and Rd were selected and hydrolyzed at a temperature of 120 oC. The results show that these polar ginsenosides can be converted to low-polar ginsenosides such as Rg2, Rg6, F4, Rk3 and Rg5 by hydrolyzing the sugar chains.

Optimizing SSR-PCR system of Panax ginseng by orthogonal design

An orthogonal design was used to optimize SSR-PCR amplification system using Panax ginseng genomic DNA as template. Four levels of five factors （DNA template, Taq DNA polymerase, Mg^2＋, primer, and dNTP） and annealing temperature have been tested separately in this system. The results demonstrated the reaction efficiency was affected by these factors. Based on the results, a stable, productive and reproducible PCR system and cycling program for amplifying a ginseng SSR locus were obtained： 20 μL system containing 1.0 U Taq DNA polymerase, 2.0 mmol·L^-1 Mg^2＋, 0.2 mmol·L^-1 dNTPs, 0.3 μmol·L^-1 SSR primer, 60 ng· μla^-1 DNA template, performed with a program of 94℃ for 5 min, 94℃ for 30 s, annealing at 56.3℃ for 30 s, 72℃ for 1 min, 37 cycles, finishing at 72℃ for 7 min, and storing at 4℃.

A new panaxadiol from the acid hydrolysate of Panax ginseng

A new panaxadiol （compound 1） was obtained from the acid hydrolysate of the total ginsenosides of Panax ginseng C. A. Meyer （Araliaceae）. On the basis of spectroscopic data and single-crystal X-ray diffraction data, its chemical structure was elucidated to be dammar-（E）-20（22）-ene-3β,12β,25 -tfiol.

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson's disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.

A new reaction system to determine nonlinear chemical fingerprint and its use in Panax ginseng identification method based on double reaction system

A new reaction system to determine nonlinear chemical fingerprint(NCF)and its use in identification method based on double reaction system was researched.Panax ginsengs,such as ginseng,American ginseng and notoginseng were identified by the method.The NCFs of the three samples of Panax ginsengs were determined through two nonlinear chemical systems,namely system 1 consisting of sample components,H2SO4,MnSO4,NaBrO3,acetone and the new system,system 2 consisting of sample components,H2SO4,(NH4)4Ce(SO4)2,NaBrO3 and citric acid.The comparison between the results determined through systems 1 and 2 shows that the speed to determine NCF through system 2 is much faster than that through system 1;for systems 1 and 2,the system similarities of the same kind of samples are≥98.09%and 99.78%,respectively,while those of different kinds of samples are≤63.04%and 86.34%,respectively.The results to identify the kinds of some samples by system similarity pattern show that both the accuracies of identification methods based on single system 1 and 2 are≥95.6%,and the average values are 97.1%and 96.3%,respectively;the accuracy of the method based on double system is≥97.8%,and the average accuracy is 99.3%.The accuracy of the method based on double system is higher than that based on any single system.

Structure Analysis of Pectin SB_(1-1) from the Root of Panax ginseng

A water-soluble pectin SB_~1-1 was isolated and purified from the root of Panax ginseng C. A. Mey. The HPLC analysis indicates that SB_~1-1 is homogenous. Its molecular weight was estimated via gel filtration to be 10000. The GC analysis indicated that it contains the monosaccharides of GalA, Gal, Ara and Rha. Their molar ratio is 2.10∶1.00∶0.12∶0.13. Partial hydrolysis with acid, pectinase treatment, periodate oxidation, Smith degradation, methylation analyses, GC/MS analyses and NMR analyses were used for the structure analyses of SB_~1-1 . The results reveal that SB_~1-1 has a lower branched structure. The main chain is composed of GalA and Gal; the inner part is α-1,4-linked-GalA; the border is 1,4-linked-Gal. Some of the 1,4-linked-GalA and 1,4-linked-Gal residues are substituted at O6. On an average, there is one branch for every ten hexose residues. The side chain is composed of 1,6-linked-Gal and 1,3,6-linked-Gal. The nonreduced end is composed of Rha, Ara and Gal. The main glycosidic link of SB_~1-1 has an α configuration.

Effects of tissue-cultured mountain ginseng （Panax ginseng CA Meyer） extract on male patients with erectile dysfunction

Korean ginseng and mountain ginseng （Panax ginseng CA Meyer） are important traditional herbal plants whose ginsenosides are generally accepted as serving to improve sexual functions, such as penile erection. We investigated the effects of tissue-cultured mountain ginseng extract （TMGE） on male patients with erectile dysfunction （ED）. A double-blind, placebo-controlled study was conducted with 143 patients experiencing ED. Over the course of 8 weeks, one group took 1 000 mg of TMGE twice a day, and the other group took 1 000 mg of placebo twice a day. The effects of the TMGE and the placebo were analyzed using the Korean version of the International Index of Erectile Function （IIEF） questionnaire. A total of 86 patients completed 8 weeks of treatment. The scores on the five domains of the IIEF after medication were significantly higher than the baseline scores in the group treated with TMGE （P 〈 0.05）, whereas no significant improvement was observed in the placebo group （P 〉 0.05）. Erectile function and overall satisfaction scores after medication were significantly higher in the TMGE group than in the placebo group （P 〈 0.05）. Erectile function of patients in the TMGE-treated group significantly improved, suggesting that TMGE could be utilized for improving erectile function in male patients.

A new compound with cytotoxic activities from the leaves of Panax ginseng C.A. Meyer

A new compound, 3,6,20（S）-trihydroxy- 12,23-epoxydammar-24-ene,6,20-di-O-β-D-glucopyranoside （1）, was isolated from the leaves of Panax ginseng C.A. Meyer, whose structural elucidation was carried out by means of spectral analysis （including IR, HR- FAB-MS and NMR）. This compound showed the moderate cytotoxic activities against U937 and HeLa cells by using the MTT method.

A New Dammarane Glycoside, Ginsenoside Ⅲ from the Flower-buds of Panax ginseng C.A. Meyer

From the dried flower-buds of Panax ginseng C.A. Meyer, a new ndnor dammaranetype triterpene saponin named ginsenoside III was iso1ated. On the basis of spectral and chemical evidence, the structure of the new saponin was elucidated as 3 -O- [β-D -glucopyranosyl (1→2 ) - βD- glucopyranosyl] - 20-O-β-D-glucopyranosyl 3 β, 12β- 20(S) -trihydroxydammar- 25 - en- 24-one.

Selecting suitable sites for mountain ginseng(Panax ginseng)cultivation by using geographically weighted logistic regression

With the well-being trends to pursue a healthy life, mountain ginseng(Panax ginseng) is rising as one of the most profitable forest products in South Korea. This study was aimed at evaluating a new methodology for identifying suitable sites for mountain ginseng cultivation in the country. Forest vegetation data were collected from 46 sites and the spatial distribution of all sites was analyzed using GIS data for topographic position, landform, solar radiation, and topographic wetness. The physical and chemical properties of the soil samples, including moisture content, p H, organic matter, total nitrogen, exchangeable cations, available phosphorous, and soil texture, were analyzed. The cultivation suitability at each site was assessed based on the environmental conditions using logistic regression(LR) and geographically weighted logistic regression(GWLR) and the results of both methods were compared. The results show that the areas with northern aspect and higher levels of solar radiation, moisture content, total nitrogen, and sand ratio are more likely to be identified as suitable sites for ginseng cultivation. In contrast to the LR, the spatial modeling with the GWLR results in an increase in the model fitness and indicates that a significant portion of spatialautocorrelation in the data decreases. A higher value of the area under the receiver operating characteristic(ROC) curve presents a better prediction accuracy of site suitability by the GWLR. The geographically weighted coefficient estimates of the model are nonstationary, and reveal that different site suitability is associated with the geographical location of the forest stands. The GWLR increases the accuracy of selecting suitable sites by considering the geographical variations in the characteristics of the cultivation sites.

后熟阶段的人参(Panax ginseng C.A.Mey)种子对脱水和贮存温度的反应

完成形态后熟和生理后熟期中的人参（PanaxyinsenyC．A．Mey）湿润种子的含水量由47％降至9．4％和5．8％，形成于籽后保存于室温，0～5℃和-196℃条件下1～3年。保存后的形态后熟于籽再低温处理，继续完成生理后熟。结果显示：完成形态后熟的种子在1～5℃保存1年后，种子胚根能萌发，但胚轴、胚芽不能正常生长，保存3年，种子丧失生活力；生理后熟期中的种子在0～5℃保存1年尚能正常出苗，出苗率为44．0％，保存3年，种胚已不能正常发育。但是在-196℃保存3年后，出苗率同保存在0～5C1年的相近．仍为43．1％。说明完成形态后熟种子还没有产生足够的脱水耐性，而经低温层积处理于生理后熟期中的种子就具有了这种耐性，并能进行低温短期或超低温长期保存。